Mining antimicrobial peptides from small open reading frames in Ciona intestinalis

Though being able to encode various kinds of bioactive peptides, small open reading frames (sORFs) are poorly annotated in many genomic data. The present study was conducted to evaluate the potential of sORFs in encoding antimicrobial peptides (AMPs) in the basal chordate model Ciona intestinalis. About 4.8 m genomic sequence was first retrieved for sORFs mining by the program sORFfinder, then the sORFs were translated into amino acid sequences for AMP prediction via CAMP server, and thereafter, ten putative AMPs were selected for expression and antimicrobial activity validation. In total, over 180 peptides deduced from the sORFs were predicted to be AMPs. Among the ten tested peptides, six were found to have significant expressed sequence tag matches, providing strong evidence for gene expression; five were proved to be active against the bacterial strains. These results indicate that many sORFs in C. intestinalis genome contain AMP information. This work can serve as an important initial step to investigate the role of sORFs in the innate defense of C. intestinalis. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

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。     

植物小开放阅读框编码肽的研究进展

小开放阅读框（small open reading frame,sORF）一般指基因组中能够编码长度在100个氨基酸左右或以内短肽的开放阅读框。它们广泛存在于植物基因组,却因编码短肽而常被基因组注释忽视。随着翻译组学和蛋白质组学测序技术的发展,具有翻译活性的sORF被证实广泛存在于植物基因组,且参与植物生长发育等重要过程的调控。该文归纳了近些年来植物领域sORF的一些研究进展,主要包括sORF的来源与分类、信息学预测方法和生物学功能等,并基于此对植物sORF未来的研究方向进行了展望。     

Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex

A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5' and 3' regions of enod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.     

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.     

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。     

Uncovering small membrane proteins in pathogenic bacteria: Regulatory functions and therapeutic potential

Bacterial small proteins (below 50 amino acids) encoded by small open reading frames (sORFs) are recognized as an emerging class of functional molecules that have been largely overlooked in the past. While some were uncovered serendipitously, global approaches have recently been developed to detect these sORFs. A large portion of small proteins appears to be hydrophobic and located in the bacterial membrane. In the present review, we describe functional small hydrophobic proteins discovered in pathogenic bacteria and report recent advances in the discovery of additional ones. Small membrane proteins contribute to bacterial adaptation to changing environments and often appear to be implicated in negative feedback regulation loops by modulating the function or stability of larger membrane proteins. A subset of these proteins belongs to toxin-antitoxin modules. We highlight the features of characterized hydrophobic small proteins that may pave the way for identification of the functional small proteins among novel sORFs discovered. Besides providing new insights into bacterial pathogenesis, identification of naturally occurring small hydrophobic proteins of pathogenic bacteria can lead to new therapeutic interventions, as recently shown with the development of synthetic peptides derived from natural small proteins that display antibacterial or antivirulence properties.     

基于8种真核生物的整合分析揭示种属特异性小蛋白的功能和进化特征

小蛋白 ( < 100个氨基酸) 广泛存在于三界生命中,具有重要生物功能．早期涉及小蛋白的研究主要集中于少量特殊物种中的蛋白质家族,以及在全基因组尺度预测短小开放读码框(sORFs)的算法开发,但并无跨真核物种的大规模组学分析来揭示小蛋白的功能和进化特征．通过对已知小蛋白和拥有短小开放读码框的基因进行全基因组尺度的计算分析,长度小于100个氨基酸的RefSeq proteins按照其序列保守性被划分为存在于所有8种真核生物、只存在于脊椎动物和只存在于哺乳动物三个进化分类中,此三个进化分类所对应的生物学功能揭示了小蛋白行使种属特异性功能的特征．进一步研究发现,大多数人类特有的小蛋白也是组织表达特异性的,并且绝大多数古老的小蛋白在人体内普遍表达．因此认为,一些真核小蛋白出现并在自然选择压力下富集,行使种属特异性功能,并且以特殊的方式进化和表达．     

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.     

Biologically active peptides: prospects for drug development

Biologically active peptides aree typified by their unbiquity of distribution, their high receptor affinity and an almost infinite diversity of structure. For these reasons, considerable effort is now being expended to elucidate the possible role of peptides in brain function. This effort has been stimulated by the discovery of a number of new endogenous peptides, such as the enkephalins, endorphins, vasoactive intestinal peptide and neurotensin. At present, there is no clearly defined role for these peptides, although they may form an important basis for the chemical coding of various brain functions, including pain, mood and memory. At present, the potential for drug development of peptide agonists remains in fairly circumscribed areas such as analgesia, pituitary hormone control, and gastrointestinal motor and secretory control. Peptide antagonists may provide a vast field for future development, although only one area, that of antifertility drugs based on LHRH antagonists, shows any promise of immediate success. Industrial research approaches to new peptide agonists and antagonists mainly rely at present on rational drug design through structural analogies. Other fruitful approaches to be considered are the screening of natural microbial and plant products and the possible application of genetic engineering techniques.     

Geometric differences in the ribosome exit tunnel impact the escape of small nascent proteins

The exit tunnel is the subcompartment of the ribosome that contains the nascent polypeptide chain and, as such, is involved in various vital functions, including regulation of translation and protein folding. As the geometry of the tunnel shows important differences across species, we focus on key geometrical features of eukaryote and prokaryote tunnels. We used a simple coarse-grained molecular dynamics model to study the role of the tunnel geometry in the post-translational escape of short proteins (short open reading frames [sORFs]) with lengths ranging from 6 to 56 amino acids. We found that the probability of escape for prokaryotes is one for all but the 12-mer chains. Moreover, proteins of this length have an extremely low escape probability in eukaryotes. A detailed examination of the associated single trajectories and energy profiles showed that these variations can be explained by the interplay between the protein configurational space and the confinement effects introduced by the constriction sites of the ribosome exit tunnel. For certain lengths, either one or both of the constriction sites can lead to the trapping of the protein in the “pocket” regions preceding these sites. As the distribution of existing sORFs indicates some bias in length that is consistent with our findings, we finally suggest that the constraints imposed by the tunnel geometry have impacted the evolution of sORFs.     

The evolution of pro-opiomelanocortin: looking for the invertebrate fingerprints

The presence and role of the pro-opiomelanocortin (POMC) gene and encoded peptides in invertebrates are here summarized and discussed. Some of the POMC-derived peptides show a significant similarity regarding their functions, suggesting their appearance before the split of protostomian–deuterostomian lineages and their maintenance during evolution. The basic mechanisms that govern the exchange of information between cells are usually well conserved, and this could have also been for POMC-derived peptides, that are mainly involved in fundamental functions such as immune and neuroendocrine responses. However, the presence and functions that POMC-derived peptides exhibit in taxonomically distant models, are not always reflected by the expected gene homology, leaving the problem of POMC evolution in invertebrates in need of additional study.     

Role of a short open reading frame in ribosome shunt on the cauliflower mosaic virus RNA leader

The pregenomic 35 S RNA of cauliflower mosaic virus (CaMV) belongs to the growing number of mRNAs known to have a complex leader sequence. The 612-nucleotide leader contains several short open reading frames (sORFs) and forms an extended hairpin structure. Downstream translation of 35 S RNA is nevertheless possible due to the ribosome shunt mechanism, by which ribosomes are directly transferred from a take-off site near the capped 5' end of the leader to a landing site near its 3' end. There they resume scanning and reach the first long open reading frame. We investigated in detail how the multiple sORFs influence ribosome migration either via shunting or linear scanning along the CaMV leader. The sORFs together constituted a major barrier for the linear ribosome migration, whereas the most 5'-proximal sORF, sORF A, in combination with sORFs B and C, played a positive role in translation downstream of the leader by diverting scanning ribosomes to the shunt route. A simplified, shunt-competent leader was constructed with the most part of the hairpin including all the sORFs except sORF A replaced by a scanning-inhibiting structure. In this leader as well as in the wild type leader, proper translation and termination of sORF A was required for efficient shunt and also for the level of shunt enhancement by a CaMV-encoded translation transactivator. sORF A could be replaced by heterologous sORFs, but a one-codon (start/stop) sORF was not functional. The results implicate that in CaMV, shunt-mediated translation requires reinitiation. The efficiency of the shunt process is influenced by translational properties of the sORF.     

智能多肽的自组装机理及其生物学应用

智能多肽是指智能响应外界刺激并做出相应回应的多肽。由于其形成过程为自发的自组装,故智能多肽又可称为自组装多肽。智能多肽的氨基酸构成使其拥有良好的生物相容性及生物可降解性,作为构筑基元拼接成为功能性材料,在新型生物材料方面展示出了广阔的应用前景。概括了智能多肽的性质、自组装机理及应用,重点阐述了它在生物能源、生物医学工程和分离工程上的应用,以期在系统认识智能多肽的基础上,发掘其应用潜能,突破开发瓶颈。     

SmProt: A Reliable Repository with Comprehensive Annotation of Small Proteins Identified from Ribosome Profiling

Small proteins specifically refer to proteins consisting of less than 100 amino acids translated from small open reading frames (sORFs), which were usually missed in previous genome annotation. The significance of small proteins has been revealed in current years, along with the discovery of their diverse functions. However, systematic annotation of small proteins is still insufficient. SmProt was specially developed to provide valuable information on small proteins for scientific community. Here we present the update of SmProt, which emphasizes reliability of translated sORFs, genetic variants in translated sORFs, disease-specific sORF translation events or sequences, and remarkably increased data volume. More components such as non-ATG translation initiation, function, and new sources are also included. SmProt incorporated 638,958 unique small proteins curated from 3,165,229 primary records, which were computationally predicted from 419 ribosome profiling (Ribo-seq) datasets or collected from literature and other sources from 370 cell lines or tissues in 8 species (Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Danio rerio, Saccharomyces cerevisiae, Caenorhabditis elegans, and Escherichia coli). In addition, small protein families identified from human microbiomes were also collected. All datasets in SmProt are free to access, and available for browse, search, and bulk downloads at http://bigdata.ibp.ac.cn/SmProt/.     

抗菌肽结构改造与人工智能研发策略

抗菌肽是一类广泛存在于生物体内的小分子肽,参与构成生物体先天免疫,可以有效抵抗病原微生物的入侵。抗菌肽具有广谱抗菌活性,且不易产生耐药性等特点,在治疗感染性疾病方面具有独特的优势,有望成为理想的抗感染药物。然而,由于部分抗菌肽尚存在稳定性差、毒性高等问题,限制了抗菌肽的广泛应用。由于人工智能算法能有效合成具有高稳定性、低毒性的抗菌肽,在探索天然抗菌肽中展现了巨大的潜力,因此本文简述了抗菌肽的抗菌机制、结构改造以及利用机器学习和深度学习等人工智能算法进行新型抗菌肽研发的优化策略,以期为抗菌肽结构优化及研发提供新思路。     

In Silico Analysis of Peptide Potential Biological Functions

Over the past decade, tools of omics technologies have generated a large amount of data in various repositories, which are of interest for meta-analysis today. Now, researchers in the field of proteomics and peptidomics focus not on sequencing, but on functions performed by molecules and metabolic interactions, in which the proteins or peptides participate. As a result of a single LC-MS/MS analysis, several thousand unique peptides can be identified, each of which may be bioactive. A classic technique for determining the peptide function is a direct experiment. Bioinformatics approaches as a preliminary analysis of potential biological functions are an important step and are able to significantly reduce time and cost of experimental verification. This article provides an overview of computational methods for predicting biological functions of peptides. Approaches based on machine learning, which are the most popular today, algorithms using structural, evolutionary, or statistical patterns, as well as methods based on molecular docking, are considered. Databases of bioactive peptides are reported, providing information necessary to construct new algorithms for predicting biological functions. Attention is paid to the characteristics of peptides, on the basis of which it is possible to draw conclusions about their bioactivity. In addition, the report provides a list of online services that may be used by researchers to analyze potential activities of peptides with which they work.     

Lipopolysaccharide (Endotoxin)-host defense antibacterial peptides interactions: Role in bacterial resistance and prevention of sepsis

Lipopolysaccharide (LPS) is the major molecular component of the outer membrane of Gram-negative bacteria and serves as a physical barrier providing the bacteria protection from its surroundings. LPS is also recognized by the immune system as a marker for the detection of bacterial pathogen invasion, responsible for the development of inflammatory response, and in extreme cases to endotoxic shock. Because of these functions, the interaction of LPS with LPS binding molecules attracts great attention. One example of such molecules are antimicrobial peptides (AMPs). These are large repertoire of gene-encoded peptides produced by living organisms of all types, which serve as part of the innate immunity protecting them from pathogen invasion. AMPs are known to interact with LPS with high affinities. The biophysical properties of AMPs and their mode of interaction with LPS determine their biological function, susceptibility of bacteria to them, as well as the ability of LPS to activate the immune system. This review will discuss recent studies on the molecular mechanisms underlying these interactions, their effects on the resistance of the bacteria to AMPs, as well as their potential to neutralize LPS-induced endotoxic shock.