GFam: a platform for automatic annotation of gene families

We have developed GFam, a platform for automatic annotation of gene/protein families. GFam provides a framework for genome initiatives and model organism resources to build domain-based families, derive meaningful functional labels and offers a seamless approach to propagate functional annotation across periodic genome updates. GFam is a hybrid approach that uses a greedy algorithm to chain component domains from InterPro annotation provided by its 12 member resources followed by a sequence-based connected component analysis of un-annotated sequence regions to derive consensus domain architecture for each sequence and subsequently generate families based on common architectures. Our integrated approach increases sequence coverage by 7.2 percentage points and residue coverage by 14.6 percentage points higher than the coverage relative to the best single-constituent database within InterPro for the proteome of Arabidopsis. The true power of GFam lies in maximizing annotation provided by the different InterPro data sources that offer resource-specific coverage for different regions of a sequence. GFam’s capability to capture higher sequence and residue coverage can be useful for genome annotation, comparative genomics and functional studies. GFam is a general-purpose software and can be used for any collection of protein sequences. The software is open source and can be obtained from http://www.paccanarolab.org/software/gfam/.     

Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases.

The polytopic membrane protein Rhomboid-1 promotes the cleavage of the membrane-anchored TGFalpha-like growth factor Spitz, allowing it to activate the Drosophila EGF receptor. Until now, the mechanism of this key signaling regulator has been obscure, but our analysis suggests that Rhomboid-1 is a novel intramembrane serine protease that directly cleaves Spitz. In accordance with the putative Rhomboid active site being in the membrane bilayer, Spitz is cleaved within its transmembrane domain, and thus is, to our knowledge, the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and our results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread.     

Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation

Unlike mammalian Toll-like Receptors, the Drosophila Toll receptor does not interact directly with microbial determinants but is rather activated upon binding a cleaved form of the cytokine-like molecule Spatzle (Spz). During the immune response, Spz is thought to be processed by secreted serine proteases (SPs) present in the hemolymph that are activated by the recognition of gram-positive bacteria or fungi . In the present study, we have used an in vivo RNAi strategy to inactivate 75 distinct Drosophila SP genes. We then screened this collection for SPs regulating the activation of the Toll pathway by gram-positive bacteria. Here, we report the identification of five novel SPs that function in an extracellular pathway linking the recognition proteins GNBP1 and PGRP-SA to Spz. Interestingly, four of these genes are also required for Toll activation by fungi, while one is specifically associated with signaling in response to gram-positive bacterial infections. These results demonstrate the existence of a common cascade of SPs upstream of Spz, integrating signals sent by various secreted recognition molecules via more specialized SPs.     

Automated annotation of Drosophila gene expression patterns using a controlled vocabulary

MOTIVATION: Regulation of gene expression in space and time directs its localization to a specific subset of cells during development. Systematic determination of the spatiotemporal dynamics of gene expression plays an important role in understanding the regulatory networks driving development. An atlas for the gene expression patterns of fruit fly Drosophila melanogaster has been created by whole-mount in situ hybridization, and it documents the dynamic changes of gene expression pattern during Drosophila embryogenesis. The spatial and temporal patterns of gene expression are integrated by anatomical terms from a controlled vocabulary linking together intermediate tissues developed from one another. Currently, the terms are assigned to patterns manually. However, the number of patterns generated by high-throughput in situ hybridization is rapidly increasing. It is, therefore, tempting to approach this problem by employing computational methods. RESULTS: In this article, we present a novel computational framework for annotating gene expression patterns using a controlled vocabulary. In the currently available high-throughput data, annotation terms are assigned to groups of patterns rather than to individual images. We propose to extract invariant features from images, and construct pyramid match kernels to measure the similarity between sets of patterns. To exploit the complementary information conveyed by different features and incorporate the correlation among patterns sharing common structures, we propose efficient convex formulations to integrate the kernels derived from various features. The proposed framework is evaluated by comparing its annotation with that of human curators, and promising performance in terms of F1 score has been reported.     

Granzymes: a family of lymphocyte granule serine proteases

Granzymes, a family of serine proteases, are expressed exclusively by cytotoxic T lymphocytes and natural killer (NK) cells, components of the immune system that protect higher organisms against viral infection and cellular transformation. Following receptor-mediated conjugate formation between a granzyme-containing cell and an infected or transformed target cell, granzymes enter the target cell via endocytosis and induce apoptosis. Granzyme B is the most powerful pro-apoptotic member of the granzyme family. Like caspases, cysteine proteases that play an important role in apoptosis, it can cleave proteins after acidic residues, especially aspartic acid. Other granzymes may serve additional functions, and some may not induce apoptosis. Granzymes have been well characterized only in human and rodents, and can be grouped into three subfamilies according to substrate specificity: members of the granzyme family that have enzymatic activity similar to the serine protease chymotrypsin are encoded by a gene cluster termed the 'chymase locus'; granzymes with trypsin-like specificities are encoded by the 'tryptase locus'; and a third subfamily cleaves after unbranched hydrophobic residues, especially methionine, and is encoded by the 'Met-ase locus'. All granzymes are synthesized as zymogens and, after clipping of the leader peptide, maximal enzymatic activity is achieved by removal of an amino-terminal dipeptide. They can all be blocked by serine protease inhibitors, and a new group of inhibitors has recently been identified - serpins, some of which are specific for granzymes. Future studies of serpins may bring insights into how cells that synthesize granzymes are protected from inadvertent cell suicide.     

The structural basis of mode of activation and functional diversity: a case study with HtrA family of serine proteases

HtrA (High temperature requirement protease A) proteins that are primarily involved in protein quality control belong to a family of serine proteases conserved from bacteria to humans. HtrAs are oligomeric proteins that share a common trimeric pyramidal architecture where each monomer comprises a serine protease domain and one or two PDZ domains. Although the overall structural integrity is well maintained and they exhibit similar mechanism of activation, subtle conformational changes and structural plasticity especially in the flexible loop regions and domain interfaces lead to differences in their active site conformation and hence in their specificity and functions.     

The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families

By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.     

ClpP: a distinctive family of cylindrical energy-dependent serine proteases

Processes maintaining protein homeostasis in the cell are governed by the activities of molecular chaperones that mainly assist in the folding of polypeptide chains and by a large class of proteases that regulate protein levels through degradation. ClpP proteases define a distinctive family of cylindrical, energy-dependent serine proteases that are highly conserved throughout bacteria and eukaryota. They typically interact with ATP-dependent AAA+ chaperones that bind and unfold target substrates and then translocate them into ClpP for degradation. Structural and functional studies have provided a detailed view of the mechanism of function of this class of proteases.     

Ruleminer: a knowledge system for supporting high-throughput protein function annotations

In this paper, we present RuleMiner, a knowledge system to facilitate a seamless integration of multi-sequence analysis tools and define profile-based rules for supporting high-throughput protein function annotations. This system consists of three essential components, Protein Function Groups (PFGs), PFG profiles and rules. The PFGs, established from an integrated analysis of current knowledge of protein functions from Swiss-Prot database and protein family-based sequence classifications, cover all possible cellular functions available in the database. The PFG profiles illustrate detailed protein features in the PFGs as in sequence conservations, the occurrences of sequence-based motifs, domains and species distributions. The rules, extracted from the PFG profiles, describe the clear relationships between these PFGs and all possible features. As a result, the RuleMiner is able to provide an enhanced capability for protein function analysis, such as results from the integrated sequence analysis tools for given proteins can be comparatively analyzed due to the clear feature-PFG relationships. Also, much needed guidance is readily available for such analysis. If the rules describe one-to-one (unique) relationships between the protein features and the PFGs, then these features can be utilized as unique functional identifiers and cellular functions of unknown proteins can be reliably determined. Otherwise, additional information has to be provided.     

The rhomboids: a near ubiquitous family of intramembrane serine proteases evolved via multiple horizontal gene transfers

Background  

The rhomboid family consists of polytopic membrane proteins, which show a level of evolutionary conservation that is unique among membrane proteins. The rhomboids are present in nearly all sequenced genomes of archaea, bacteria and eukaryotes, with the exception of several species with small genomes. On the basis of experimental studies with the developmental regulator Rhomboid from Drosophila and the AarA protein from the bacterium Providencia stuartii, the rhomboids are thought to be intramembrane serine proteases whose signaling function is conserved in eukaryotes and prokaryotes.     

Two gene families clustered in a small region of the Drosophila genome

Three Drosophila genes that are clustered within 8 X 10(3) bases of DNA at the chromosomal region 44D have been identified and mapped, and the gene cluster entirely sequenced. The three genes are 55 to 60% homologous in DNA sequence. One gene contains an intron in its 5'-proximal protein coding sequence while the other two have none at this position; similarly, another gene has an intron in its 3'-proximal protein coding sequence which is not found in the other genes. All three genes are abundantly expressed together in Drosophila first, second, and early third instar larval stages and in adults, but they are not abundantly expressed in either embryonic, late third instar larval, or pupal stages. This gene family lies 11 X 10(3) bases away from another cluster containing four Drosophila larval cuticle protein genes plus a pseudogene. The cuticle genes are all abundantly expressed throughout third instar larval development. Thus, at least seven protein-coding genes and one pseudogene lie within 27 X 10(3) bases of DNA. Moreover, two small gene families can lie adjacent on a chromosome and exhibit different patterns of developmental regulation, even though individual genes within each clustered family are co-ordinately expressed.     

Dynamics of the ClpP serine protease: A model for self-compartmentalized proteases

Abstract

ClpP is a highly conserved serine protease present in most bacterial species and in the mitochondria of mammalian cells. It forms a cylindrical tetradecameric complex arranged into two stacked heptamers. The two heptameric rings of ClpP enclose a roughly spherical proteolytic chamber of about 51 Å in diameter with 14 Ser–His–Asp proteolytic active sites. ClpP typically forms complexes with unfoldase chaperones of the AAA+ superfamily. Chaperones dock on one or both ends of the ClpP double ring cylindrical structure. Dynamics in the ClpP structure is critical for its function. Polypeptides targeted for degradation by ClpP are initially recognized by the AAA+ chaperones. Polypeptides are unfolded by the chaperones and then translocated through the ClpP axial pores, present on both ends of the ClpP cylinder, into the ClpP catalytic chamber. The axial pores of ClpP are gated by dynamic axial loops that restrict or allow substrate entry. As a processive protease, ClpP degrades substrates to generate peptides of about 7–8 residues. Based on structural, biochemical and theoretical studies, the exit of these polypeptides from the proteolytic chamber is proposed to be mediated by the dynamics of the ClpP oligomer. The ClpP cylinder has been found to exist in at least three conformations, extended, compact and compressed, that seem to represent different states of ClpP during its proteolytic functional cycle. In this review, we discuss the link between ClpP dynamics and its activity. We propose that such dynamics also exist in other cylindrical proteases such as HslV and the proteasome.