A mystery pathway in prokaryotes

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

1. 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.
2. Rv2411c (split as DUF404+DUF407 in Pfam 25): A circularly permuted peptide ligase of the ATP-grasp fold.
3. Rv2409c, Rv2569c: Transglutaminases that could serve either as a peptidase or a classical transglutaminase.
4. Rv2568c (DUF2248 in Pfam 25): A metallopeptidase-family peptidase.
5. Rv2567: An inactive circularly permuted ATP-grasp fused to the Alpha-E domain.
6. Rv2566 (Transglut+DUF2126 in Pfam 25): A transglutaminase fused to a circularly permuted peptide ligase of the COOH-NH2 ligase superfamily.
7. 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

1. 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.
2. 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).
3. 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...

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

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.

Free SAMPylation Musings

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

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

The fellowship of the RING: Bacteria have them too!

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.

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

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 yeast Ubiquitin network

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

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.

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).

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. For latest updates on pupylation click here

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.

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

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