Comparative analysis of editosome proteins in trypanosomatids

Detailed comparisons of 16 editosome proteins from Trypanosoma brucei, Trypanosoma cruzi and Leishmania major identified protein motifs associated with catalysis and protein or nucleic acid interactions that suggest their functions in RNA editing. Five related proteins with RNase III-like motifs also contain a U1-like zinc finger and either dsRBM or Pumilio motifs. These proteins may provide the endoribonuclease function in editing. Two other related proteins, at least one of which is associated with U-specific 3′ exonuclease activity, contain two putative nuclease motifs. Thus, editosomes contain a plethora of nucleases or proteins presumably derived from nucleases. Five additional related proteins, three of which have zinc fingers, each contain a motif associated with an OB fold; the TUTases have C-terminal folds reminiscent of RNA binding motifs, thus indicating the presence of numerous nucleic acid and/or protein binding domains, as do the two RNA ligases and a RNA helicase, which provide for additional catalytic steps in editing. These data indicate that trypanosomatid RNA editing is orchestrated by a variety of domains for catalysis, molecular interaction and structure. These domains are generally conserved within other protein families, but some are found in novel combinations in the editosome proteins.     

Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus

Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.     

Principles and Methods in Computational Membrane Protein Design

After decades of progress in computational protein design, the design of proteins folding and functioning in lipid membranes appears today as the next frontier. Some notable successes in the de novo design of simplified model membrane protein systems have helped articulate fundamental principles of protein folding, architecture and interaction in the hydrophobic lipid environment. These principles are reviewed here, together with the computational methods and approaches that were used to identify them. We provide an overview of the methodological innovations in the generation of new protein structures and functions and in the development of membrane-specific energy functions. We highlight the opportunities offered by new machine learning approaches applied to protein design, and by new experimental characterization techniques applied to membrane proteins. Although membrane protein design is in its infancy, it appears more reachable than previously thought.     

The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.     

The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.     

Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins

Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.     

Oceanic Metagenomics: The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.     

Buffer Interference with Protein Dynamics: A Case Study on Human Liver Fatty Acid Binding Protein

Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding protein (hLFABP). Rather than affecting the structure of hLFABP, we found that the dynamics of hLFABP, which were previously proposed to be relevant to its functions, were significantly affected by the binding of hLFABP with MES. Buffer interference with protein dynamics was also demonstrated with Bis-Tris buffer, which is quite different from MES and fatty acids in terms of their molecular structures and properties. This result, to our knowledge, is the first published report on buffer interference with protein dynamics on a microsecond to millisecond timescale and could represent a generic problem in the studies of functionally relevant protein dynamics. Although being a fortuity, our finding of buffer-induced changes in protein dynamics offers a clue to how hLFABP accommodates its ligands.     

Cyclin-Dependent Kinase-Like Function Is Shared by the Beta- and Gamma- Subset of the Conserved Herpesvirus Protein Kinases

The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5)), is a kinase that phosphorylates the cellular retinoblastoma (Rb) tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks). A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been termed the conserved herpesvirus protein kinases (CHPKs) to distinguish them from other human herpesvirus-encoded kinases. To determine if the Cdk-like activities of UL97 were shared by all of the CHPKs, we individually expressed epitope-tagged alleles of each protein in human Saos-2 cells to test for Rb phosphorylation, human U-2 OS cells to monitor nuclear lamina disruption and lamin A phosphorylation, or S. cerevisiae cdc28-13 mutant cells to directly assay for Cdk function. We found that the ability to phosphorylate Rb and lamin A, and to disrupt the nuclear lamina, was shared by all CHPKs from the beta- and gamma-herpesvirus families, but not by their alpha-herpesvirus homologs. Similarly, all but one of the beta and gamma CHPKs displayed bona fide Cdk activity in S. cerevisiae, while the alpha proteins did not. Thus, we have identified novel virally-encoded Cdk-like kinases, a nomenclature we abbreviate as v-Cdks. Interestingly, we found that other, non-Cdk-related activities reported for UL97 (dispersion of promyelocytic leukemia protein nuclear bodies (PML-NBs) and disruption of cytoplasmic or nuclear aggresomes) showed weak conservation among the CHPKs that, in general, did not segregate to specific viral families. Therefore, the genomic and evolutionary conservation of these kinases has not been fully maintained at the functional level. Our data indicate that these related kinases, some of which are targets of approved or developmental antiviral drugs, are likely to serve both overlapping and non-overlapping functions during viral infections.     

Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks

Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular context and integrated approaches for understanding proteome evolution. We feel that the development of λ may be a valuable addition to the toolbox applied to understand the molecular basis of evolution.     

Plant FtsZ1 and FtsZ2 expressed in a eukaryotic host: GTPase activity and self-assembly

Plants and algae contain the FtsZ1 and FtsZ2 protein families that perform specific, non-redundant functions in plastid division. In vitro studies of chloroplast division have been hampered by the lack of a suitable expression system. Here we report the expression and purification of FtsZ1-1 and FtsZ2-1 from Arabidopsis thaliana using a eukaryotic host. Specific GTPase activities were determined and found to be different for FtsZ1-1 vs. FtsZ2-1. The purified proteins readily assembled into previously unreported assembly products named type-I and -II filaments. In contrast to bacterial FtsZ, the Arabidopsis proteins do not form bundled sheets in the presence of Ca²⁺.     

The Crystal Structure of the Streptococcal Collagen-like Protein 2 Globular Domain from Invasive M3-type Group A Streptococcus Shows Significant Similarity to Immunomodulatory HIV Protein gp41

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.     

A structural metagenomics pipeline for examining the gut microbiome

Metagenomic sequencing data provide a rich resource from which to expand our understanding of differential protein functions involved in human health. Here, we outline a pipeline that combines microbial whole genome sequencing with protein structure data to yield a structural metagenomics-informed atlas of microbial enzyme families of interest. Visualizing metagenomics data through a structural lens facilitates downstream studies including targeted inhibition and probe-based proteomics to define at the molecular level how different enzyme orthologs impact in vivo function. Application of this pipeline to gut microbial enzymes like glucuronidases, TMA lyases, and bile salt hydrolases is expected to pinpoint their involvement in health and disease and may aid in the development of therapeutics that target specific enzymes within the microbiome.     

Comprehensive classification of nucleotidyltransferase fold proteins: identification of novel families and their representatives in human

This article presents a comprehensive review of large and highly diverse superfamily of nucleotidyltransferase fold proteins by providing a global picture about their evolutionary history, sequence-structure diversity and fulfilled functional roles. Using top-of-the-line homology detection method combined with transitive searches and fold recognition, we revised the realm of these superfamily in numerous databases of catalogued protein families and structures, and identified 10 new families of nucleotidyltransferase fold. These families include hundreds of previously uncharacterized and various poorly annotated proteins such as Fukutin/LICD, NFAT, FAM46, Mab-21 and NRAP. Some of these proteins seem to play novel important roles, not observed before for this superfamily, such as regulation of gene expression or choline incorporation into cell membrane. Importantly, within newly detected families we identified 25 novel superfamily members in human genome. Among these newly assigned members are proteins known to be involved in congenital muscular dystrophy, neurological diseases and retinal pigmentosa what sheds some new light on the molecular background of these genetic disorders. Twelve of new human nucleotidyltransferase fold proteins belong to Mab-21 family known to be involved in organogenesis and development. The determination of specific biological functions of these newly detected proteins remains a challenging task.     

囊泡转运蛋白SM蛋白家族的研究进展

真核细胞中含有多种不同功能的转运囊泡。虽然转运途径和携带物质各异,但细胞转运的基本分子机制却呈现出高度相似性和保守性。大多数转运途径都需要一种SNARE（Soluble NSF Attachment Protein Receptor）蛋白质复合体介导转运膜泡与靶膜的融合。同时,另一个蛋白家族,Secl／Muncl8蛋白（SM蛋白）也在囊泡运输中发挥重要作用。但是相比于对SNARE蛋白的认识的一致性,在不同的研究中SM蛋白的功能及其与SNARE复合体的相互作用方式却不尽相同。以下综述近年来有关SM蛋白结构和功能的研究进展,并归纳SM蛋白分子的作用机制、功能以及应用。     

Protein folds, functions and evolution.

The evolution of proteins and their functions is reviewed from a structural perspective in the light of the current database. Protein domain families segregate unequally between the three major classes, the 32 different architectures and almost 700 folds observed to date. We find that the number of new topologies is still increasing, although 25 new structures are now determined for each new topology. The corresponding analysis and classification of function is only just beginning, fuelled by the genome data. The structural data revealed unexpected conservations and divergence of function both within and between families. The next five years will see the compilation of a definitive dictionary of protein families and their related functions, based on structural data which reveals relationships hidden at the sequence level. Such information will provide the foundation to build a better understanding of the molecular basis of biological complexity and hopefully to facilitate rational molecular design.     

Do protein-protein interaction databases identify moonlighting proteins?

One of the most striking results of the human (and mammalian) genomes is the low number of protein-coding genes. To-date, the main molecular mechanism to increase the number of different protein isoforms and functions is alternative splicing. However, a less-known way to increase the number of protein functions is the existence of multifunctional, multitask, or "moonlighting", proteins. By and large, moonlighting proteins are experimentally disclosed by serendipity. Proteomics is becoming one of the very active areas of biomedical research, which permits researchers to identify previously unseen connections among proteins and pathways. In principle, protein-protein interaction (PPI) databases should contain information on moonlighting proteins and could provide suggestions to further analysis in order to prove the multifunctionality. As far as we know, nobody has verified whether PPI databases actually disclose moonlighting proteins. In the present work we check whether well-established moonlighting proteins present in PPI databases connect with their known partners and, therefore, a careful inspection of these databases could help to suggest their different functions. The results of our research suggest that PPI databases could be a valuable tool to suggest multifunctionality.