New direction on the function of GRAS proteins in gibberellin signaling

Gibberellin GA-1

Gibberellins (GAs) are key plant hormones that regulate various aspects of growth and development of land plants and have been at the center of the “green revolution”. In angiosperms practically every aspect of plant life including seed germination, elongation growth, and flowering are influenced by the action of GAs. In at least some angiosperms, they have a key role in surviving certain stress conditions such as saline environments and cold. In ferns certain GAs (e.g. GA1) or related compounds (e.g. antheridic acid) induce male gametophyte development, might repress the female gametophytes and also play a role in spore germination. Commercially GAs are used in a wide range of applications such as to promote growth of fruit crops, to increase sugar yield in sugar cane and, stimulate malting of barley during beer production. Did you know that almost all seedless grapes and sweet bing cherries are treated with GA derivatives to increase their size?  GAs are also used in “fruit cosmetics” to prevent the undesirable russeting in apples. The next time you visit a plant nursery, note that GA inhibitors are often used to retard growth of nursery plants. As you can imagine given its remarkable uses, the GA pathway is one of the most intensely studied in plant biology and agriculture. It is in this regard that a new story has emerged from research in our group.

Russeting in apples

Research in the past 20 years or so has shown that some of the key players in the GA response pathway  are members of the GRAS family of proteins, which includes proteins such as GA insensitive (GAI), Repressor of GA1-3 (RGA1/DELLA), Short-root, Scarecrow (SCR), and Nodulation Signal Pathway 1 and 2 (NSP1 and NSP2). For a variety of historical reasons, including incorrect domain prediction, GRAS protein were generally believed to function as conventional transcription factors. The original reports that described them as transcription factors were based on flawed recognition of features such as coiled-coil regions, resulting in comparisons between them and bZIP proteins. This was compounded by another erroneous piece of sequence analysis which led to the idea that they might be plant equivalents of the STAT transcription factors (which have P53/cytochrome f fold DNA-binding domains) known from animal and amoebozoans. Consequently, almost all efforts to understand this family have been spent in testing hypotheses arising from this perspective. However, the evidence that GRAS proteins bind DNA is not very rigorous, with several GRAS proteins failing to display the purported DNA-binding activity despite sharing a conserved structure, raising questions about their mode of action and function.

Our recent studies help clarify the situation. We showed that the GRAS family actually belongs to the Rossmann-fold methyltransferase superfamily. We establish that the GRAS family first emerged in bacteria and plant versions represent a case of lateral gene transfer prior to the radiation of land plants. We further show that all bacterial, and a subset of plant GRAS proteins are likely to function as small molecule methylases, but the remaining plant members have lost one or more AdoMet (SAM)-binding residues while preserving their substrate-binding residues. Thus, based on sequence- structure analysis, combined with functional evidence, we predict that GRAS proteins might either modify or bind small molecules, which might include GAs or their derivatives.

Our results have thus falsified the previously-published relationships that were proposed for the GRAS proteins, and more importantly throw a completely new spin on their mode of action in the context of  GA binding or modification. One delicious possibility is that the active versions function as methylases that might modify certain GAs or their derivatives, whereas inactive versions act as GA binding proteins (Experimentalists  take note). While a GA receptor belonging to the alpha/beta hydrolase superfamily has been described previously, the functional evidence suggests that not all aspects of the GA signaling are channelized via that receptor. Hence, the possibility of direct interaction between a GA or its modified derivative with the GRAS methylase domain remains open and a potentially important avenue for signaling. In addition, very little is known of the fate and prevalence of GA methylation which is a mechanism of GA deactivation in angiosperms. The currently characterized GA methylases (GAMT1 and GAMT2) which are also Rossmann-fold methylases belonging to a radiation of plant methylases of ultimately bacterial origin, includes enzymes that methylate carboxy, hydroxyl and amino groups in synthesis of plant metabolites  like caffeine, theobromine, methyl salicylate, and methyl jasmonate among others. In Arabidopsis, these are primarily expressed in the siliques (fruits) including the seeds and are believed to deactivate GAs via methylation and subsequent degradation during the maturation of seeds. One possibility is that such a methylation dependent control of GAs also occurs in other parts and other developmental processes via the action of GRAS family methylases. The possibility of the inactive versions of the GRAS proteins binding methylated or other modified GAs is also an avenue for possible functional studies.

It should be noted that our phylogenetic analysis (see figure above) suggests that the GRAS superfamily was delivered to plants via a single lateral transfer from bacterial prior to the diversification of land plants --  this ancestral plant GRAS protein underwent a lineage-specific expansion into 13 distinct well-supported clades that contained at least one representative from bryophytes, lycopodiophytes and angiosperms. At face value, assuming a direct GA-related role for the GRAS family, this would suggest that the GA-like molecules were already functional in the early history of land plants. This clearly goes contrary to certain suggestions of plant evolutionists that GA-like molecules were absent in bryophytes like Physcomitrella, but supports recent experimental results suggesting a role for GA-like molecules in caulonema formation, growth direction of protonemata, and spore germination these mosses (Hayashi et al). Our findings suggest that the predicted small-molecule binding/modifying activity would extend to the base of land plants and could have bearing on the enigma of the role of GA-like molecules in basal land plants. For more details, you can read our paper here.

Sensory domains in bacterial signal transduction

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

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

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

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.
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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