Research Highlights of the Aravind group

On the origins of the bacterial transcription apparatus

2012-01-21T22:58:00.001-05:00

Many years ago, a little after the first RNA polymerase structures were solved, we obtained several remarkable insights into the core transcription apparatus of life. We were the first to show that the RNA polymerase subunits, cognates of the bacterial beta and betaprime subunits, contain recognizable, evolutionarily conserved domains and that each of these subunits contribute a double-psi beta barrel domain to the active site. We also showed that the polymerase subunits accreted several other domains in a lineage-specific manner, which differ between the archaeo-eukaryotic and the bacterial subunits, and even within the bacterial versions. Our study also established the common origin of the RNA-dependent RNA polymerase involved in RNAi and the cellular DNA-dependent RNA polymerases (Click to access [Reference1] [Reference 2]).

Recently, we conducted a reanalysis of the bacterial transcription apparatus and from this study emerged several new insights that have refined or redefined our thinking on the origins of the transcriptional apparatus [Click to read]. Some of these new findings were discussed at greater length with a leading researcher in the field of transcription and a part of the correspondence is reproduced below as questions and answers.

Question: One of the new points uncovered in this study is the shared evolutionary ancestry of the archaeo-eukaryotic TFIIB and the bacterial sigma factor, based on structural similarity of the cognate HTH domains that interact with similar sites on the archaeo-eukaryotic and bacterial RNAP, respectively. Is the homology in any way reflected on the sequence level?"

Answer: The simple answer to the question is yes -- we can detect using different sequence profile methods statistically significant sequence similarity between the TFIIB and Sigma HTHs. In conclusion there is no doubt about their evolutionary relatedness and descent from a common ancestor (for example a comparisons of the HMMs of archaeal TFIIB orthologs with Sigma70-like superfamily using profile-profile comparisons; e.g. HHpred; gives p=1.8e-6 and probability of 86% and many more such lines of support). .

Question: Could the similarity between the transcription factor-RNAP interactions in the bacterial holo-RNAP and the RNAPII-TFIIB / RNAP-TFB complexes be a case of convergent evolution?

Answer: Several aspects of the interactions of the bacterial and archaeo-eukaryotic RNAPs are very likely to be convergent and we have no counter-argument in this regard. The main point is the orthology of sigma and TFIIB despite being distantly related (which seems likely now to us).

Question: The current consensus in the field is that there are no real sigma homologs in archaea, or eukaryotes. It is argued that the LUCA RNAP could have initiated in a transcription factor-independent manner, and that the sigma and TFIIB/TFB-related factors emerged in evolution following the split of the bacterial and archaeo-eukaryotic lineages.

Answer with a bit of history from LA): Many moons ago in our early days of sequence analysis we had studied the HTHs in considerable depth. One thing that became clear was that all these HTHs, be it sigma or TFIIB certainly shared a common origin (a view articulated in these papers that we wrote several years later pmid: 10556324 [Click to access]and another in 2005 pmid: 15808743 [Click to access]). As a result of these investigations it became clear that TFIIB (of course including TFB)/cyclin/RB and sigma are *real homologs*, but throughout that period the issue remained as to whether they were *real orthologs*. The reason being many other basal HTHs also show significant similarity to each. Of course, we could rule out things like TFIIE wHTH and MBF-like 4-helical HTHs from contending for ortholog-hood with sigma because they belong to different lineages of HTHs that have their own clear-cut bacterial cognates. But sigma remained unclear. In course of the above mentioned papers, I took a stance that indeed sigma and TFIIB, while being genuine homologs, were independent recruitments as basal TFs which interacted with the RNAP. But since 2005 we got an opportunity to understand the RNA polymerase evolution better using the template of our earlier studies on these proteins (12553882 [Click to access], 15194191 [Click to access]) aided by the various versions from diverse selfish elements that offered potential evolutionary intermediates. So in conclusion it became clear that they began as RNAPs that could have initiated transcription factor-independently, especially given that they lacked any specially adaptation to interact with TFs or had inbuilt HTH domains that might have substituted for the TF. But the beta cognates of the RNAPs of cellular life were unified by one striking synapomorphy in the form of the insertion of the SBHM within the catalytic DPBB domain that could not have been convergence. The emergence of this insert would indicate the emergence of interactions of a DNA-bound TF as it plays this role in all the three superkingdomains of life and is absent in the RdRP-like RNA polymerases (e.g. YonO) and RNAPs of selfish elements such as the NCgl1702-type RNAPs. This, taken together with the homology of the sigma and TFIIB, and the fact they have double HTHs, made us reconsider our former position and accept the more simple explanation of sigma and TFIIB being orthologs, albeit distant in sequence. Of course this divergence in sequence is not surprising with lot of independent action happening around them such as emergence of TBP in the archaeo-eukaryotic lineage etc.

Question: If the primordial ribozyme RNAP evolved into the extant multisubunit RNAP by recruiting a dimeric DPBB protein cofactor which usurped the active site, and over time increased the subunit complexity to result in the extant multisubunit RNAP, where does that leave the single subunit enzymes? Did they emerge later, earlier, or at the same time? Different members of extant single subunit nucleic acid polymerases have all these activities (RNAPs, DNAPs, RT etc.). Assuming that they would have predated multisubunit RNAPs, when did the change of guard occur, and for what functional reasons/selective advantages?

Answer: Currently we can list the following major independent inventions of RNA polymerase activity:
Within the RRM-like fold or the classical palm-containing polymerases: 1.1) The RNA viral RdRPs; 1.2) the THG1 (5'->3')-CRISPR-like RNA polymerases (at least some are RdRPs) and the 1.3) Phage T7-like RNAPs. Within the RRM-like fold with a flange: 2) archaeo-eukaryotic type primases. Within the TOPRIM fold: 3) DNAG-like primases. Within the pol-beta fold: 4) CCA-adding enzyme-poly A polymerase-like. Within the DPBB fold: 5) The double barrel RdRPs and DdRPs.

While there were many inventions of RNA polymerases, the following observations seem to hold: The RNAPs in the group 1.1 are the main replicative enzymes that replicate RNA in independent replicons. While the double-psi barrel RdRPs replicate small RNAs in the eukaryotic RNAi system, there is no evidence currently for them being dominant replicative enzymes of large replicons. The RdRPs in group 1.1 are further closely related to the replicative reverse transcriptases, which appear to have a single origin. On the other hand, representatives from 1.1, 1.3, 2, 3 and 5 can be associated with replication in the context of the synthesis of the RNA primer for DNA replication. Additionally, the primpols from group 2 can replicate DNA after initiating it with a RNA polymerase activity for priming. We are of the opinion that indeed RNA was more likely the primary nucleic acid (supported by: 1) its catalytic and replicative capacity; 2) its association with polypeptide templating, and 3) the priming problem making DNA a difficult starting genome. This conclusion, combined with the above observations regarding the RNAPs of group 1 and the relationship to RTs, leads us to propose that the polymerases from the 1.1. group were the first to emerge. They enabled the rise of DNA genomes with the origin reverse transcribing ability as they radiated. The emergence of DNA in turn offered a new niche for RNA polymerases due to the priming problem. This selective force appears to resulted in the emergence of multiple RNA primer synthesizing enzymes (early representatives of 1.3, 2, 3 and 5) as evidenced by the above observations. Even at this stage it is possible that there was a reverse transcribing intermediate in replication, which also helped solve the transcription problem for DNA replicons. The rise of large DNA replicons appears to have placed the pressure for transcription-specific RNAPs. This unique niche appears to have favored two major groups of RNA polymerases -- 1.3 and 5, but in the lineage leading to the cellular replicons 5 seems to have dominated. We suspect that the elements of the architecture of the double-psi beta barrel polymerases allowed them to be more effective transcription enzymes due to: 1) their ability to initiate transcription at internal sites independently of a replication origin signal for which the other enzyme were optimized; 2) their offering interfaces for regulation -- in particular the distinctive bihelical extension preceded by two extended segments forming a standalone haripin in beta-prime. The latest analysis of the evolution of double-psi beta barrel RNAPs suggests that they two began as a fusion of two DPBBs in a single polypeptide followed by a split prior to LUCA.

Question: Could these accretions have been responsible for improved regulatory potential or higher fidelity? In that context it is noteworthy than no single subunit RNAP can 'backtrack' and undergo transcript cleavage.

Answer: The addition of subunits, basal TFs and SBHMs and other domains do clearly point in the direction of continuous evolution favoring higher fidelity and regulatory potential. In particular it might have helped provide robustness to this central cellular system in face of mutational "attack" -- over-engineering.The last point of the question is of note and might have been a selective force in the later evolution of the RNAPs.

Question: Since the RNAP are predicted to have their origins as ribozymes and went through an RNA-protein stage, why is the ribosome apparently slower in losing its RNA components, as compared to nucleic acid polymerases.

Answer: First, regarding the ribosome where the RNA plays a role in peptidyltransfer: We have recently extensively studied the emergence of peptide bond forming activity in protein enzymes (pmid: 20023723 [Click to access], 20678224 {Click to access]). There were at least 11 independent inventions of peptide ligase activity, but an examination of each of these suggest that they are unable to handle the reaction in an amino acid independent manner. This inability of the protein peptide ligases might have allowed the RNA to persist. Further, a look at the other ancient ribozyme RNAse P suggests that shape selective recognition of nucleic acid structures, which is a feature it shares with the ribosomal RNAs might be a key factor that cannot be entirely reproduced by proteins. In these cases the ribozymes certainly would persist. Further RNA is also a better scaffold than proteins in certain contexts and it continues to be used as such in contexts like the eukaryotic Polycomb RNAs and HOTAIR. So, we do not see a need for RNA to be displaced in every case. Our original ribozyme displacement hypothesis was based on the observations like: 1) Several of the ancient enzymes are homologs of non-enzymatic ancient domains that bind RNA and 2) In cases like RNAseP, the protein component increases the catalytic rate of the ribozyme by potentially increasing local affinity metal ion. This offers a pre-adaptation for the protein acquiring metal-binding dependent catalysis. Now, given the new information on the evolution of the doublepsi beta barrel RNAPs, it appears that the RdRP activity might be a secondary innovation. Hence, it is conceivable the DPBB domains were merely nucleic acid binding cofactors in an already protein dominant world and its associated nucleic acid might not have had any catalytic activity. It is becoming increasingly likely that a RNA only world was probably never there (i.e. independent of proteins) and early RNAs at best had restricted catalytic capabilities in the RNA world. It is even possible that right from the beginning the basic reciprocal catalytic cycle involved early RNAs catalyzing peptide-bond formation and protein synthesis (precursor of the ribosome) and the proteins in turn catalyzing the formation of the phosphodiester bond and RNA synthesis.

Question: What about the evolutionary origins of the TBP fold, and of TBP itself? The single fold itself can be found in RNaseHIII and DNA glycosylases but it has not been demonstrated to mediate any direct interactions with DNA or DNA, that emerged later, with TBP in the archaeo-eukaryotic lineage. What happened before that, did the LUCA RNAP initiate TFIIB-sigma dependent?

Answer: TBP belongs to the larger helix-grip fold (pmid: 11276083 [Click to access]) that includes proteins with various binding capabilities. When we first showed the relationship between TBP and the RNAseHIII N-terminal domain in 2001 (pmid:11582786 [Click to access]), it was the closest to TBP within the helix grip fold. However, since then we found another member of the fold, CCTBP that is as related as the one in RNAseHIII to TBP (PMID: 19089947). Both these are much closer to TBP than the version in the DNA glycosylases. Hence, the evolution of TBP is to be understood in the context of these related domains. Of these the CCTBP is involved in sulfotransfer along with ubiquitin like proteins. The evidence does suggest that the RNAseHIII TBP domain might interact with DNA-RNA hybrid molecules. Hence, it appears that during the radiation of the TBP family it acquired very distinct activities, but the one associated with primer degradation or RNA-based DNA restriction is a more likely candidate for precursor of TBP the basal TF than CCTBP, which is associated with distinct metabolic activities. However, this might change if a nucleic acid binding activity is demonstrated for the CCTBP domain.

A mystery pathway in prokaryotes

2011-10-29T19:17:00.001-04:00

Computational studies of proteins have greatly contributed to our understanding of the biology of a species or a system. In many instances, computational analyses have solved tricky biochemical problems (e.g. the biochemistry of pupylation), or have uncovered unexpected systems or pathways (e.g. the prokaryotic cognates of the eukaryotic ubiquitin pathway), or solved long-standing mysteries (e.g. the principal transcription factors of apicomplexa), or clarified difficult evolutionary problems (e.g. the extent of lateral transfer between prokaryotes, the evolutionary origins of the AID/APOBEC deaminases). Yet there are instances, when the biochemistry of most parts of a system are easily identifiable, but the biology remains an unsolved puzzle. Recently, we uncovered one such widespread system present in most lineages of proteobacteria, actinobacteria, spirochaetes, cyanobacteria, chlamydiae and chloroflexi and also some crenarchaea. As the system is present in Mycobacterium tuberculosis, we shall use the Mycobacterial gene names as representative identifiers. The basic system consists of

Rv2410c (DUF403 in Pfam 25) : An alpha-helical protein,called Alpha-E that contains an internal duplication with each repeat possessing conserved ER motifs. Click here to access a multiple alignment.
Rv2411c (split as DUF404+DUF407 in Pfam 25): A circularly permuted peptide ligase of the ATP-grasp fold.
Rv2409c, Rv2569c: Transglutaminases that could serve either as a peptidase or a classical transglutaminase.
Rv2568c (DUF2248 in Pfam 25): A metallopeptidase-family peptidase.
Rv2567: An inactive circularly permuted ATP-grasp fused to the Alpha-E domain.
Rv2566 (Transglut+DUF2126 in Pfam 25): A transglutaminase fused to a circularly permuted peptide ligase of the COOH-NH2 ligase superfamily.
Some species additionally contain an NTN hydrolase related to the proteasomal peptidase (called Anbu in one study) in the gene neighborhoods (not Mycobacterium) and amidotransferases of the GAT-I family. Click here to access all operons.

Thus, these systems together include two active peptide ligases, 5 distinct types peptidase-like proteins (2 transglutaminases, Zincin-like metallopeptidase, the GAT-I domain and a NTN peptidase) , the mystery Alpha-E protein and an inactive peptide ligase that may be fused to the mystery Alpha-E domain. In any case all systems minimally contain at least one peptide ligase, the Alpha-E protein and one peptidase-like domain. The only evidence for its biological context comes from experiments in Pseudomonas putida where the transglutaminase is highly expressed upon nitrogen starvation. Several protein/peptide conjugation systems contain peptide ligases (e.g. the ubiquitin transferring enzymes, the Pup ligases) as well as deconjugating emzymes (e.g. JAB deubiquitinase and Dop depupylase) in the same gene context (For a comprehensive set of examples, read our paper on amidoligases).
However, assembling the pieces of the puzzle together, we can be sure of a few things

This is not involved in amino acid or glutathione biosynthesis. The species containing this system typically have intact pathways for glutathione or amino acid biosynthesis. Also there are no other genes suggestive of metabolic function in the neighborhood.
It is not involved in the biosynthesis of a distinctive secondary metabolite such as an antibiotic or siderophore, for it lacks characteristic associations seen in these systems (see examples in our study of such systems).
There is no evidence of a small protein that is conjugated to a target as in ubiquitination or pupylation.

Gene neighborhoods of the novel system described in this post

Thus the system appears to be a novel peptide transfer/peptidase system with the Alpha-E protein playing a central role. We postulate that the ATP-grasp and COOH-NH2 ligase in this system catalyze two distinct peptide bond formations. It is tempting to speculate that the Alpha-E protein with the highly conserved ER motifs serve as a substrate for elongation of a peptide via the gammacarboxylate of its side chain. This proposal is consistent with the use of glutamate side chains as substrates in eukaryotic proteins such as tubulin by peptide tagging ATP-grasp enzymes.The presence of two peptidase genes in most of these operons suggests that two successive peptidase reactions are necessary for removal of the peptide product.
Alternatively, the transglutaminase superfamily protein might indeed function in cross-linking the peptide to lysine side chains or other amino groups. Thus, the weight of the contextual evidence supports a role for this widespread conserved gene-neighborhood in peptide synthesis; the resulting peptide could be added as a tag to the unique Alpha-E protein in this system.Such a tag could either regulate the assembly of complexes of the alpha-E domain protein via cross-linking or its interactions (e.g. as in tubulin) or serve as an amino acid storage mechanism. Yet, as you can see, certain details of this interesting pathway are in need of further investigation, but its widespread presence suggests that an important and exciting piece of biology awaits creative experimentalists...

Bacterial O-antigens, capsules, and cell-surface polysaccharides: not just all-sugar

2011-10-29T01:23:00.001-04:00

You probably heard of Escherichia coli O104:H4, which caused a devastating outbreak of an enterohemorrhagic disease in many European countries this year. Did you ever wonder what the O and H in the name represent? In the pre-genome sequence era, enterobacteria were usually distinguished based on the type of their polymorphic surface antigens by a process called serotyping. In this, antibodies that specifically recognized a distinct type of surface antigen were used to identify the bacterial serotype. This was an extraordinarily successful tool in epidemiological studies. In enterobacteria, the polymorphic surface molecules are typically a surface lipopolysaccharide (O-antigen), flagellar proteins (H antigen) and/or the capsular polysaccharide (K-antigen). Thus the O104:H4 in the E.coli strain name refers to the type numbers of the O and H antigens respectively. E. coli has about 700 serotypes combined from some 180 O-antigens, 70 K-antigens and 54 H-antigens. Salmonella has about 2500 serotypes! Below we highlight a new twist to the O-antigen structure that we recently uncovered in our study on peptide ligases.

Let us study the Lipopolysaccharide (LPS), of which the O-antigen is a component, in some more detail (see figure below).The LPS is comprised of four parts. 1) Lipid A, a lipid anchor that forms the outer monolayer of the outer membrane and anchors the LPS, 2) an inner core composed of characteristic sugars such as Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) and a heptose, 3) an outer core typically containing hexose sugars, and finally (4) the O-antigen repeats that exhibit variations in the type and arrangement of the sugar residues within the O-unit of LPS (see figure below). Some O-antigens have repeats of 3-5 sugar units, others are branched with 4-6 sugar units. Also present are unusual sugars only seen in these surface antigens.The number of such repeats also greatly vary (See the O-antigen database). Estimates suggest that there are about a million LPS molecules sticking out from the outer membrane per E. coli cell. The variations are a means for the bacterium to escape the surveillance of the host immune system and function as a virulence factor. Additionally, the antigens might vary to avoid bacteriophages that target the O-antigen for attaching and invading the bacterial cell. The genes involved in the biosynthesis of the O-antigen are present in a large gene cluster and not unexpectedly show great variations between various O-antigen types. Many of these are involved in the biosynthesis and export of the sugar units in the LPS.

O-antigen structure (from Raetz and Whitfield)

In a recent study, we noticed a somewhat unexpected presence in these gene neighborhoods-- peptide ligases. The proteins encoded by the E.coli/Shigella wfdG and wfdR O-antigen cluster genes (incorrectly labeled as glycosyltransferases) are members of the ATP-grasp superfamily of peptide ligases. Members of this family are present widely across bacteria, e.g. firmicutes, actinobacteria, proteobacteria, spirochaetes, bacteroidetes, fusobacteria and cyanobacteria. Interestingly, they are also present in the capsular biosynthesis locus of Streptococcus pneumoniae (e.g. wcyv). In general, this family of peptide ligases are combined with genes that encode proteins involved in biosynthesis of cell surface polysaccharides. In some instances members of this family are fused to other domains such as glycosyltransferases and the capsular biosynthesis-type PP2A-fold phosphatases. Often these neighborhoods encode multiple paralogous copies of ATP-grasps (access the operons here). Pioneering studies in Proteus and Providencia (e.g. Kocharova et al. and Kondakova et al) have shown that sugars of the cell surface O-antigen are further aminoacylated by D- and L-aspartic acid residues. Given the presence of ATP-grasp genes in these operons, we predict that they would catalyze the ligation of amino acids to sugar moieties in these polymers, as observed in these studies.

One other cell surface polysaccharide with known sugar-amino acid conjugates is teichuronopeptide, a highly acidic copolymer of glucuronic acid and amino acids such as glutamate that contributes to alkaliphily of organisms such as Bacillus halodurans. Experimental studies by Aono had implicated the TupA gene in the biosynthesis of this product but the mode of action was not understood until we unified TupA to the same family of ATP-grasps (TupA-like) present in the O-antigen and capsule biosynthesis loci. We predict that this is the ligase required for synthesis of the polyglutamate portion of the teichuronopeptide. The Teichuronopeptide synthesis locus additionally contains three paralogous ATP-grasp genes (see operons here). A comparable combination of gene neighborhoods is also seen in alkali resistant bacteria such as Dethiobacter alkaliphilus and Oceanobacillus, and the polycyclic aromatic hydrocarbon degrading Mycobacterium sp. JLS. This suggests that the teichuronopeptide-like polymer might have been an important solution to the problem of high alkaline or salt conditions. The lateral transfer of this neighborhod might have been important in the emergence of alkali resistance in various distantly related bacteria.

Teichuronopeptide unit

The wide phyletic distribution of this ATP-grasp-centered and related operons suggests that sugar/sugar acid and amino acid conjugates are a common feature of the capsules and other distinctive cell surface polymers of a large number of bacteria. The presence of up to four ATP-grasp genes in some of these operons suggests peptide chains with complexity comparable to the peptide linkages in peptidoglycan might be present in some of these polymers. This throws an exciting twist to the composition of the cell surface polysaccharides of bacteria. The nature and type of amino acids in these various species would definitely be of great interest and importance to bacteriology and epidemiology. You can access our paper here and browse the extensive supplement here.

The remarkable story of the mutagenic AID/APOBECs and other deaminases

2011-09-03T16:34:00.003-04:00

The nucleotide and nucleic acid deaminases such as CDD1, ADAR, TadA/Tad2, AID, APOBEC and DYW catalyze deamination of nucleotide or nucleic acid bases in a wide range of contexts. Of these, some of the most remarkable ones are the nucleic acid deaminases such as AID/APOBEC, ADAR and DYW that modify bases in nucleic acids in a range of contexts such as organellar RNA editing, hypermutation of viruses and the generation of hypervariability in proteins involved in adaptive immunity. One characteristic of these that was most mystifying was the limited phyletic distribution of some families and their rapid evolution.In a comprehensive sequence-structure analysis of the deaminase superfamily, we now uncover several aspects that were previously unclear, including the overall history of the fold with respect to protein superfamilies such as the JAB peptidases. We report several new families of which the most remarkable are deaminases that serve as toxins in bacterial polymorphic toxin systems. Several new and interesting candidates in eukaryotes are also identified. Watch this space for key highlights. For now you can read the paper. Click here to access the paper. There is an extensive supplement available here.

Do bacterial pathogens secrete protein methylases to modify eukaryotic chromatin?

2011-07-04T23:39:00.011-04:00

Figure: Hartmanella grasping a Legionella (source microbe world).

In eukaryotes members of the DOT1 family rather exclusively modify H3K79, and processively methylate it to give rise to mono-, di-, and trimethylated forms. DOT1-catalyzed methylation is rather distinctive in that it is a modification that targets a residue right within the globular histone fold, rather than lysines in low-complexity tails. Unlike the histone methylations catalyzed by the PRMT family, methylation catalyzed by the DOT1 family appears to have a predominantly negative effect on gene expression across eukaryotes. Studies in mammals indicate that DOT1 is part of a large protein complex, including two pairs of paralogous proteins, all of which give rise to fusion proteins arising from chromosomal translocations in mixed lineage leukemia (MLL): (1) ENL and AF9/MLLT3, both similar to TAF14, with a N-terminal YEATS domain and a C-terminal BrC domain and (2) AF17/MLLT6 and AF10/MLLT10, both with two N-terminal PHD fingers and C-terminal AT-hook motifs.

Studies in Saccharomyces cerevisiae, supported by studies in other eukaryotes, suggest distinct roles for the di- and trimethylated forms of H3 generated by Dot1, which occur on largely mutually exclusive sets of genes. H3K79me3 occurs predominantly within the gene body (i.e., protein-coding sequence), and is largely absent in promoters and intergenic regions. This form has been associated with genes that are transcriptionally less active, and is explicitly excluded from the nucleosomes associated with the most highly expressed genes: 50% of the genes generating just 1–4 mRNAs per hour are enriched in nucleosomes showing this modification, in contrast to just 2% of the genes giving rise to >50 mRNAs per hour. The increased processivity of DOT1 in catalyzing trimethylation appears to depend on prior monoubiquitination of histone H2BK123 by the Rad6/Bre1 ubiquitinating complex. Unlike H3K79me3 levels, which do not vary greatly over the cell cycle, H3K79me2 levels change significantly with the cell-cycle, being lowest in G1 and elevated during the G2/M progression. Further, H3K79me2 is not restricted to the gene bodies and is also seen in intergenic regions, including promoters. Moreover, the genes associated with this modification tend to be transcriptionally inactive during the G2/M phase, when its levels are elevated. In trypanosomes, which possess three DOT1 paralogs, two have been functionally characterized. The first, DOT1A, mainly catalyzes H3K79me2 formation in a cell-cycle dependent manner, whereas the other paralog DOT1B appears to be involved in subtelomeric gene-silencing associated with antigenic variation in trypanosomes. In mammals, DOT1 appears to regulate heterochromatin formation at telomeric and centromeric regions, consistent with the observations in yeast and trypanosomes. In addition to its role in silencing and heterochromatin organization, other observations suggest that DOT1 methylation regulates multiple aspects of DNA repair, such as base excision repair, Rad9-mediated checkpoint function, and negative regulation of the action of the translesion repair polymerases.

A big question to us has been when and how this big player in chromatin modification emerged in eukaryotes. With the exception of the basal eukaryotes Trichomonas and Giardia, DOT1 orthologs are present in all other major eukaryotic lineages for which genome sequences are available. However, within those lineages there are certain notable instances of gene loss—while the basal plant lineages such as the chlorophyte algae and lycopodiophytes have one or more DOT1 paralogs, they have been completely lost in the crown-group land plants such as angiosperms. Within animals and fungi, typically only a single DOT1 paralog is seen and they display a largely vertical pattern of evolution. However, in the caenorhabditiform nematodes there has been a notable lineage-specific expansion (LSE) of DOT1, with at least five paralogs in Caenorhabditis elegans. It seems important to study the potential functional compartmentalization of these newly emergent DOT1 versions in this organism. Phylogenetic analysis also suggests that the precursor of DOT1A and DOT1B in trypanosomes appears to have been acquired via lateral transfer from the animal lineage (click here to access a tree from our Supplementary material). Following this transfer, it appears to have acquired an N-terminal Zn-chelating domain with four conserved cysteines, and was then duplicated to yield two functionally distinct paralogs. In microbial eukaryotes such as chlorophyte algae, stramenopiles, apicomplexans, ciliates, and trypanosomes, there appear to have been multiple lateral transfer events that have disseminated DOT1 paralogs between distantly related lineages. Consequently, some of these eukaryotes have multiple DOT1 paralogs, with particularly notable complements of three or more paralogs seen in certain stramenopiles and trypanosomes (the third trypanosome DOT1 paralog is distinct from the previously studied DOT1A and DOT1B). This presence of multiple DOT1 paralogs is rather different from the situation seen in most animals and fungi, raising the possibility that some of them might have evolved distinct substrate specificities or may regulate H3K79 methylation in alternative signaling or developmental contexts.

Thus our studies raise two key issues:

1) While DOT1 seems to perform immensely important roles across the model eukaryotes, it was probably not present in the earliest branches of eukarya, and was acquired only prior to the separation of the parabasalids and diplomonads from other eukaryotes.

2) In many eukaryotes the multiplicity of DOT1s suggests that histone methylations catalyzed by the multiple paralogous forms might have a much richer contextual “meaning” than what is seen in model systems.

This leads to the question: So, after all where did DOT1 come from?

We discovered that DOT1 is nested within a vast radiation of bacterial methylases that are involved in the synthesis of secondary metabolites such as mycolic acids in mycobacteria (including the mycolic acid cyclopropane synthases), polyether antibiotics such as nigericin (e.g., NigE of Streptomyces sp. DSM4137) and as yet uncharacterized compounds in Micromonospora (gi: 288794127; in the same operon as a SnoaL-like polyketide cyclase). Further, gene neighborhood analysis suggests that several members in this bacterial radiation (e.g., gi: 294507034 from Salinibacter ruber) are specified by a conserved operon along with an amino acid transporter. It is conceivable that these versions are involved in the utilization of particular amino acids, or metabolites derived from them (Click here to access the operons). Thus, it appears the DOT1-like group arose as part of the radiation of methylases involved in generating diversity among secondary metabolites by adding of specific methyl groups to these metabolites – a common strategy used in the arms race between antibiotic producers and their intended victims. Alternative some of them were used by bacteria probably as a strategy to utilize amines or amino acids by methylating them. Some of these bacterial forms, such as those seen in Legionella, myxobacteria, and Protochlamydia, are particular close to the eukaryotic forms and share conserved sequence motifs in both the N-terminal extended element and in the loop between strand-6 and strand-7 (e.g., the conserved aromatic residue, click here [svg] or here [txt] to access an alignment of Dot1 and its homologs). Most interestingly, of the bacterial versions closest to the eukaryotic forms, some are encoded by intracellular pathogens or endosymbionts: These include the causative agent of Legionnaires' disease and Pontiac fever, i.e. Legionella. In addition to infecting animals Legionella is a very versatile endoparasite that infects amoebozoans like Hartmannella and Acanthamoeba, heteroloboseans like Naegleria and ciliates like Tetrahymena. DOT1 homologs are also seen in Protochlamydia a dedicated bacterial endosymbiont of Acanthamoeba. Given that it was laterally exchanged between distantly related endo- symbionts/parasites points to its importance for this mode of life.

This raises the issue of whether DOT1 is used to regulate the eukaryotic host’s behavior by modifying its histones. In support of this contention we found that these versions have signal peptides that are likely to allow their secretion into the host cells. Second, unlike the other bacterial versions they also lack operonic associations with secondary metabolite biosynthesis. Hence, it would be of great interest for experimental works to test our prediction to see if these bacterial DOT1s play a role in regulating host behavior via histone methylation comparable to the endogenous DOT1. Importantly, this observation also suggests that DOT1 was originally acquired by eukaryotes from their intracellular bacterial symbionts/parasites. The other bacteria with such DOT1 homologs are the myxobacteria – we suggest that these might enable them to play in the “big league” i.e. compete with environmental eukaryotes by deploying this secreted DOT1, among other proteins as a potential toxin. You can read a detailed account of protein methylases here. Also feel free to browse the extensive supplement.

Free SAMPylation Musings

2011-06-25T14:04:00.003-04:00

SAMPylation was recently introduced by the Maupin-Furlow group at the University of Florida as a novel protein conjugation system in archaea with parallels to the ubiquitin conjugation system. SAMPylation has gotten quite a bit of attention. Attachment of SAMP1 and SAMP2 (members of the ThiS/MoaD clade, which is the basal lineage of the larger ubiquitin-like clade of beta-grasp domains) to a target protein via an E1-like ligase might be interpreted as representing a rudimentary form of the classical eukaryotic Ubiquitin (Ub) and Ubiquitin-like (Ubl) conjugation system, which attaches Ub/Ubl domains to targets via a tri-ligase enzyme cascade initiated by an E1-like ligase and completed by the E2 and E3 ligases. In forthcoming articles, we review origins of SAMPylation (among other issues) in the context of other prokaryotic protein tagging systems and the eukaryotic ubiquitin system. Given the burgeoning interest in SAMPylation, however, we’ll preview these articles with a set of extended thoughts specific to the origins of SAMPylation.

1) Phyletic distributions and phylogenetic affinities of SAMP1/SAMP2
The SAMP2 protein is clearly a member of a small branch of the ThiS clade, which is restricted to euryarchaeota, with substantial representation in the haloarchaea as well as some methanoarchaea. The SAMP1 protein, on the other hand, is clearly a member of the MoaD clade; however, the phylogenetic relationships of the MoaD clade are complicated by the repeated occurrence of gene duplications and horizontal gene transfer (HGT) events. Recently, phylogenetic relationships between bacterial genomes have been described in terms of a “thicket” rather than a linear tree due to rampant genome duplication and exchange through HGT. Analogously, one can think of the MoaD gene family as the MoaD gene “thicket”. This leads to a poor picture of the divisions among the multiple functional roles ascribed to the MoaD family at large, including the incursion of a protein tagging role in SAMP1.

2) Multiple functional roles for the MoaD homolog thicket and the ThiS clade
Despite difficulties in assigning function to uncharacterized members of the MoaD family, a wide range of functional roles have been identified for MoaD-like proteins. In fact, it is no longer appropriate to view the MoaD and ThiS families exclusively as sulfur carriers for molybdenum and thiamine cofactor biosynthesis, respectively. As well as roles for both families in SAMPylation, they are also involved in sulfur transfer during siderophore-like compound and cysteine biosynthesis and tRNA thiolation. Our group predicts additional functions for these families in tungsten cofactor biosynthesis and perhaps a tagging role in recruitment to the ClpAP complex via association with a ClpS domain. As an added twist, some MoaD/ThiS domains have the demonstrated ability to carry out more than a single functional role. The second study on SAMPylation from the Maupin-Furlow group demonstrates that in Haloferrax, the SAMP1 protein is indispensible for MoCo/WCo biosynthesis to the point that the primary role for SAMP1 is indeed be akin to the classical MoaDs to which it is closely related. Meanwhile, SAMP2 appears to be important in tRNA thiolation as well as conjugation (Comparable to Urm1). Notably, all of these functional roles are thought to be performed in conjunction with an E1-like ligase domain.

The takeaway from these observations is that the ThiS/MoaD-E1 enzyme combination has proven to be an extremely adaptable platform for acquisition of novel functional roles, primarily in the context of sulfur incorporation but also for protein tagging (as seen now in SAMPylation, Urmylation, and Ubiquitination).

3) Independent emergence of multiple protein tagging systems in prokaryotes
SAMPylation is the latest in a string of newly-discovered prokaryotic tagging systems, perhaps most well-known is the co-translational tmRNA-based peptide tag. More recently the PUPylation system has been discovered. PUPylation involves attachment of the Pup protein to target proteins via the PafA ligase, unrelated to the SAMP/Ub tag/ligase counterparts. Most relevant to this discussion is the phyletic distribution of PUPylation, detailed in our past work. PUPylation-like systems are sporadically present in a range of bacterial lineages; however, these systems very clearly emerged first in actinobacteria before spreading to other lineages via HGT. This limited distribution very much resembles the distribution of SAMP2ylation.

In addition to PUPylation, several other tagging systems are known to prokaryotes: 1) our group has pioneered in the identification of ubiquitin-like systems containing the Ub tri-ligase (E1, E2, and E3-like ligase) complement in bacteria, while another group recently identified a similar system after sequencing the archaeon Candidatus Caldiarchaeum subterraneum. 2) N-end rule arginyl/leucyl ligation. 3) Convergently-emergent protein tagging systems in diverse prokaryotes predicted in a separate paper from our group. 4) Predicted conjugation of certain members of the YukD family. Given this list, it appears tagging systems have emerged multiple times in prokaryotes. The mode of emergence of many of these tagging systems also shares commonalities, having frequently emerged first in a particular lineage of prokaryotes, only to be transferred later to additional sporadic representatives of diverse lineages via HGT.

4) SAMPylation is condition-specific
To this point, SAMP2 has been demonstrated to form conjugates with substrate proteins (and itself) only in response to two conditions: nitrogen and oxygen depletion. SAMP1 conjugation is largely restricted to substantial nitrogen depletion.

Summary and outstanding questions
The above observations begin to clarify the relationship between SAMPylation and other prokaryotic tagging systems. Phyletic distributions and peripheral or condition-specific functional roles seem to suggest prokaryotic tagging systems in general have secondarily emerged within differentiated lineages, with the ThiS/MoaD/Ub-E1 functional connection seemingly particularly suited for acquisition of tagging roles. This description is particularly apt for the SAMP2ylation system which appears to be a genuine, condition-specific adaptation restricted to a small group of archaea. One key consequence of this observation: it is highly unlikely that SAMP2ylation represents a precursor for classical Ubiquitination. The greater mystery, however, lies with SAMP1ylation. At one extreme, SAMP1ylation could occur in all MoaD family members across bacteria and archaea, directing tagged proteins for proteosomal degradation. Given the broad distribution of MoaD and the relatively restricted distribution of core proteasomal subunits this seems questionable, particularly for bacterial MoaD-like domains. At the other extreme, MoaD family members are only conjugated in genomes capable of performing SAMP2ylation; a scenario implying conjugation of SAMP1 under certain conditions was adapted from the SAMP2ylation apparatus. Addressing key points outlined below surrounding SAMP1ylation would help immensely in clarifying the Ubiquitination/SAMPylation relationship.

Q1) What is the extent to which SAMP1 is used as a modifier?
The MoaD clade thicket (of which SAMP1 is a member) has been adapted to a vast and varied range of functional roles. Further analysis of SAMP1ylation in the MoaD clade is needed to define the precise conditions and functional contexts in which SAMP1ylation, if any, is observed amongst the set of all MoaD clade proteins.

Q2) Is there a relationship between the extreme conditions required to initiate SAMPylation and the likely functional roles for SAMP1 (and SAMP2)?
SAMP1, and not SAMP2, is linked to proteosomal degradation due to the accumulation of SAMP1ylated substrates in proteosomal subunit mutant strains. Why does extreme nitrogen depletion signal for sudden degradation of massive amounts of cellular protein? As an alternative theory, could extreme nitrogen depletion cause the UbaA protein to become more “promiscuous” in recognition of ThiS/MoaD-like substrates for conjugation? In a similar vein, the function of SAMP2ylation is still not entirely clear, although it has been linked to conjugation of proteins predicted to be involved in tRNA thiolation. Is SAMP2ylation acting as a negative feedback loop regulating the primary sulfur-carrier functional role for SAMP2 in tRNA thiolation? It remains to be seen if these conjugates are subject to deSAMPylation by some of the JAB domain proteins encoded in these genomes. This might better help in clarifying the above questions.

Q3) What happens when there is overlap between SAMP1ylation and other potential tagging systems, i.e. in bacteria?
If all MoaD-like proteins are capable, to some degree, of being conjugated to other proteins under the right conditions, this would result in the overlap of SAMPylation with other protein tagging systems including some specifically involved in targeting proteins for proteasomal-based destruction. This is most notable in the actinobacteria, where unlike the overlap between PUPylation and prokaryotic ubiquitin-like conjugation systems, which appear to occupy distinct functional niches (discussed in another post here), SAMP1ylation would directly functionally overlap with PUPylation.

Final Thoughts
The recent characterization of diverse tagging systems strongly suggests protein ligation emerged several times in prokaryotic evolution. Several of these systems are known or predicted to be coupled to proteasomal degradation, specifically suggesting multiple, distinct inventions of protein tagging-directed degradation. Many tagging systems appear to have emerged later in prokaryotic evolution within certain terminal branches of the bacterial tree followed by wider dissemination to additional lineages, in varying degrees, via HGT. In particular, more research into the generality of of SAMP1ylation should be investigated in greater detail across bacteria and archaea. In the absence of support of for its generality, and given the lineage-specific nature of SAMP2, the current balance of evidence, cannot rule out, and might in fact support a scenario where SAMPylation via E1-only conjugation systems represents a separate development. This type of conjugation, especially SAMP2lyation, might have emerged in parallel to the elaboration of more complete prokaryotic ubiquitination systems that contain just E2 or both E2 and E3 ligases, rather than being precursors of these systems.

On the other hand, evidence from eukaryotes for conjugation of Urm1 suggests that indeed a protein conjugation capability might have been latent right from the ancestral Ub-like beta-grasps domains that participated in ancient biosynthetic sulfur insertion functions. It is possible that this capability was an “unintended” consequence of the chemistry of sulfotransfer, primarily coming to fore under certain special conditions, not necessarily in a functional sense – i.e., it might be viewed as a Gouldian spandrel. However, in certain organisms it was exapted to different degrees and functionally “channelized” as a protein tag. Indeed, protein tags are a necessary prerequisite for targeted protein degradation and provide an additional advantage a regulatory mechanism – these provide a “niches” into which the Ubl-E1 dependent conjugation systems could diversify as protein tags. We had earlier discussed how distinct protein tagging systems for targeting proteins for degradation are basal and nearly universal across two of the three superkingdoms of life – the tmRNA-based system in bacteria and the Ub-based system in eukaryotes. Only the archaea, which ancestrally share the proteasome with eukaryotes seemingly lack such a system. From this viewpoint, evidence for cognates of SAMPylation across archaea, based on the more widely distributed MoaD clade, certainly need to be further investigated.

Additional Reading: Click on the pubmed ids to access the references

Experimental characterization of SAMPlylation in Marlin-Furlow lab, 20054389, 21368171
Discovery of PUPylation in the Darwin lab 18832610
Evolutionary history of the PUPylation system 18980670
Evolution of Ubl pathways in prokaryotes 16859499 21547297 21169198
Higher-order relationships between five-stranded beta-grasp domain-containing protein families including ThiS, MoaD, Urm1, Ub/Ubl 17605815
Functional linkages between E1 and ThiS/MoaD, Ub/Ubl 19089947

DNA N6-adenine methyltransferases in eukaryotes

2011-06-14T18:34:00.014-04:00

The N6 methylated adenine base (N6A) in DNA is widely present in prokaryotes but relatively uncommon in eukaryotes. However, right from the earliest days, DNA N6A methylation has been identified in several eukaryotic lineages including ciliates, chlorophyte algae and dinoflagellates. In some species 0.5-10% of the adenines in the genome may be modified. Ciliates in particular have been studied as a model for the regulatory role of adenine methylation (Click here to read articles on the same). Until recently, the identity of the eukaryotic adenine methyltransferases was a mystery. A study in plants, reported a potential adenine methyltransferase, but it turns out to be an RNA methylase of the TRM11 family.

The N6A methylases and the related N4C methylases can be distinguished by a characteristic sequence signature in the active site (loop after strand-4) of the form [NDS]PP[YFW] (see structure above). However, this signature is present in both RNA and DNA adenine methylases and several RNA methylases are highly conserved across life. The classical DNA N6A methylases (N6A-MTase or Dam), appear to have emerged from the HemK-RsmC-RsmD clade of RNA methylases (see figure from our previous study). Most prokaryotic N6A DNA methylases are found in R–M systems, which have been widely disseminated via lateral transfer across distantly related lineages, but in some instances such as in the E. coli Dam protein and in the Caulobacter CcrM, they have been taken up for host function. N4C is another amine group base methylation seen in DNA, but sequence analysis reveals that they have emerged from the N6A methylases on multiple occasions independently.

In a recent study, we now report at least six distinct families of DNA N6-adenine methylases (N6A-MTase or Dam) in eukaryotes. Several of these are specified by different types of mobile elements. For example the Dictyostelium DIRS-1-like retrotransposon element encodes a N6A methylase. This element is widely disseminated across eukaryotes and expanded in several distantly-related organisms. However, except certain chlorophyte algae the methylases in most other genomes are inactive. This suggests that in most other species the methylase domain is likely to only function as a DNA-binding regulatory protein instead of a methylase. Another family found only in Trichomonas includes N6A-DNA methylases that are often fused to phage structural proteins. The CrRem1-like LTR-containing retrotransposons of Chlamydomonas encode a polyprotein with the methylase fused to C-terminal aspartyl protease and reverse transcriptase domains. Another predicted mobile element seen in chlorophytes and certain chythrid fungi contains a Dam methylase that is fused to a ParB-like HTH domain. The pervasive presence of Dam-like methylases associated with distinct groups of transposons suggests that they might act in cis to control their own gene expression and mobility through methylation of specific adenines within themselves or in their vicinity.

We also report eukaryotic N6A methylases that appear to be cellular enzymes with a role in chromatin organization. One of these, found across chlorophyte algae is a multidomain protein with the N6A methylase domain fused to one or more N-terminal BMB/PWWP and C-terminal PHD-X/ZF-CW domains. Additionally, they often contain PHD finger domains N-terminal to the methylase domain. The accessory domains function as adaptors that read the histone code. The architectural syntax suggests that these enzymes localize to specific regions of chromatin where the histones bear modified marks such as trimethylated lysines, which is followed by localized N6A or N4C methylation.

Most of these eukaryotic methylases are obviously related to the DNA N6A-methylases of mobile elements of prokaryotes, suggesting their derivation from prokaryotic mobile elements. The situation in ciliates, which are a model system for studying the regulatory role of N6A methylation in eukaryotes is quite different and interesting. Sequence searches failed to reveal any obvious DNA N6-MTase candidates. However, ciliates have a distinctive paralogous version of the Ime4-like family of adenine methylases, which is fused to N-terminal ZZ Zn-fingers, a domain of the treble-clef fold also found in chromatin proteins such as Ada2 and CBP/p300. Given that all ciliates studied to date show substantial N6mA in DNA, and have no other candidate methylases to catalyze this reaction, we suggest that these ZZ-domain containing methylases indeed perform this function. Orthologous methylases of this ciliate version are found in amoeboflagellates like Naegleria and the rhodophyte alga Cyanidioschyzon, suggesting a possibly wider distribution for this form of adenine methylation across eukaryotes.

The Ime4/MunI-like methylases have several interesting aspects. They are circularly permuted. Bacterial versions are part of Restriction-modification systems, suggesting that these are DNA methylases. In contrast, the classical eukaryotic IME/MT-A70 methylases, which are derived from bacterial methylases, are RNA methylases. Although ciliates have the regular IME4-like version, the presence of this distinct paralog and its architecture suggests a return to DNA methylation in these species!!

You can read about this in our detailed study on DNA methylation-demethylation systems (Click here).

DNA modifications in life

2011-06-14T18:08:00.002-04:00

Inspired by Gommers-Ampt and Borst.

New roles for the Clag family in apicomplexan biology

2011-06-11T23:00:00.006-04:00

The Clag family is comprised of gigantic membrane proteins, which are found in all vertebrate parasitic apicomplexan lineages except the basal Cryptosporidium. These proteins have been implicated in many aspects of parasite biology. Phylogenetic analysis suggests that there is one ancient lineage of the Clag family typified by the rhoptry neck protein Ron2, which is conserved across the “crown group” apicomplexans (Click here for a detailed review on apicomplexan adhesion modules and surface proteins). This protein localizes to the rhoptry neck and is delivered to the host cell during invasion. Once delivered to the host cell, Ron2 along with few other parasite secreted rhoptry neck proteins creates a “receptor” complex on the host cell surface. This receptor complex recognizes the APPLE domain protein Ama1 delivered via the micronemes and anchored on the parasite cell surface. This interaction then leads to the formation of the moving junction which is a hallmark of the apicomplexan invasion process. Indeed the phyletic patterns of the Ron2 and the APPLE domain proteins precisely match suggesting that they emerged coevally as part of the invasion strategy used by crown group apicomplexans. From these ancestral Ron2 proteins of the moving junction complex were derived in the Plasmodium lineage the RopH1 or Clag proteins. There are 5 paralogs of the Clag/RopH1 protein in Plasmodium falciparum, several of which have been previously shown to be exported via the rhoptries into the host cell. Each Clag associates with two other proteins RhopH2 and RhopH3 to give rise to multiple (up to 5) RhopH complexes that apparently differ in the type of Clag they possess. Phylogenetic analysis of the Clag family suggests that the RhopH1 (i.e. Clags proper) are a Plasmodium-specific clade that branched off from the Ron2s before the divergence of the P.vivax, P.yoelii/chabaudi and P.falciparum lineages. Even in the common ancestor of these lineages the Clags proper had already split into two distinct lineages: 1) The group-1 Clags which are typified by the Clag9. These are thus far found only in a single copy in each species of Plasmodium studied to date. 2) The group-2 Clags typified by Clag2, Clag3.1/2, and Clag8. These appear to undergo lineage specific expansions in some species. Thus, all the P. falciparum Clags in group-2 are part of lineage specific expansion of 4 paralogs with Clag3.1 and Clag3.2 being alternatively expressed genes encoding different versions of the Clag3 gene. Similarly, in P. vivax there is duplication with two distinct group-2 Clags. In conclusion, it appears that the RhopH complexes are likely to be of distinct groups depending on the group of Clag they contain (e.g. Clag9 type RhopH or Clag2/8/3.1/2 type RhopH).

Early studies on the Clag9 protein implicated it in the binding of infected erythrocytes to the endothelial receptor CD36. Subsequently, the RhopH complex has been shown to be important for successful growth of P.falciparum in the host; however, the exact role of this Plasmodium-specific offshoot of the Ron2 lineage has been shrouded in mystery. They do not appear to form part of the moving junction complex with the Rons, but form distinct complexes on the erythrocyte membrane. This was the state of affairs until an unexpected turn of events connected the Clags (or at least Clag3) to an arduous quest that has been spearheaded by Sanjay Desai for almost two decades. Sanjay Desai’s work has been central to the identification of a key nutrient uptake mechanism for the parasite at the host cell membrane. This is believed to occur via the plasmodial surface anion channel (PSAC). However, its molecular basis had remained unknown. Due to the technological and methodological advances in the Desai lab this channel activity was finally mapped to single parasite locus on chromosome 3, which turned out to be none other than the gene encoding Clag3. This suggested that the membrane spanning segments of Clag3 might very well constitute the structural elements of this anion channel. Previously, a mutant version of the channel, which conferred resistance to Plasmodium against the broad spectrum actinobacterial peptide inhibitor of serine and cysteine peptidases, leupeptin (N-Acetyl-leucyl-leucyl-arginine aldehyde) had been identified. Sequencing revealed that this A1210T mutation mapped to the main conserved TM helix we had predicted at the C-terminus of the protein. This conserved TM helix has a distinctive glycine in most of the Clags suggesting that it might adopt a pi-helix conformation that might be critical for channel activity. Treatment with the peptidase pronase E significantly reduced sorbitol uptake via this channel. Mapping of the peptidase site revealed that it cleaved the protein in the predicted exposed region (which hypervariable among Clags, probably due to positive selection), just upstream of this conserved helix. This supports our prediction based on sequence analysis that the helix is likely to form a transmembrane channel in conjunction with the further N-terminal TM regions. Indeed, the further N-terminal helices also show an atypical composition for TM regions suggesting that this might contribute to the formation of a peculiar intramembrane structure that typifies the channel.

This leads to the question as to whether there might be general implications for the whole Clag family (i.e. including the Ron2s). The TM regions are not particularly conserved between the Clags proper and the Ron2s. This suggests that they might not have similar roles as far as channel activity goes. However, they do share extensive similarity in their N-terminal alpha-super helical region. This, coupled with their shared tendency to translocate to the host membrane suggests that they might still possess similar functions. This extended helical region might act as a scaffold for assembly of the Ron complex in the formation of the moving junction, whereas in the case of the Clags it might contribute to the formation of the RhopH complex via interaction with the other RhopH proteins. Thus it appears that the original principles in assembly of the Ron complex were reused for the assembly of a distinct host membrane complex in the Plasmodium lineages. It is conceivable that the ancestral Ron complex also performed special roles in reorganizing the host membrane during moving junction formation via its TM helices. This may have indeed been reused in the phenomenon of nutrient uptake by the Clags. In light of this it would be interesting to study if the Toxoplasma-specific paralogs of Ron2 have been adapted for a similar function. It would also be of interest to understand how the innovation of the Ron2 complex made the invasion mechanism of the crown group apicomplexans different from that of the more basal forms like Cryptosporidium. For complete details on this study, click here.

Tête-à-tête with TETs-- Methylcytosine hydroxylation and demethylation

2011-05-31T17:09:00.037-04:00

The report of the existence of a stable hydroxymethylcytosine modification in animals and the discovery of the enzymes that catalyze this modification represents one of the great triumphs of collaborative research. It has veritably opened a new angle to epigenetic regulation and our pioneering paper in the field has over 200 citations in just under two years. Scientific papers often conceal the history, drama and effort that goes into discovery and this study too had its fair share.

Our interest in this family was kindled by the discovery by Dr. Piet Borst's group of the enzymes that catalyze an unusual base modification in kinetoplastids. Euglenozoans and their parasitic relatives the kinetoplastids contain a distinct modified thymidine derivative in their DNA; base J. Unlike several phages, where modified nucleotides are incorporated during replication, base J conversion occurs on DNA and its synthesis was shown to involve two steps. First, thymidine is hydroxylated to hydroxymethyluracil and this is then glucosylated by a glucosyltransferase to give Base J.

Two proteins, JBP1 and JBP2, were shown to be important for base J synthesis and threading analysis revealed a common hydroxylase domain of the 2-oxoglutarate and iron dependent dioxygenases (2OGFeDO) superfamily in both of them, which suggested a 2OGFeDO-like mechanism that catalyzed the first step. You can read about the discovery of base J here.

Given our long-term interest in this superfamily (see an early study), we investigated this domain using sensitive sequence analysis methods and we noticed that it was also present in bacteria, phages, fungi, Naegleria and in the animal TET proteins (e.g. CxxC6), mutations in which were known to cause various myeloid cancers. Interestingly, in animals the domain was fused to the CXXC domain that is known to discriminate methyl cytosine containing nucleotides in DNA. This gave us a clue that the TET proteins modified DNA in situ like the JBP1/2 proteins, and perhaps at cytidine instead of thymidine. Given its hydroxylase function, we speculated that it might be involved in a DNA demethylation pathway through a hydroxyl intermediate, in a 2007 paper in the International Journal of Parasitology. However, there were too many unanswered questions and a hydroxylation-based C5-demethylation of cytosine was unprecedented in vivo (although it can be achieved in vitro), as compared to N-6 demethylation through a hydroxylase intermediate (e.g. as in the AlkB reaction). Experimental investigations, thus, were key to further understanding the biochemistry and functional role of this domain in animals.

This was when Dr. Anjana Rao's group at Harvard joined the effort in trying to unravel the function of the animal homologs containing the JBP1/2-like dioxygenase domain. Dealing with these proteins is no joke as they are gigantic. They range in size from 1600-2300 amino acids. Further, TET1, TET2 and TET3 contain a distinct cysteine rich domain inserted into the N-terminal part of the 2OGFeDO domain and TET1 and TET3 also contain an N-terminal CXXC domain which is separated from the cysteine-rich and 2OGFeDO domains by a long low complexity region (Click here for a text alignment or here for a colored version). The heroic efforts of Mamta Tahiliani resulted in the purification of these proteins and the development of an in vitro system to assay their activity. Overexpressing TET1 or just a minimal version with the 2OGFeDO domain showed a decrease in 5mC levels and the presence of an distinct TLC band. This was also reproducible in the in vitro assay. In collaboration with Dr. David Liu's group the distinct nucleotide species was shown to be hydroxymethylcytosine. Hydroxymethylcytosine research was prominent in the heydays of phage biology, it now had a second birth.

The study was published in two parts that were released almost simultaneously. A comprehensive theoretical analysis of the superfamily, its phyletic distribution, evolutionary history and contextual associations was published in Cell Cycle and the experimental studies were published in Science. TET proteins are now implicated in embryonic stem cell re-programming, cell lineage specification, reprogramming the paternal genome, fine-tuning transcription and opposing aberrant methylation. Our analysis provided the first biochemical leads towards understanding the basis of myelomas caused by mutations in the TET proteins. The list is only going to increase over time. While the animal proteins have received much press, there are several gems in the waiting in other eukaryotes. More on this in the next post.

The fellowship of the RING: Bacteria have them too!

2011-05-06T06:43:00.028-04:00

If there is one lesson to learn from comparative genomics, it is to never underestimate the bacteria. Bacteria are verily the engines of protein diversity and have provided some of the most remarkable insights on the origins of various pathways and systems. In our next release on the ubiquitin-mediated signaling/ tagging/ protein turnover pathway, we address the origins and roles of several treble clef domains, including the RING finger, found in the eukaryotic ubiquitin pathway. This study also answers some tricky questions raised in a previous post.

Q1. Do any species have the entire complement of both the ubiquitin and pupylation based protein turnover/tagging system?

Indeed, some actinobacteria such as Frankia and planctomycetes such as Pirellula staleyi contain both the entire complement of the ubiquitin system and pupylation. The list of species with both systems is only expected to grow as more such genomes are sequenced. The discovery of the Frankia RING finger was a bit complicated. It turns out that the RING finger is encoded in the opposite strand to the sequence that is submitted to the database and whats more the RING is sandwiched between an E1 and E2. For details, peruse our supplement.

Q2. If so, how is the labor of protein turnover divided between pupylation and ubiquitination?

This far we can only make a reasonable guess, but this is where experiments will reveal more. There is, however, a curious aspect to the prokaryotic RING-finger containing Ub-systems. Many of the core Ub-pathway proteins have transmembrane (TM) helices. In Frankia, both the JAB deubiquitinase and the RING are fused to transmembrane helices. In Pirellula, the RING finger while containing a TM helix is also fused to a distinct domain prototyped by DUF3137, a potential solute sensor. This suggests that a major fraction of the prokaryotic RING domains might have functions related to either regulation or modification of membrane-associated proteins. While the pupylation system of actinobacteria is cytoplasmic, the Pirellula Pup ligase, like other planctomycete versions, has 4 transmembrane helices inserted within the core domain, suggesting that in some species, pupylation has a strong membrane component. Study the architectures and associations of these Ub-systems here

Q3. In light of the discovery in Caldiarchaeum, what can we say about the origins of the eukaryotic Ub-system?

The Caldiarchaeum Ub-system is remarkable in that each of its Ub-system components is very closely related in sequence and structure to the corresponding eukaryotic version. For example, the E1 protein of this archaeon contains a C-terminal Ub-fold UFD domain (that was only detected in eukaryotes to date). However, this far no other archaeon contains a complete complement of the Ub-system. Further bacterial versions of the complete Ub-system are also sporadically distributed, suggesting a strong component of lateral transfer in the dissemination of this system across prokaryotes. Therefore, we cannot be certain if the eukaryotic Ub-system emerged from a Caldiarchaeum-like system in the archaeal symbiont during eukaryogenesis. Indeed, such systems might be present in as yet un-sampled bacteria suggesting that it is not unlikely that eukaryotes acquired such a system from the primary bacterial symbiont or even via an independent lateral transfer of the operon from yet another prokaryote.

Based on this and our previous study on the ubiquitin system, we can now confidently state that systems resembling eukaryotic Ub-conjugation systems were put together to different degrees in prokaryotes during the diversification of various biosynthetic and regulatory pathways. With regards to the proteasomal association, while the core proteasomal apparatus is of archaeal origin, it is also present in various bacteria of which some possess the complete core Ub-system. Hence, it is possible that this connection between the Ub system and the proteasome developed either in bacteria or archaea, and was merely retained in eukaryotes which vertically inherited their core proteasomal complex from the archaea.

Wait.. there much more to this story. Feel free to access the paper here and browse through the extensive supplement.

Diatom shapes and biomineralization: a chromatin connection?

2011-04-30T16:25:00.052-04:00

Diatoms are fascinating eukaryotes. The belong to a distinct lineage, the stramenopiles. Peek into any water sample through a microscope and you will be sure to find them, with a great diversity of beautiful shapes and a peculiar cell wall. Apparently, diatoms were test objects for microscopic lenses in the 19th century. The cell wall is mostly composed of silica and is called a frustule. The frustule is composed of two "valves" (imagine a petridish with its cover), the upper valve is called the epitheca and is slightly larger (the petridish cover) than the lower one, the hypotheca (the petridish base). These are joined together by girdle bands. During cell division, one daughter cell inherits the hypotheca and the other the epitheca, and they both become the epitheca of the new cell. Thus one daughter is always smaller than the parent, but we wont get into how this is resolved. Immediately after cell division, a new valve is formed and biomineralized. Silica is precipitated in a specialized vesicle and this involves a remarkable group of proteins called the silaffins. So remarkable that it is worth examining a sequence of one of them.

MKLTAIFPLLFTAVGYCAAQSIADLAAANLSTEDSKSAQLISADSSDDASDSSVESVDAASSDVSGSSVESVDVSGSSLESVDVSGSSLESVDDSSEDSEEEELRILSSKKSGSYYSYGTKKSGSYSGYSTKKSASRRILSSKKSGSYSGYSTKKSGSRRILSSKKSGSYSGSKGSKRRILSSKKSGSYSGSKGSKRRNLSSKKSGSYSGSKGSKRRILSSKKSGSYSGSKGSKRRNLSSKKSGSYSGSKGSKRRILSGGLRGSM

Beyond the signal peptide sequence, the sequence is of low compositional complexity with lysine and serine residues dominating the sequence landscape. The lysine residues are heavily modified by oligo N-methyl propylamine units, hydroxylation and methylation, while the serines are mostly phosphorylated. An interesting point of note is that these modifications appear to follow a certain code based on the sequence of neighboring residues and might show species-specific differences (click to read). All this makes the silaffin a very cationic polypeptide. At acidic pH, the above conditions in the silaffins is a perfect environment for precipitating silica particles. Take a solution of silicic acid and add silaffins, and lo and behold, silica precipitates. You can read about this remarkable study by Kroger, Deutzmann and Sumper here. Further addition of free long-chain polyamines (another major component of the cell wall) and phosphates to a solution of silicic acid results in the precipitation of silica in honeycomb-like hexagonal nanopatterns resembling those seen in the diatom cell wall (click to read). So what does all this have to do with chromatin?

Above figure taken from Sumper et al. Angew Chem Int Ed Engl. 2007;46(44):8405-8.

Recently, while surveying the distribution of chromatin domains involved in protein methylation and demethylation in eukaryotes, we encountered a distinct lineage-specific expansion of SET domain proteins in the diatoms (Click here to access these proteins). Some of these, inPhaeodactylum and Fragilariopsis are fused to a spermidine synthase-like Rossmann fold domain (e.g. the Phaeodactylum protein PHATRDRAFT_42788), and one protein inFragilariopsis is further fused to a methylated lysine-binding chromo domain (Note: in the model Thalassiosira pseudonana, the orthologous spermidine synthase domain, THAPSDRAFT_21371, appears to be a solo domain). Normally, one might speculate that these proteins are involved in chromatin function. The only problem is that these diatom proteins are all secreted!

The SET domains are protein methyltransferases, and transfer methyl groups to the epsilon-amino group of lysines on proteins. The most well characterized SET domains are those that modify lysine residues in histone tails. This far, SET domain containing protein are not known to be secreted. The classical spermidine synthases transfer propylamine groups to putrescine. Putting all this together for these diatoms proteins, it is rather obvious that these secreted SET domain containing proteins are the ones most likely to catalyze lysine methylation in silaffins. The spermidine synthase domains would then catalyze transfer of propylamine units to lysines, and perhaps also contribute to the formation of free polyamines. There are at least 3 families of secreted hydroxylases in diatoms that we reported in our study on various hydroxylases (for example,the Syn9-gp54 family of 2OGFeDO dependent hydroxylases; click here). We predict that among these are the enzymes that hydroxylate lysine residues. In a sense, the silaffin modifying code is like the histone code, with species-specific differences in the diatoms. Further, silaffin sequences are very different between the completely sequence diatoms. These in turn could be the basis of the various shapes of diatoms. Think about all this when you look at a diatom under a microscope the next time. You can read about this discovery on the silaffin modifying proteins and other protein methyltransferases and demethylases here.

Where Pupylation and Ubiquitination co-occur, who does what?

2011-04-20T08:23:00.020-04:00

Recently, the authors sequencing the archaeon Caldiarchaeum unearthed a remarkable operon with the entire core of the ubiquitin system containing genes encoding Ubiquitin, E1,E2,E3 and the deubiquitinase of the JAB family(also called MPN1) (Click here to read). These proteins are remarkable in that they share several sequence features with their eukaryotic counterparts. Further, the archaeon, like all members of its clade, contains the archaeo-eukaryotic proteasomal degradation system, suggesting the presence of the basic complement of the ubiquitin-based protein turnover system.

Great progress has also been made on the biochemistry of the other peptide-tag based protein turnover system; the pupylation pathway. A large number of substrates have now been reported and we also know that the Pup-ligase (PafA) paralog (also called Dop) which in some species deamidates glutamine, is a depupylase (Click here to read).

The consequence of these studies raises some interesting possibilities and questions.

1. Do any species have the entire complement of both the ubiquitin and pupylation based protein turnover/tagging system?

2. If so, how is the labor of protein turnover divided between pupylation and ubiquitination?

3. In light of the discovery in Caldiarchaeum, what can we say about the origins of the Ub-system?

Answers to these questions are in the following post. Click here to access the answers.

The dark side of amino acyl tRNA synthetases - The case of cyclodipeptide synthases

2011-02-08T11:21:00.032-05:00

It is textbook knowledge that amino acyl tRNA synthetases (AAtRS) catalyze the esterification of an amino acid with its cognate tRNA. The charged tRNAs then incorporates the cognate amino acid into a growing polypetide during translation. However, increasing evidence from recent studies point to non-ribosomal roles for these ancient enzymes and charged tRNAs, such as in peptide metabolite biosynthesis and cell wall biosynthesis. Recently, we reported a few such distinct families and contexts, one of which is described below.

Cyclodipeptides are fascinating molecules formed by the condensation of two amino acids resulting in the formation of a diketopiperazine. Natural cyclopeptides have diverse roles as antibacterials, antifungals, antitumor and immuno-suppressive agents. Most cyclopeptides are synthesized by giant multi-domain proteins called non-ribosomal peptide synthetases. However, in an exciting recent study, a new family of enzymes that catalyze cyclopeptide formation was reported. The study showed that a distinct enzyme AlbC catalyzes the formation of the cyclodipeptide Albonoursin. AlbC was unusual in that it required charged amino acids in the form of aminoacylated tRNAs as substrates, thus defining a hitherto unreported enzyme family. The same study also reported other related enzymes involved in the synthesis of the cyclodipeptides pulcherriminic acid ( a red pigment) in Bacillus subtilis and cYY (cyclodityrosine) in Mycobacterium tuberculosis. This novel enzymatic family was called the cyclodipeptide synthase (CDPS). Click here to go to the article.

Using, sensitive sequence analysis methods, we showed that the AlbC -like CDPSs are related to Class I aminoacyl tRNA synthetases such as tyrosine tRNA synthetase and tryptophan tRNA synthetase. The CDPSs however lack the key amino acids required for ATPase activity (called the HIGH motifs) normally seen in Class I AAtRS. They instead contain a distinct constellation of residues that we predict are required to form an amide linkage ( as in cyclodipeptides) from aminoacylated tRNAs, as opposed to the adenylation followed by ester formation seen in ancestral AAtRS (See figure below). Thus these enzymes bind aminoacylated tRNAs like classical AAtRSs but catalyze a completely different reaction. The crystal structure of the Mycobacterium tuberculosis cyclodityrosine synthetase, published subsequent to our publication, confirmed our prediction. You can access this study here and the New and Views about these discoveries.

Sequence searches with CDPSs revealed them to be present in a wide phyletic range of free-living bacteria, and also in intracellular parasitic bacteria such as Legionella, Rickettsiella and certain chlamydiae. In the latter organisms, the products of the CDPSs may be involved in the survival in or manipulation of host cells. We also detected CDPSs in pathogenic fungi (where it may have a bioactive effect on its host) and in the annelid Platynereis, where the CDPS is induced as part of an antibacterial innate immune response. Thus cyclodipeptides might play a role as endogenously encoded antibiotics in the immune response of certain animals.

Contextual analysis suggests that these enzymes are found in the neighborhood of cytochrome P450s, nitroreductases, 2OGFeDO-like hydroxylases, and other peptide ligases that might further modify the cyclodipeptide (some of this has been shown experimentally). Yet another fascinating context, seen in Actinosynnema mirum and Streptomyces sp AA4, is the presence of CDPSs in the neighborhood of enzymes that suggests that it might also use aminoacyl-coAs as substrates. Get ready for a whole new world of cyclodipeptides in the coming years. You can access our article here. Feel free to peruse the extensive supplementary material and watch this space for more.

A 40 year old mystery: The identity of the wybutosine hydroxylase and other questions

2010-12-31T13:52:00.023-05:00

Figure: Occurrence of Y-base (wybutosine) in the literature using Google Ngram

Modifiedbases are particularly prevalent at position 37 of tRNA. Being adjacent to the anticodon, these modifications stabilize mRNA–tRNApairing and assist maintenance of the reading frame during translation. One such complex modified base found at this position in eukaroytic phenylalanine tRNA synthetase is wybutosine (also called Y-base or yW). Over the past 40 years various studies identified the intermediates and enzymes involved in its biosynthesis. Additionally, it was shown that precursors of this modification pathway are present in archaea, suggesting an archaeal origin for this modification.

Although wybutosine is detected in diverse eukaryotes, this base position in tRNA^Phe shows considerable variation. For example, tRNA^Phe in yeast contains wyosine in the same position whereas flies only have 1-methylguanosine. This type of variation can be attributed to gene loss, given that 1-methylguanosine and wyosine are precursors in the wybutosine biosynthesis pathway. This is also supported by the phyletic distribution of the enzymes of this biosynthesis pathway in these organisms. In contrast, mammalian liver extracts, and Geotrichum were shown to contain a further modification; hydroxy/hydroperoxywybutosine, suggesting the presence of a distinct enzyme that catalyzes this step. Until recently, the identity of this hydroxylase was not known.

Using a combination of sequence and contextual analysis, we identified the enzymatic domain involved the biosynthesis of hydroxy/hydroperoxywybutosine. The domain is often fused to enzymes involved in the biosynthesis ofwybutosine precursors and also occurs as a stand-alone domain in metazoans (e.g. C2orf60 in humans). What is remarkable is that it turned out to be a member of the JOR(jumonji-related)/JmjC superfamily. Members of this superfamily are normally characterized as hydroxylases of proteins or histone demethylases. This is the first example of an RNA substrate for a member this superfamily. A few months after our publication, this prediction was experimentally confirmed. The JOR/JmjC belongs to a lineage of protein called the 2-oxoglutarate Fe (II) dependent dioxygenases or 2OGFeDO. In turn, the core of this lineage belong to the double stranded beta helix (DSBH) fold.

This discovery actually unraveled more questions--

Q1. Are RNA substrates ancestral to the JOR/JmjC superfamily or were they derived only in the wybutosine hydroxylase family?

Answer: The use of an RNA substrate as in the wybutosine hydroxylase appears to be a derived condition in this superfamily of proteins.

Q2. What are the inter-relationships between the various JOR/JmjC families?

Answer: All studies until now only used eukaryotic members for phylogenetic reconstruction of evolutionary relationships between various JOR/JmjC families. Using a comprehensive sequence, structure and phylogenetic based approach that included bacterial sequences, we show that the eukaryotes contain 17 major lineages of JOR/JmjC proteins, that were in turn acquired on three distinct occasions from bacteria. Thus, the major groups of JOR/JmjC appear to have diversified in bacteria followed by a transfer of at least one member from each of the three clades to the eukaryotes, prior to the divergence of the heterolobosean-kinetoplastid clade and the remaining eukaryotes. The three major clades are named the histone demethylase-like, FIH1/yW-hydroxylase-like and the MINA/No66-like clade respectively.

In addressing this, we went a few steps further and were able to classify the entire double-stranded beta-helix fold. Here's an interactive site where you can play with our classification. This led to some interesting hypotheses about their evolution.

Q3. What were the roles of the bacterial ancestors of the eukaryotic JOR/JmjC?

Answer: Quite consistently, we observe that bacterial representatives of this superfamily are coded by gene clusters involved in biosynthesis of secondary metabolites, such as pyoverdine-like siderophores and peptide antibiotics. These gene clusters often encode multiple functionally linked dioxygenases, tryptophan halogenase-like oxidoreductases and other enzymes involved in non-ribosomal peptide biosynthesis and modification. Actually, some of these contexts are quite remarkable. For example, in the Synechococcus phage Syn9 one of these gene clusters encodes 10 tandem dioxygenases including the MINA/No66 homolog, Syn9-gp49. The remaining nine dioxygenases belong to the classical 2OGFeDO superfamily. Analysis of these nine dioxygenases suggests that they are all not closely related. They belong to at least five distinct families, including one distinguished by a fusion to tetratricopeptide repeats.

Q4. Are there any other distinct substrates predicted for the eukaryotic JOR/JmjC superfamily?

Answer: Did you know that there are members of the JOR/JmjC family that are membrane associated or secreted? In each of the 3 major clades in eukaryotes, we detected secreted or membrane associated proteins with JOR/JmjC domains. Some members of the FIH1 clade are fused to sulfotransferases. A distinct lineage-specific expansion of MINA/No66 like JOR/JmjC in Monosiga comprises of receptor-like proteins with extracellular JOR/JmjC domains. All of these proteins combine a JOR/JmjC domain with one or more of several extracellular domains such as cysteine-rich GCC2/3 repeats, immunoglobulin, disintegrin or SUSHI domains and with intracellular SH2 or tyrosine kinase domains. These extracellular proteins appear have been recruited for modifying cell surface proteins, probably as hydroxylases similar to leprecan and the prolyl hydroxylase. Further, the receptor-like proteins in Monosiga could also function as sensors of redox conditions that signal via intracellular tyrosine phosphorylation pathways.

Finally, a common misconception is that the N-terminal region of the JOR/JmjC domain, called JmjN in the literature is a distinct domain. Structural analysis shows this to be conserved in all members of the JOR/JmjC superfamily. Further the domain has no independent existence and merely represents a structural extension of the DSBH fold.

Wait, there is much more.... You can read about it here in detail. Feel free to browse the comprehensive supplement.

UMA and MABP domains in receptor endocytosis and endosomal sorting

2010-06-15T17:41:00.005-04:00

Elucidating interactions between ESCRT complexes and membrane proteins or lipids is critical to understand endosomal trafficking. We identified two domains with potential significance for this process. The MABP (MVB12-associated domain beta prism domain)found in animal ESCRT-I subunit MVB12, Crag, a regulator of protein sorting, and bacterial pore-forming proteins might mediate novel membrane interactions in trafficking. The UMA domain found in MVB12 and UBAP1/2 helps define a novel class of adaptor domains that might recruit diverse targets to ESCRT-I.

A key aspect of eukaryotic intracellular trafficking is the sorting of cell-surface proteins into multi-vesicular endosomes or bodies (MVBs), which eventually fuse with the lysosome, where they are degraded by lipases and peptidases. This is the primary mechanism for down-regulation of signaling via transmembrane receptors and removal of misfolded or defective membrane proteins. This mechanism is also utilized by several viruses (e.g. HIV-1) to facilitate budding of their virions from the cell-membrane. Studies on animal and fungal models have shown that the process depends on an intricate series of interactions that is initiated via ubiquitination (typically one or more mono-ubiquitinations) of the cytoplasmic tails of membrane proteins by specific E3 ubiquitin (Ub) ligases. The ubiquitinated membrane proteins are then captured into endosomes by the ESCRT system and prevented from being recycled back to the plasma membrane via the retrograde trafficking system. The ESCRT system also folds the endosomal membranes into invaginations that are concentrated in these ubiqutinated targets and catalyzes their abscission into intra-luminal-vesicles inside the endosome. This largely seals the fate of these membrane proteins as targets for lysosomal degradation. The ESCRT system itself comprises of 4 major protein complexes, ESCRT0 to ESCRT-III, which are successively involved in the above-described steps. ESCRT-0 with proteins bearing multiple Ub-binding modules is the primary sensor for the subset of membrane proteins that are ubiquitinated. Both ESCRT-I and ESCRT-II have proteins with a single Ub-binding domain and appear to be the successive recipients of the ubiqutinated cargo initially sensed by ESCRT-0. The ESCRT-II complex also contains lipid-binding modules and is likely to initiate the invagination of the endosomal membrane. ESCRT-III, which includes the conserved AAA+ ATPase VPS4 as a component, mediates the final abscission of the invaginated membrane to form the ILV. In this relay the ESCRT-I complex is a central player, for it acts as a bridge between the initial sensor of the ubiquitinated targets and membrane-binding ESCRT-II.

Domain architectures of the UMA and MABP domains/ Structure of the MABP domain

ESCRT-I is comprised of three major subunits that are conserved between yeast and animals, namely the inactive E2-ligase protein TSG101/VPS23, Vps28 and Vps37. Additionally, both fungal and animal ESCRT-Is contain a fourth subunit termed MVB12; however, the MVB12 subunits from the two lineages were found to be not homologous. The animal MVB12 was shown to be critical for receptor endocy-tosis and also virus release. Given its key role in receptor down-regulation we were interested in understanding if the lack of the homology with the fungal MVB12 might reflect the emergence of novel interactions related to the expansion of receptor-mediated signaling in animals.

Identification of the MABP and UMA domains throws light on two key aspects of vesicular trafficking. Firstly, the MABP domain could be a common denominator in the recognition of specific membrane-associated features by a functionally diverse set of traf-ficking proteins in eukaryotes and bacterial proteins involved in pore formation and cell-wall interaction. The prediction that the diverse metazoan UMA domain proteins are alternative MVB12-like proteins implies that the recruitment of ESCRT-I to endosomal structures could occur via diverse mechanisms, including the poss-ible direct recognition of membranes by the MABP domain, inte-raction with ubiquitinated peptides or other protein-protein interac-tions. The UMA domain is currently only detectable in metazoans and appears unrelated to the yeast MVB12 protein. This is consistent with the vast expansion of diverse signaling receptors such as receptor tyrosine kinases, ion channels and 7TM receptors in the metazoan lineage and indicates the emergence of a dedicated receptor attenuating system that functions via ESCRT-I. Intriguingly, we found that plants (e.g. ArabidopsisAT5G53330) have a conserved protein that has a series of C-terminal UBA domains closely related to those found in UBAP-1/2. While we failed to find statistically significant similarity between the N-terminal region of these plant proteins and the UMA domain, they share a few tantalizing sequence patterns. Hence, it cannot be ruled out that these plant proteins contain a region remotely related to the UMA domain and perform a comparable function in relation to the ESCRT complex.

While certain core components of the ESCRT complex (e.g. VPS4 and MIT domains of ESCRT-III) have been traced to archaea, the MABP domain is not currently found in any archaea. Instead it is found in several bacteria, suggesting that the eukaryotes could have potentially acquired it early in their evolution from a bacterial precursor. Thus, the eukaryotic vesicular trafficking system appears to have been pieced together from different components acquired from both archaeal and bacterial precursors.

Sensory domains in bacterial signal transduction

2010-03-18T16:04:00.002-04:00

If your are interested in the structure, evolutionary history and phyletic patterns of domains involved in bacterial signaling then you may peruse the following comprehensive overview by us:

Sensory domains in bacterial signal transduction

Chemical network analysis reveals a system of chromatin and trafficking level backups in the cell

2009-12-22T18:46:00.004-05:00

Chemical genetics in yeast has shown great potential for clarifying the pharmacology of various drugs. Investigating these results from a systems perspective has uncovered many facets of natural chemical tolerance, but many cellular interactions of chemicals still remain poorly understood. We integrated several independent chemical genetics datasets with protein–protein interactions and a comprehensive collection of yeast protein complexes. We found potential targets and mode of action of certain poorly understood compounds. Intriguingly, the majority of the complexes in our network probably perform indirect roles in countering deleterious effects of chemicals. We propose that they form underlying buffering system that has been so far over-looked. Such complexes are basically composed of two classes: chromatin and vesicular dynamics. The former set of complexes seems to act by setting up or maintaining transcriptional programs necessary to protect the cell against chemical effects. On the other hand, the latter include specific vesicle tethering complexes, indicating that different chemicals might be routed via different points in the intracellular trafficking system. We propose a general operational similarity between these complexes and molecular capacitors (e.g. the chaperone Hsp90). Both have a key role in increasing the systems robustness, although at different levels, through buffering stress and mutation, respectively. It is therefore conceivable that some of these complexes identified here might have roles in molding the evolution of chemical resistance and response.

The yeast Ubiquitin network

2009-12-22T18:36:00.004-05:00

In this paper we assembled a comprehensive network of the ubiquitin system (Ub-system), namely ubiquitin-like proteins, their conjugation and deconjugation apparatus, binding partners and the proteasomal system. To achieve the best possible network, we integrated numerous ubiquitination/sumoylation datasets with public protein/genetic interaction databases. To aid in the data analysis, we devised two novel representations, the rank plot to understand the functional diversification of different components and the clique-specific point-wise mutual-information network to identify significant interactions in the Ub-system. Using these representations, we found supporting evidence for the functional diversification of SUMO-dependent Ub-ligases. We also identify novel components of SCF complexes, receptors in the ERAD system and a key role for Sus1 in coordinating multiple Ub-related processes in chromatin dynamics. We also identified several modified transcription factors, suggesting an extensive regulatory impact of the Ub system in the cellular networks. Furthermore, the dynamics of the Ub-network suggests that Ub and SUMO modifications might function cooperatively with transcription control in regulating cell-cycle-stage-specific complexes and in reinforcing periodicities in gene expression. Combined with evolutionary information, the structure of this network helps in understanding the lineage-specific expansion of SCF complexes with a potential role in pathogen response and the origin of the ERAD and ESCRT systems.

To read more go here

Peptide tagging and modification systems in prokaryotes: exploring the base of the iceberg

2009-12-22T17:25:00.009-05:00

In a previous study, we had uncovered several prokaryotic systems encoding homologs of E1, E2 and Ub, pointing to the presence of ubiquitination-like systems in prokaryotes. We had also described several poorly characterized peptide modification systems involving E1-like enzymes. More recently, we uncovered the enzymatic basis of pupylation, a peptide tagging system found in some bacteria. In a comprehensive study, we now describe over 20 novel systems involved in peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. These systems involve one or more enzymes of the ATP-grasp, the COOH-NH2 ligase and acetyltransferase fold.

Some highlights include:

1. A potential peptide-tagging system involving a circularly permuted ATP-grasp domain and circularly permuted glutamine synthetase-like ligase, an NTN-hydrolase fold peptidase and a
novel alpha helical domain.

2. The elucidation of the biochemical mechanism of key steps involved in the synthesis of various peptide antibiotics and cell surface polysaccharides like teichuronopeptides.

3. Discovering the first prokaryotic orthologs of the eukaryotic tyrosine tubulin ligases (TTL).

Watch this space for further details about some of these systems. For now, you may read the article here.

ASRAH: new family of small molecule-binding domains

2009-09-24T22:08:00.007-04:00

The Anabaena sensory rhodopsin transducer is a small protein that is co-expressed with the bacterial sensory rhodopsin (ASR) and has been proposed to act as the transducer for this light-activated sensor. In a recent paper in Biology Direct we apply in-depth sequence and genome context analysis and demonstrate this protein is likely to bind small molecules, probably carbohydrates, and is also homologous to domains found in tandem in secreted proteins from several bacterial clades. We propose that the solo versions of this domain, whose homologs are usually associated with sugar-related enzymes, might represent a new kind of cytoplasmic sensor for sugars levels and, as such, regulate a diverse range of sugar metabolism operons and the light sensory behavior in Anabaena.

Reconstructing the Yeast Ubiquitin network

2009-04-02T13:09:00.001-04:00

Watch this space for more details. For now, please read the full manuscript.

Reconstructing the ubiquitin network - cross-talk with other systems and identification of novel functions. Venancio TM, Balaji S, Iyer LM, Aravind L. Genome Biol. 2009 Mar 30;10 (Click here to read).

SZY-20: A centrosomal protein with RNA binding function

2009-01-14T12:22:00.008-05:00

Microtubules are organized by the centrosome, a dynamic organelle that exhibits changes in both size and number during the cell cycle. The maintenance of appropriate centrosome size is critical for proper cell division and partitioning of biomolecules and organelles between the daughter cells. However the exact mechanism by which this process is regulated is unclear.

In a collaborative study with Dr. Kevin O'Connell of the NIDDK, we showed that SZY-20, a predicted RNA-binding protein, plays a critical role in limiting centrosome size in the nematode worm C. elegans. Homologs of SZY-20 are present throughout eukaryotes pointing to conserved role for this protein. SZY-20 localizes in part to centrosomes and in its absence centrosomes possess increased levels of centriolar and pericentriolar components including gamma-tubulin and the centriole duplication factors ZYG-1 and SPD-2. These enlarged centrosomes possess normal centrioles, nucleate more microtubules, and fail to properly direct a number of microtubule-dependent processes. Depletion of ZYG-1 restores normal centrosome size and function to szy-20 mutants, whereas loss of szy-20 suppresses the centrosome duplication defects in both zyg-1 and spd-2 mutants. Our results thus describe a pathway that determines centrosome size and implicate centriole duplication factors in this process. Computational analysis showed that SZY-20 contains a two novel protein domains the SUZ and SUZ-C domain which are predicted to be respectively critical for RNA-binding and targeting of ribonucleoprotein complexes. It was also shown to be a part of a large complex of RNA-binding proteins. Mutagenesis of conserved residues in these domains result in loss of SZY-20 function and loss of ability for form RNA-protein complexes. The presence of a RNA-binding domain in SZY-20, a centrosomal protein, suggests that it might be key for partitioning of RNA during cell division.
For more details, click here.

Host response against Cerebral Malaria - Insights from microarray and sequence analysis

2009-01-14T12:14:00.005-05:00

Cerebral malaria is a primary cause of malaria-associated deaths, especially in sub-Saharan Africa. There is very poor understanding of the molecular profile of the progression from Plasmodium falciparum regular malaria to cerebral malaria. This hampers the development of prognostic tools for this condition. To this end, in collaboration with Dr. Sanjai Kumar of FDA, we used the Plasmodium berghei ANKA murine model of experimental cerebral malaria and high-density oligonucleotide microarray analyses to identify host molecules that are strongly associated with the clinical symptoms of this condition.

Comparative expression analyses were performed with C57BL/6 mice, which have an experimental cerebral malaria (ECM)-susceptible phenotype, and with mice that have ECM-resistant phenotypes: CD8 knockout and perforin knockout mice on the C57BL/6 background and BALB/c mice. These analyses allowed the identification of more than 200 host molecules (a majority of which had not been identified previously) with altered expression patterns in the brain that are strongly associated with the manifestation of ECM. Among these host molecules, brain samples from mice with ECM expressed significantly higher levels of p21, metallothionein, and hemoglobin alpha1 proteins by Western blot analysis than mice unaffected by ECM. The higher expression of hemoglobin alpha1 in the brain may be associated with ECM and could be a source of excess heme, a molecule that is considered to trigger the pathogenesis of CM. Our studies greatly enhance the repertoire of host molecules for use as diagnostics and novel therapeutics in CM.

Click here to read the paper.

DBC1 - an inactive enzyme that functions as a signal integrator

2009-01-14T11:49:00.007-05:00

Deleted in Breast Cancer-1 (DBC1) and its paralog CARP-1 are large multi-domain proteins, with a nuclear or perinuclear localization, and a role in promoting apoptosis upon processing by caspases. Recent studies on human DBC1 show that it is a specific inhibitor of the sirtuin-type deacetylase, Sirt1, which deacetylates histones and p53. However, the exact mechanism of action of these proteins has largely remained mysterious.

Using sensitive computational methods we showed that the central conserved globular domain present in the DBC1 and CARP-1 is a catalytically inactive version of the Nudix hydrolase (MutT) domain. Given that Nudix domains are known to bind nucleoside diphosphate sugars and NAD, we predict that this domain in DBC1 and its homologs binds NAD metabolites such as ADP-ribose. Hence, we developed a model that DBC1 and its homologs are likely to regulate the activity of SIRT1 or related deacetylases by sensing the soluble products or substrates of the NAD-dependent deacetylation reaction. The complex domain architectures of the members of the DBC1 family, which include fusions to the RNA-binding S1-like domain, the DNA-binding SAP domain and EF-hand domains, suggest that they are likely to function as integrators of distinct regulatory signals including chromatin protein modification, soluble compounds in NAD metabolism, apoptotic stimuli and RNA recognition.

You can read the open access version of this paper here

A challenge for the biochemist- the priming problem

2008-11-30T23:32:00.036-05:00

All cellular life forms and many DNA viruses,phages and plasmidsuse a primase to synthesize a short RNAprimer with a free 3'OH group that is subsequently elongatedby a DNA polymerase. The existence of this process is one of the most fascinating unsolved problems. Essentially, it is unclear why DNA polymerases require a primer to initiate DNA replication, while RNA polymerases do not. From an evolutionary perspective, although DNA polymerases have evolved on multiple occasions independently, and a variety of independent solutions have evolved to address the priming problem, in no case is the DNA polymerase rid of a primer. Could this reflect something more fundamental?

First, let us review the known solutions to the priming problem.

In bacteria and archaeo-eukaryotes (a term clubbing archaea and eukaryotes) that possess DNA polymerases of unrelated folds, two unrelated families of primases synthesize RNA primers. Bacteria possess a DNAG-like primase of the Toprim fold, whereas in a comprehensive sequence-structure analysis, we showed that the archaeo-eukaryotic primase (AEP) belongs to the RRM fold (click here to read).
In certain plasmids and phages/viruses, a primpol protein performs both the RNA polymerase (for primer synthesis) and DNA polymerase (for DNA synthesis) activities. Primpols belong to two distinct families, one that is experimentally confirmed and belongs to the archaeo-eukaryotic primase superfamily, and the second, which awaits experimental verification, belongs to the TV-Pol family.
Retroelements (including retroviruses and other reverse-transcriptase based DNA mobile elements) prime DNA replication by using a tRNA that provides a free 3' OH that is used for elongation by the reverse transcriptase (RNA-dependent DNA polymerase).
In adenoviruses and the φ29 family of bacteriophages,a hydroxyl group is provided by the side-chain of an amino acid of the genome attached terminal protein to which nucleotides are added by the DNA polymerase to form a new strand.
Another solution to the priming problem is seen in several families of DNA viruses, such as parvoviruses, geminiviruses and circoviruses, and many phages and plasmids. All of these replicate their DNA by rolling circle replication (RCR). Here, the RCR endonuclease (RCRE) creates a nick in one of the DNA strands. The 5' end of the nicked strand is transferred to a tyrosine residue on the nuclease, and the free 3' OH group is elongated by a DNA polymerase for the new strand synthesis.
The only known exception of a DNA polymerase lacking a primer, is the reverse transcriptase of the Mauriceville plasmid that uses the 3' tRNA-like structure of the parent mRNA/pRNA to de novo synthesize a daughter DNA strand.

Thus although DNA polymerases have evolved on multiple occasions independently, they don't seem to have rid themselves of the need for a primer.

Hypothesis. In our study on the evolutionary history of the archaeo-eukaryotic primases, we speculated that these observations suggests a strong constraint against ‘invention’ of de novo initiation of DNA synthesis which, probably, stems from fundamental chemical differences between ribo- and deoxyribonucleotides, rather than a frozen evolutionary accident that maintains a primer. We proposed that the inefficiency in de novo DNA synthesis by DNA polymerases may, at least in part, be due to a competing futile reaction of 3'->5' nucleotide cyclization while using deoxyribonucleotides. Given the tendency of diverse, unrelated RNA polymerases to initiate de novo strand synthesis, it seems likely that this problem does not arise with ribonucleotides. As support for this hypothesis we note that the DNA cyclases are specifically related to DNA polymerases and have evolved from the latter on multiple occasions independently.

To the best of our knowledge this hypothesis has not yet been tested. You can read about our comprehensive study on the archaeo-eukaryotic primases by clicking here.

One protein family, many insights: The TV-pol story

2008-11-25T17:21:00.015-05:00

Using sensitive sequence analysis methods, we recently discovered and characterized a divergent member of the DNA polymerase I superfamily /Superfamily A DNA polymerase that we denote the Transposon-Virus polymerase (TV-Pol) family. These proteins are found in a wide range of bacteria and their prophages, phages, the chloroplast of the alga Nephroselmis, and in the Sputnik virus (virophage of Mimivirus).

Using gene neighborhood analysis we show that the TV-pol genes are components of mobile elements and might be involved in replicative transposition. As evidence, we detected a recent transposition event in Brucella melitensis 16M, of a transposon with a direct repeat that contains the TV-Pol gene, and a γδ-resolvase.More specifically, based on their frequent fusion to D5-helicases (e.g. V13 of the Sputnik virus) we speculate that TV-Pol proteins are primase-polymerases (primpols), like some members of the archaeo-eukaryotic primases (To learn more about AEP-like primpols click here).

Additional interesting insights

Superfamily A DNA polymerases contain a HTH domain.Upon defining the structural core of the DNA polymerase I superfamily, we noted that these proteins are distinguished by the presence of a HTH-domain within the fingers of the RRM-like palm domain. This HTH contains the highly conserved RxxxK motif characteristic of this superfamily, and potentially interacts with the elongating daughter strand.
The thumb, palm and fingers probably existed as independent polypeptides at an early point in the evolution of the superfamily A polymerases. Given the presence of distinct globular folds in the fingers (HTH) and palm domain (RRM) and also the displacement of the coiled coil thumb by a distinct globular domain in a TV-Pol of Gemmata obscuriglobus, an early stage in the evolution of this superfamily can be conceived where these three units were present on different polypeptides and then fused to give the Superfamily A DNA polymerases.
The predicted primpol activity of the TV-Pols throws light on the origins of the T7-like RNA polymerases. The T7-like DNA-dependent RNA polymerases are members of the superfamily A DNA polymerases. Their origins can now be understood in light of the discovery of TV-pols, where a primpol ancestor that had both DNA and RNA polymerase activity might have possibly contributed to the T7-like RNA polymerases. This view is also supported by experimental studies that have shown some members of the T7-like RNA polymerases to function as primases.
The Sputnik virophage could have evolved from a mobile element. Based on the gene contexts of the TV-Pol gene in the Sputnik virophage, we speculate that the virus may have arose from a a TV-Pol containing transposase, which subsequently acquired a DNA-packaging HerA-FtsK ATPase and virion proteins from a distinct viral source.

You can read the open access version of this study by clicking here.

Prokaryotic orthologs of Pac2 and the ancestral proteasomal chaperone

2008-11-05T19:23:00.004-05:00

Studies on the eukaryotic proteasome have revealed that several chaperones are required for it successful assembly. Proteasome assembly chaperones (PACs) work in a sequence, with PAC1-PAC2 dimer and the PAC3-PAC4 dimer acting on a subunits to form the heptameric a-rings. Then the 6 ß subunits come in to form a half proteosome complex with the a-rings, releasing the Pac3-Pac4 chaperones; the place of the 7th subunit is occupied by the Ump1 chaperone. Finally the 7th subunit is added which dimerizes the two half proteasomes to complete the structure. At this point the proteasome is activated by autocatalytic maturation of the ß subunits. It then degrades Pac1-Pac2 and Ump1. While the origin of the proteasome itself is traced back to the simple proteasomal precursor found in the archaea, the origin of the chaperone system remained mysterious. Thus far none of the chaperones have been found in archaea or the bacterial proteasomal systems acquired from the archaea.

We discovered that an ortholog of the eukaryotic PAC2 (e.g. Corynebacterium cg2106, PBD: 2p90) is often present in the vicinity of the actinobacterial Pup-proteasome gene-neighborhoods and some archaeal proteasomal ATPase gene-neighborhoods. Most bacteria and archaea encode two Pac2 paralogs. The structure of Cg2106 suggests that PAC2 forms a trimeric torroid. Hence it might provide a scaffold for assembly of proteasomal peptidase subunits. As none of the other eukaryotic proteasomal chaperones have orthologs in archaea or bacteria, this protein is likely to represent the ancestral chaperone of the proteasome. Thus, for the first time we find evidence for a eukaryote-type proteasomal assembly process in the prokaryotes, which possibly operated on the a subunits even there.
For more details, you can read the open access version of the paper. Click here to access it. You can also access the Pac2 alignment and operons in our supplementary material to the above paper. Please click here to access it.

What is the biochemistry of Pupylation?

2008-10-31T16:15:00.017-04:00

Recently, a remarkable study showed that Mycobacteria have a distinct "ubiquitin-like" system in which a small protein, Pup, is transferred to the ε-amino groups of lysines in target proteins. These experiments also implicated a gene neighbor, the PafA protein, in this activity. How this was mediated was a mystery.

Using sensitive sequence and structure analysis methods, we unified the PafA proteins to the glutamine synthetase (or carboxylate-amine/ammonia ligase) superfamily. In particular the PafA proteins are closer to the γ-glutamyl-cysteine synthetases. This unification provides a simple explanation for the reaction mechanism of Pupylation by PafA (the Pup ligase).

First the Pup ligase catalyzes an ATP-dependent phosphorylation of the γ-carboxylate of glutamate followed by ligation with the ε-amino group of lysines in target proteins with the formation of an amide linkage.

In Pups with a terminal glutamine instead of a glutamate (e.g. Mycobacterial Pup), the glutamine is first deamidated and converted to glutamate. Given the similar chemistry, we propose that this reaction too might be catalyzed by the Pup ligase. Our analysis suggests that pupylation is a bacterial innovation that emerged from proteins involved in amino acid (glutamine) and cofactor (glutathione) biosynthesis. The parallels with the ubiquitination system are striking in which the ubiquitin system evolved in bacteria from a system involved in cofactor (Moco) and amino acid (cysteine) biosynthesis. Thus the similiarities in pupylation and ubiquitination represent a remarkable case of convergent evolution.

Additional points of interest

Pup is predicted to be a α-helical protein with an extended tail and is not related to ubiquitin.
The pupylation system is present in most actinobacteria, and also sporadically in verrucomicrobia, nitrospirae, deltaproteobacteria and planctomycetes. In all cases both Pup and the Pup-ligase are immediate gene neighbors.
Barring a few exceptions, gene neighborhoods reveal two paralogs of Pup ligases suggesting that they function as heterodimers. In species with only one copy, they would function as homodimers. Note Mycobacteria have two copies of PafA corresponding to genes Rv2097c and Rv2112c.
Gene neighborhoods also reveal that the actinobacterial pupylation genes are neighbors of the archaeal-type proteasomal AAA+ ATPases and proteases (NTN hydrolase superfamily) in line with prior studies that in these bacteria pupylated proteins are targeted for degradation. However, this may not be always so. The Pup ligases of deltaproteobacteria and planctomycetes are remarkable in that they have 4 transmembrane helices inserted within the core domain and are also neighbors of membrane proteins. In these bacteria, the pupylation system might target membrane proteins.
We also detected the prokaryotic homolog of the proteasomal chaperone PAC2 in the gene neighborhood of some actinobacterial Pupylation genes. This is the first report of a prokaryotic proteasomal chaperone and given the absence of other proteasomal chaperone subunits, it appears that PAC2 is the most ancient proteasomal chaperone (see the separate blog on PAC2).
Could other members of this family catalyze analogous reactions? In this quest, we detected two other previously uncharacterized families of proteins that belong to the glutamine synthetase superfamily. However, their domain contexts and gene neighborhoods suggest that they may be involved in glutathione or related peptide secondary metabolites biosynthesis.

For more details, you can read the open access version of the paper. Click here to access it. For latest updates on pupylation click here

The Evolution of Chromatin Proteins and Prediction of Novel Factors in Chromatin Dynamics

2008-10-30T15:14:00.009-04:00

An overview of recent results on the analysis of chromatin proteins across eukaryotes. Please view slide show below

Chromatin Meeting

View SlideShare presentation or Upload your own. (tags: dna rna)

Uncovering the origins and diversity of the E1-superfamily of proteins

2008-10-15T11:24:00.000-04:00

The E1-superfamily of proteins are central to ubiquitin (Ub) conjugation, biosynthesis of cysteine, thiamine and MoCo and several secondary metabolites. Yet the diversity and evolutionary history of these proteins was poorly understood. Recently, we undertook a comprehensive study of the E1 superfamily and uncovered several interesting and surprising insights.

Watch this page for a more detailed summary. For now, please click here to access the full article.

Transcriptional regulatory networks: Pitfalls and Surprises

2008-06-04T15:56:00.011-04:00

The use of high-throughput methods to reconstruct the transcriptional regulatory program of a species is an exciting development of the post-genomic era. At least three distinct methods based on unrelated principles are in vogue and include reconstructions of the transcriptional program by 1) over-expression of transcription factors; 2) deletion of transcription factors; and 3) large-scale Chromatin immunoprecipitation-chip (ChIP-chip) experiments. From these data, a transcriptional regulatory network (TRN) can be constructed where a node represents a transcription factor (TF) or target gene (TG) and an edge is made between a TF and a TG if the TF has a regulatory effect on a TG, or if the TF binds the promoter of the TG.

Recently, we along with Dr. Madan Babu of MRC-UK, compared the transcriptional regulatory networks of the budding yeast, Saccharomyces cerevisiae, reconstructed from the above datasets and reached some surprising conclusions.

Despite sharing a significant number of TFs, the overlap in the regulatory interactions between the networks is strikingly small (<5%).
The network structure differs between the different TRN reconstructions. For example, the number of TGs regulated by a given TF shows a power-law distribution in the ChIP-chip and TF deletion networks. In contrast the same distribution in the TF over-expression network shows a central tendency.
Although the ChIP-chip and TF deletion networks show a similar global structure, only about a quarter of the total number of hubs (top 20% of TFs with the greatest number of TGs) are shared between the two networks.
We also show that unintended consequences of experimental design, may significantly influence the TRNs. For example, in the ChIP-chip network, telomeric looping effect, or the interaction of chromosome ends with diverse TFs in the inner nuclear envelope may have contributed to the unusually large number of TFs bound to the promoters of several subtelomeric TGs. An analysis of the gene over-expression TRN revealed that several TGs that are regulated by a large number of TFs were components of stress response pathway/s pertaining to protein unfolding and oxidative damage. One such set of proteins that are expressed are the homologs of human DJ1 implicated in protein aggregation defect in Parkinson's disease. Thus over-expression of TFs may cause an increase in mis-folded polypeptides triggering the expression of proteins involved in a specific stress response pathway. Similarly, in the gene deletion TRNs, most of the top hubs have regulatory interactions with ribosomal components. Thus altering the expression of these proteins may alter the stoichiometry of the ribosomal components which in turn may indirectly affect the expression of several genes.

Our study shows that each of these TRN reconstructions captures a different aspect of the transcriptional regulatory program, Secondary effects caused by experimental design may significantly influence the network and future high-throughput experiments must subtract such additional effects to obtain more accurate TRNs. Finally, high-throughput methods may throw unexpected and novel insights into specific biological phenomena.
For more details read the full article here.

Lachrymation and START domains

2008-04-09T11:50:00.011-04:00

Lachrymation caused while cutting onions is due to the well known volatile lachrymatory factor (LF) propanthial S-oxide released by cutting (injuring) the onion bulb. The lachrymatory factor irritates the corneal nerve endings causing tears in the eyes. Recently, genetically modified "tear-free" onions were developed by a collaborative effort between House Foods Corporation (Japan) and Crop & Food Research (New Zealand). In these onions, the lachrymatory factor synthase, responsible for LF synthesis is shut down using RNAi. These onions retain the flavor of the alliums but do not cause lachrymation while being processed.

The lachrymatory factor synthase is a member of the Birch allergen-like START domains. Members of this family are greatly expanded in plants, and in an early study on the unification of various START domains, we noticed over 55 copies of this family in Arabidopsis. Other experimentally characterized members of this family include the cytokinin-specific binding protein from mung bean, the birch allergen which also has ribonuclease activity, the stress induced protein PR10, and the major latex proteins. The wide spectrum of ligand binding and enzymatic activities of these proteins suggest that the plant-specific expansion corresponds to adaptations to binding or modifying various small molecules. In this study, we had predicted a critical role for certain residues in the upper rim of the helix-grip structure of the Birch allergen-like START domains for enzymatic activity.

The START superfamily in turn comprises a wide range of ligand binding and enzymatic domains, such as the lipid-binding classical START domains, the polyketide cyclases and aromatases that are greatly expanded in actinomycetes and involved in secondary metabolite synthesis, and the Birch allergen-like START domains that have both ligand binding domains and enzymes. Many of these proteins are poorly characterized. The START domain is a rare case of adaptation of a ligand binding protein fold for both enzymatic and non-enzymatic activity.

You can read more about this study by clicking here.

The BEN domain: A novel module in chromatin function and DNA viruses

2008-02-15T22:46:00.006-05:00

The BEN domain is an α helical domain detected in diverse animal transcription factors and chromatin proteins, including BANP/SMAR1, NAC1, Drosophila mod(mdg4) isoform C and the vertebrate sex combs in midleg-like-1. The domain is also found in chordopoxviruses (E5R) and in several polydnavirus proteins and is among the few proteins with a known domain in the latter viruses.

Architectural diversity. The BEN domain is often found in multiple tandem copies. Our analysis suggests that these tandem copy containing proteins arose on multiple occasions independently in evolution. This suggests an inherent property ofthe domain to form multimeric assemblies. In addition BEN domains are fused to a variety of chromatin associated domains such as POZ, MCAFN, C4DM, C2H2 fingers and SAM, and also to the RNaseT2 domain in polydnaviruses.
Functional predictions. Experimental studies suggest that the BEN domain is involved in protein-protein interactions. However, contextual analysis points to a possible role in DNA binding. This is inferred from the fusion of the BEN domain N-terminal to the C4DM in insects, and by its presence in an isoform of mod(mdg4)and the Broad complex loci in the same exon position as other DNA binding domains.

Viral BEN domains: The chordopoxviral protein E5R, a lateral transfer of the vertebrate KIAA1553, is an abundant early virosome protein, and it may be involved in organizing viral DNA during replication. Interestingly, the Molluscipox virus E5R ortholog appears to be a secondary displacement of the viral E5R by the host KIAA1553. The polydnaviruses have one to many copies of proteins with the BEN domain. Some versions are additionally fused to RNaseT2. This suggests a possible role in RNA processing, or perhaps in modifying host function.
Click here to access the paper.

RAGNYA : a novel fold found in functionally diverse nucleic acid, nucleotide & peptide-binding proteins

2007-09-30T11:46:00.000-04:00

One of our principal research objectives is to derive a natural classification of the protein universe by unifying diverse protein superfamilies. However, the detection of relationships between these superfamilies is often non-trivial due to extensive divergence or variations, like circular permutations, in their structural scaffolds. This is particularly prevalent in numerous small folds involved in binding of nucleic-acids/nucleotides. One such alpha+beta fold that we recently identified was the RAGNYA fold that includes a diverse group of proteins principally involved in nucleic acid, nucleotide or peptide interactions. Members of the fold include the Ribosomal proteins L3 and L1, the GYF domain, DNA-recombination proteins of the NinB family from caudate bacteriophages, the C-terminal DNA-interacting domain of the Y-family DNA polymerases, the uncharacterized enzyme AMMECR1, the siRNA silencing repressor of tombusviruses, tRNA Wybutosine biosynthesis enzyme Tyw3p, DNA/RNA ligases and related nucleotidyltransferases and the Enhancer of rudimentary proteins. This fold exhibits three distinct circularly permuted versions and is composed of an internal repeat of a unit with two-strands and a helix. We show that despite considerable structural diversity in the fold, its representatives show a common mode of nucleic acid or nucleotide interaction via the exposed face of the sheet.
Click here to read the paper

Unraveling the DOMON and DM13 domains

2007-09-27T12:11:00.000-04:00

We and others had previously reported the DOMON (also called DoH) domain in several extracellular proteins from animals and plants, such as Dopamine beta- monooxygenase and SDR2. However, very little was known of its function in these contexts. Using sensitive sequence and structure comparison methods, we show that the DOMON domains are small molecule binding domains of the immunoglobulin fold that bind heme or sugars through a common mode. The presence of the heme-binding DOMON domain in several extracellular animals proteins suggest that they may be involved in yet unidentified redox reactions potentially related to protein hydroxylation or oxidative cross-linking. Interestingly, the classical vertebrate Dopamine beta-monooxygenase and the arthropod and nematode tyramine-beta-hydroxylase lack the heme coordinating motifs, although closely related proteins such as MOXD1 appear to retain heme binding. We also report the first prokaryotic members of this superfamily that include the gamma subunit of Ethyl benzene dehydrogenase (the cytochrome domain) and CbsA/cytochrome b558/556, and provide a detailed evolutionary history.

The uncharacterized DM13 domain also appears to be of prokaryotic origin and contains a highly conserved cysteine residue that could potentially be involved in redox reactions.

Click here to read the full manuscript

The NYN domains: novel predicted RNAses with a PIN domain-like fold

2007-09-25T12:19:00.000-04:00

We have shown that the Mut-7C module contains a PIN domain RNAse combined with a Zinc ribbon. Thus Mut-7 is a “double-headed” nuclease with a PIN and a 3’->5’ nuclease domain fused together.
Click here to read the paper

The signaling helix: a common functional theme in diverse signaling proteins

2007-09-25T12:15:00.000-04:00

The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood. We identified a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S)-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. We suggest that it functions as a switch that prevents constitutive activation of linked downstream signaling domains. However, upon occurrence of specific conformational changes due to binding of ligand or other sensory inputs in a linked upstream domain it transmits the signal to the downstream domain.
Click here to read the paper

Insights into chronic HCV treatment with PEG-IFN-alpha and ribavirin

2007-09-25T12:00:00.000-04:00

With Mani Subramanian and Vijay Balan’s groups we studied the global transcriptional profile during the first 4 weeks of treatment of human chronic hepatitis C patients with pegylated interferon alfa (PEG-IFN-alpha). Novel transcription factors potentially involved in secondary gene regulation cascades, a potential dsRNA receptor with a RNA helicase domain related to that found in the HELICARD protein and members of the ubiquitin signaling pathways, including a novel predicted deubiquitinating peptidase were all identified as being up-regulated upon treatment with IFN. This predicted peptidase is a highly derived version of the APG4 family of papain-like peptidases and contains a catalytic histidine that is in entirely different location from that found in the regular APG4-like proteins. The overall findings provide new light on possible physiological effects of IFN-alpha, new downstream signaling pathways and open lines of investigations on the mode of action of PEG-IFN-alpha combination therapy.
Click here to read the paper

Small ligand binding beta-grasp domains and the origins of Vitamin B12 uptake in animals

2007-09-17T15:51:00.000-04:00

We recently showed that the Vitamin B12 binding proteins typified by transcobalamin, required for B12 uptake in animals, has been derived through lateral transfer from the Gram-positive bacteria prior to the divergence of the extant animal lineages. These proteins contain a novel version of the b-grasp (ubiquitin-like) fold that has been adapted for binding small molecules like B12. In bacteria and archaea is shows a rich diversity of architectures including fusions to Helix-turn-helix domains in one-component transcription factors
Click here to read the paper

Ub-like conjugation systems in prokaryotes

2006-08-25T12:22:00.000-04:00

Hitherto it was believed ubiquitin conjugations systems were a unique possession of eukaryotes. We have recently shown that the Ub conjugation systems had their origins in prokaryotes and are widely distributed in several bacterial lineages. This bacterial system appears to have included E1 and E2-like Ub-conjugating enzymes and JAB domain peptidases. Some of them also appear to participate, like their relatives ThiS and MoaD, in sulfur incorporation reactions (in siderophore biosynthesis). In this study we also characterized a group of proteins with multiple tandem Ub-like domains that are likely to be conjugated as a “poly-ubiquitin” in certain bacteria.
Click here to read the paper