ASRAH: new family of small molecule-binding domains

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

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

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

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

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

All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a short RNA primer with a free 3' OH group that is subsequently elongated by 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.

1. 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).
   
2. 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.
   
3. 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).
   
4. 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.
5. 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.
   
6. 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

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

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?

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.

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

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

Chromatin Meeting

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

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

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

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

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 of the 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

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

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

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

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

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

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

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