Comparative genomic analysis identifies small open reading frames (sORFs) with peptide-encoding features in avian 16S rDNA

The mitochondrial genome (mt-DNA) functional repertoire has recently been enriched in mammals by the identification of functional small open reading frames (sORFs) embedded in ribosomal DNAs. Through comparative genomic analyses the presence of putatively functional sORFs was investigated in birds. Alignment of available avian mt-DNA sequences revealed highly conserved regions containing four putative sORFs that presented low insertion/deletion polymorphism rate (<0.1%) and preserved in frame start/stop codons in >80% of species. Detected sORFs included avian homologs of human Humanin and Short-Humanin-Like-Peptide 6 and two new sORFs not yet described in mammals. The amino-acid sequences of the four putative encoded peptides were strongly conserved among birds, with amino-acid p-distances (5.6 to 25.4%) similar to those calculated for typical avian mt-DNA-encoded proteins (14.8%). Conservation resulted from either drastic conservation of the nucleotide sequence or negative selection pressure. These data extend to birds the possibility that mitochondrial rDNA may encode small bioactive peptides.     

微生物小开放阅读框:小蛋白及翻译调控研究进展

小开放阅读框(small open reading frame, sORF)广泛存在于不同生物基因组中,由于其序列短,以及编码的产物小蛋白(smallprotein,或称微蛋白;microprotein或迷你蛋白miniprotein)检测困难等原因,小开放阅读框长期未得到充分注释和研究。近年来,随着高通量测序、翻译组和质谱分析等技术的不断发展,在不同生物中发现大量新的小开放阅读框,其编码的小蛋白及介导的翻译调控已应用于药物开发及植物抗病机理等研究。但是,目前对微生物的小开放阅读框相关研究和应用还相对有限。本文综述了小开放阅读框编码产物小蛋白的发现和鉴定,以及上游开放阅读框(upstream open reading frame, uORF)对mRNA翻译调控等最新研究进展,重点介绍了微生物基因组中小开放阅读框的鉴定和功能研究进展,为深入认识微生物中小开放阅读框的功能和作用机制,以及植物和动物等高等其他生物的小蛋白和翻译调控相关研究提供参考。     

Identification and characterization of hydrophobic Escherichia coli virulence proteins by liquid chromatography-electrospray ionization mass spectrometry

Virulence of enterotoxicogenic Escherichia coli is mediated by rodlike, rigid, highly hydrophobic proteins designated fimbriae or colonization factors (CFs). More than 20 different colonization factors have been described so far using predominantly immunological and genetic methods. To characterize these hydrophobic proteins by liquid chromatography-mass spectrometry (LC-MS), different methodologies were explored. A novel LC-MS method was developed using hexafluoroisopropanol to maintain the hydrophobic proteins in solution. In addition, these proteins were digested with cyanogen bromide and peptide mapping by LC-MS was established. This technique was particularly useful in identification of closely related CFs. Both LC-MS and peptide mapping methodologies were found to be useful in characterizing highly hydrophobic CFs of E. coli. To search for molecular weights of mature proteins in the National Center for Biotechnology Information (NCBI) database, a new feature was developed and its applicability tested. The identification of a class of pathogenic virulence proteins, either intact or digested, is possible with molecular weight database searching.     

Identification of bacterial small non-coding RNAs: experimental approaches

Almost 140 bacterial small RNAs (sRNAs; sometimes referred to as non-coding RNAs) have been discovered in the past six years. The majority of these sRNAs were discovered in Escherichia coli, and a smaller subset was characterized in other bacteria, many of which were pathogenic. Many of these genes were identified as a result of systematic screens using computational prediction of sRNAs and experimental-based approaches, including microarray and shotgun cloning. A smaller number of sRNAs were discovered by direct labeling or by functional genetic screens. Many of the discovered genes, ranging in size from 50 to 500 nucleotides, are conserved and located in intergenic regions, in-between open reading frames. The expression of many of these genes is growth phase dependent or stress related. As each search employed specific parameters, this led to the identification of genes with distinct characteristics. Consequently, unique sRNAs such as those that are species-specific, sRNA genes that are transcribed under unique conditions or genes located on the antisense strand of protein-encoding genes, were probably missed.     

Identification and characterization of sORF-encoded polypeptides

Abstract

Molecular biology, genomics and proteomics methods have been utilized to reveal a non-annotated class of endogenous polypeptides (small proteins and peptides) encoded by short open reading frames (sORFs), or small open reading frames (smORFs). We refer to these polypeptides as s(m)ORF-encoded polypeptides or SEPs. The early SEPs were identified via genetic screens, and many of the RNAs that contain s(m)ORFs were originally considered to be non-coding; however, elegant work in bacteria and flies demonstrated that these s(m)ORFs code for functional polypeptides as small as 11-amino acids in length. The discovery of these initial SEPs led to search for these molecules using methods such as ribosome profiling and proteomics, which have revealed the existence of many SEPs, including novel human SEPs. Unlike screens, omics methods do not necessarily link a SEP to a cellular or biological function, but functional genomic and proteomic strategies have demonstrated that at least some of these newly discovered SEPs have biochemical and cellular functions. Here, we provide an overview of these results and discuss the future directions in this emerging field.     

YidC,a newly defined evolutionarily conserved protein,mediates membrane protein assembly in bacteria

Membranes contain proteins that catalyze a variety of reactions, which lead to the selective permeability of the membrane. For membrane proteins to function as receptors, transporters, channels, and ATPases, they must be targeted to their correct membrane and inserted into the lipid bilayer. Recently, a new membrane component called YidC was discovered that mediates the insertion of proteins into membranes in bacteria. YidC homologs also exist in mitochondria and chloroplasts. Depletion of YidC from the cell interferes with the insertion of membrane proteins that insert both dependent and independent of the SecYEG/SecDFYajC machinery. YidC directly interacts with membrane proteins during the membrane protein insertion process and assists in the folding of the hydrophobic regions into the membrane bilayer. The chloroplast and bacterial YidC homologs are truly functional homologs because the chloroplast homolog Alb3 functionally complements the bacterial YidC depletion strain. The role of YidC in the membrane insertion pathway will be reviewed.     

小蛋白质富集法鉴定酿酒酵母“漏检蛋白”

小蛋白质 (Small proteins,SPs) 是由小开放阅读框 (Short open reading frames,sORFs) 编码长度小于100个氨基酸的多肽。研究发现小蛋白质参与了基因表达调控、细胞信号转导和代谢等重要生物学过程。然而,生命体中大多数的已注释小蛋白质尚缺少蛋白水平存在的实验证据,被称为漏检蛋白 (Missing proteins,MPs)。小蛋白质的高效鉴定是其功能研究的前提,也有助于挖掘“漏检蛋白”。文中采用小蛋白质富集策略鉴定到72个酵母小蛋白质,验证9个“漏检蛋白”,发现低分子量、高疏水性、膜结合、弱密码子使用偏性及不稳定性是蛋白漏检的主要原因,对进一步的技术优化具有指导意义。     

Analysis of known bacterial protein vaccine antigens reveals biased physical properties and amino acid composition

Many vaccines have been developed from live attenuated forms of bacterial pathogens or from killed bacterial cells. However, an increased awareness of the potential for transient side-effects following vaccination has prompted an increased emphasis on the use of sub-unit vaccines, rather than those based on whole bacterial cells. The identification of vaccine sub-units is often a lengthy process and bioinformatics approaches have recently been used to identify candidate protein vaccine antigens. Such methods ultimately offer the promise of a more rapid advance towards preclinical studies with vaccines. We have compared the properties of known bacterial vaccine antigens against randomly selected proteins and identified differences in the make-up of these two groups. A computer algorithm that exploits these differences allows the identification of potential vaccine antigen candidates from pathogenic bacteria on the basis of their amino acid composition, a property inherently associated with sub-cellular location.     

Crystallization of the membrane protein hVDAC1 produced in cell-free system

Structural studies of membrane proteins are in constant evolution with the development of new improvements for their expression, purification, stabilization and crystallization. However, none of these methods still provides a universal approach to solve the structure of membrane proteins. Here we describe the crystallization of the human voltage-dependent anion channel-1 produced by a bacterial cell-free expression system. While VDAC structures have been recently solved, we propose an alternative strategy for producing the recombinant protein, which can be applied to other membrane proteins reluctant to expression, purification and crystallization by classical approaches. Despite a lot of efforts to crystallize a cell-free expressed membrane protein, this study is to our knowledge one of the first reports of a successful crystallization. Focusing on expression in a soluble and functional state, in a detergent environment, is the key to get crystals. Although the diffraction of VDAC crystals is limited, the simplicity and the rapidity to set-up and optimize this technology are drastic advantages in comparison to other methods.     

Proteomics characterization of outer membrane vesicles from the extraintestinal pathogenic Escherichia coli DeltatolR IHE3034 mutant

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.     

The archaeal cell cycle: current issues

The recently discovered structural similarities between the archaeal Orc1/Cdc6 and bacterial DnaA initiator proteins for chromosome replication have exciting implications for cell cycle regulation. Together with current attempts to identify archaeal chromosome replication origins, the information is likely to yield fundamental insights into replication control in both archaea and eukaryotes within the near future. Several proteins that affect, or are likely to affect, chromatin structure and genome segregation in archaea have been described recently, including Sph1 and 2, ScpA and B, Sir2, Alba and Rio1p. Important insights into the properties of the MinD and FtsZ cell division proteins, and of putative cytoskeletal elements, have recently been gained in bacteria. As these proteins also are present among archaea, it is likely that the new information will also be essential for understanding archaeal genome segregation and cell division. A series of interesting cell cycle issues has been brought to light through the discovery of the novel Nanoarchaeota phylum, and these are outlined briefly. Exciting areas for extended cell cycle investigations of archaea are identified, including termination of chromosome replication, application of in situ cytological techniques for localization of cell cycle proteins and the regulatory roles of GTP-binding proteins and small RNAs.     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.     

Genomic analysis of secretion systems

Secretion of proteins into the extracellular environment is important to almost all bacteria, and in particular mediates interactions between pathogenic or symbiotic bacteria with their eukaryotic hosts. The accumulation of bacterial genome sequence data in the past few years has provided great insights into the distribution and function of these secretion systems. Three systems are responsible for secretion of proteins across the bacterial cytoplasmic membrane: Sec, SRP and Tat. Many novel examples of systems for transport across the Gram-negative bacterial cell envelope have been discovered through genome sequencing and surveys, including many novel type III secretion systems and autotransporters. Similarly, genomic data mining has revealed many new potential secretion substrates and identified unsuspected domains in secretion-associated proteins. Interestingly, genomic analyses have also hinted at the existence of a dedicated protein secretion system in Gram-positive bacteria, targeting members of the WXG100/ESAT-6 family of proteins, and have revealed an unexpectedly wide distribution of sortase-driven protein-targeting systems.     

Microproteins—Discovery,structure, and function

Advances in proteogenomic technologies have revealed hundreds to thousands of translated small open reading frames (sORFs) that encode microproteins in genomes across evolutionary space. While many microproteins have now been shown to play critical roles in biology and human disease, a majority of recently identified microproteins have little or no experimental evidence regarding their functionality. Computational tools have some limitations for analysis of short, poorly conserved microprotein sequences, so additional approaches are needed to determine the role of each member of this recently discovered polypeptide class. A currently underexplored avenue in the study of microproteins is structure prediction and determination, which delivers a depth of functional information. In this review, we provide a brief overview of microprotein discovery methods, then examine examples of microprotein structures (and, conversely, intrinsic disorder) that have been experimentally determined using crystallography, cryo-electron microscopy, and NMR, which provide insight into their molecular functions and mechanisms. Additionally, we discuss examples of predicted microprotein structures that have provided insight or context regarding their function. Analysis of microprotein structure at the angstrom level, and confirmation of predicted structures, therefore, has potential to identify translated microproteins that are of biological importance and to provide molecular mechanism for their in vivo roles.     

Influence of oligomerization state on the structural properties of invasion plasmid antigen B from Shigella flexneri in the presence and absence of phospholipid membranes

Shigella flexneri causes bacillary dysentery, an important cause of mortality among children in the developing world. Shigella secretes effector proteins via its type III secretion system (T3SS) to promote bacterial uptake into human colonic epithelial cells. The T3SS basal body spans the bacterial cell envelope anchoring a surface‐exposed needle. A pentamer of invasion plasmid antigen D lies at the nascent needle tip and invasion plasmid antigen B (IpaB) is recruited into the needle tip complex on exposure to bile salts. From here, IpaB forms a translocon pore in the host cell membrane. Although the mechanism by which IpaB inserts into the membrane is unknown, it was recently shown that recombinant IpaB can exist as either a monomer or tetramer. Both of these forms of IpaB associate with membranes, however, only the tetramer forms pores in liposomes. To reveal differences between these membrane‐binding events, Cys mutations were introduced throughout IpaB, allowing site‐specific fluorescence labeling. Fluorescence quenching was used to determine the influence of oligomerization and/or membrane association on the accessibility of different IpaB regions to small solutes. The data show that the hydrophobic region of tetrameric IpaB is more accessible to solvent relative to the monomer. The hydrophobic region appears to promote membrane interaction for both forms of IpaB, however, more of the hydrophobic region is protected from solvent for the tetramer after membrane association. Limited proteolysis demonstrated that changes in IpaB's oligomeric state may determine the manner by which it associates with phospholipid membranes and the subsequent outcome of this association. Proteins 2014; 82:3013–3022. © 2014 Wiley Periodicals, Inc.     

Mimicking small G-proteins: an emerging theme from the bacterial virulence arsenal

The identification of the Ras superfamily of small molecular weight GTPases (G-proteins) has opened up new fields in cancer biology, immunity and infectious disease research. Because of their ubiquitous role in cellular homeostasis, small G-proteins are common targets for several pathogens, including bacteria. It is well known that pathogenic bacteria have evolved virulence factors that chemically modify GTPases or directly mimic the activities of key regulatory proteins. However, recent studies now suggest that bacterial 'effector' proteins can also mimic the activities of Ras small G-proteins despite their lack of guanine nucleotide binding or GTPase enzymatic activity. The study of these unique pathogenic strategies continues to reveal novel mechanistic insights into host cellular communication networks and the role of small G-protein signalling during human infectious disease.     

Global gene expression analysis reveals reduced abundance of putative microRNA targets in human prostate tumours

Background

The availability of hundreds of bacterial genomes allowed a comparative genomic study of the Type VI Secretion System (T6SS), recently discovered as being involved in pathogenesis. By combining comparative and phylogenetic approaches using more than 500 prokaryotic genomes, we characterized the global T6SS genetic structure in terms of conservation, evolution and genomic organization.

Results

This genome wide analysis allowed the identification of a set of 13 proteins constituting the T6SS protein core and a set of conserved accessory proteins. 176 T6SS loci (encompassing 92 different bacteria) were identified and their comparison revealed that T6SS-encoded genes have a specific conserved genetic organization. Phylogenetic reconstruction based on the core genes showed that lateral transfer of the T6SS is probably its major way of dissemination among pathogenic and non-pathogenic bacteria. Furthermore, the sequence analysis of the VgrG proteins, proposed to be exported in a T6SS-dependent way, confirmed that some C-terminal regions possess domains showing similarities with adhesins or proteins with enzymatic functions.

Conclusion

The core of T6SS is composed of 13 proteins, conserved in both pathogenic and non-pathogenic bacteria. Subclasses of T6SS differ in regulatory and accessory protein content suggesting that T6SS has evolved to adapt to various microenvironments and specialized functions. Based on these results, new functional hypotheses concerning the assembly and function of T6SS proteins are proposed.     

Membrane translocation of folded proteins

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.     

The thylakoid delta pH-dependent pathway machinery facilitates RR-independent N-tail protein integration

The thylakoidal DeltapH-dependent and bacterial twin arginine transport systems are structurally and functionally related protein export machineries. These recently discovered systems have been shown to transport folded proteins but are not known to assemble integral membrane proteins. We determined the translocation pathway of a thylakoidal FtsH homologue, plastid fusion/protein translocation factor, which is synthesized with a chloroplast-targeting peptide, a hydrophobic signal peptide, and a hydrophobic membrane anchor. The twin arginine motif in its signal peptide and its sole integration requirement of a DeltapH suggested that plastid fusion/protein translocation factor employs the DeltapH pathway. Surprisingly, changing the twin arginine to twin lysine or deleting the signal peptide did not abrogate integration capability or characteristics. Nevertheless, three criteria argue that all three forms require the DeltapH pathway for integration. First, integration was competed by an authentic DeltapH pathway precursor. Second, antibodies to DeltapH pathway component Hcf106 specifically inhibited integration. Finally, chloroplasts from the hcf106 null mutant were unable to integrate Pftf into their thylakoids. Thus, DeltapH pathway machinery facilitates both signal peptide-directed and N-tail-mediated membrane integration and does not strictly require the twin arginine motif.     

Proteomics-driven identification of short open reading frame-encoded peptides

Accumulating evidence has shown that a large number of short open reading frames (sORFs) also have the ability to encode proteins. The discovery of sORFs opens up a new research area, leading to the identification and functional study of sORF encoded peptides (SEPs) at the omics level. Besides bioinformatics prediction and ribosomal profiling, mass spectrometry (MS) has become a significant tool as it directly detects the sequence of SEPs. Though MS-based proteomics methods have proved to be effective for qualitative and quantitative analysis of SEPs, the detection of SEPs is still a great challenge due to their low abundance and short sequence. To illustrate the progress in method development, we described and discussed the main steps of large-scale proteomics identification of SEPs, including SEP extraction and enrichment, MS detection, data processing and quality control, quantification, and function prediction and validation methods.