Protein splicing and its applications

Protein splicing elements, termed inteins, provide a fertile source for innovative biotechnology tools. First harnessed for protein purification, inteins are now used to express cytotoxic proteins, to segmentally modify or label proteins, to cyclize proteins or peptides, to study structure-activity relationships and to generate reactive polypeptide termini in expressed proteins for an expanding list of chemoselective reactions, including protein ligation.     

Protein splicing: its discovery and structural insight into novel chemical mechanisms

Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self-catalytically excised out from a nascent protein precursor and the two flanking sequences (N- and C-exteins) are ligated to produce two mature enzymes. This unique reaction was first discovered from studies of the structure and expression of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and yet comprises two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Subsequent studies have demonstrated that protein splicing is not unique for the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its precise reaction mechanisms from a viewpoint of structure-directed chemistry, a series of crystal structural studies has been carried out with the use of splicing-inactive and slowly spliceable precursors of VMA1 recombinants. One precursor structure revealed that the N-terminal junction of the introduced extein polypeptide forms an intermediate containing a five-membered thiazolidine ring. The other precursor structures showed spliced products with a linkage between the N- and C-extein segments. This article summarizes biochemical and structural studies on a self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation.     

Protein splicing

Protein splicing is a posttranslational process that results in excision of an internal protein region (intein) and ligation of its flanking sequences (exteins). As distinguished from other variants of protein processing, protein splicing does not require cofactors of enzymes. Protein splicing is catalyzed by an internal domain (so-called Hint domain) of the intein itself. The review considers the main regularities and molecular mechanisms of the process, as well as the functions of Hint domains in other protein families (Hh proteins, bacterial BIL domains, etc.). Studies of protein splicing are of importance from both theoretical and applied viewpoints. For instance, comparisons of the inteins found in different domains of life illustrate the role of horizontal transfer in intein spreading. A possible role of inteins in regulating several cell processes is discussed on the basis of recent data.     

Protein splicing

Inteins are internal polypeptide sequences that are posttranslationally excised from a protein precursor by a self-catalyzed protein-splicing reaction. Most of inteins consist of N- and C-terminal protein splicing domain and central endonuclease domain. The endonuclease domain can initiate mobility of the intein gene, this process being named intein homing. This review is focused on the recent data about the structure and function of inteins. Main intein-mediated protein-engineering applications, such as protein purification, ligation and cyclization, new forms of biosensors, are presented.     

Protein splicing

Protein splicing is a post-translational autocatalytic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). This process doesn't require any cofactors or enzymes, which distinguish it from other variants of protein processing. Protein splicing is catalyzed by Hint-domain - internal intein's domain. In review the main molecular mechanisms of this process are described. Function of analogous Hint-domains of other proteins families (Hh-proteins, BIL-domains etc) are considered. The analysis of inteins characteristic to different branches of life illustrates the role of horizontal transfer in intein's distribution and evolution. A possible role of inteins in regulation of different cell processes is discussed.     

Biological applications of protein splicing

Protein splicing is a naturally occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer.     

Protein disulfide isomerases in neurodegeneration: From disease mechanisms to biomedical applications

Protein disulfide isomerases (PDIs) are a family of foldases and chaperones primarily located at the endoplasmic reticulum that catalyze the formation and isomerization of disulfide bonds thereby facilitating protein folding. PDIs also perform important physiological functions in protein quality control, cell death, and cell signaling. Protein misfolding is involved in the etiology of the most common neurodegenerative diseases, including Alzheimer, Parkinson, amyotrophic lateral sclerosis, Prion-related disorders, among others. Accumulating evidence indicate altered expression of PDIs as a prominent and common feature of these neurodegenerative conditions. Here we overview most recent advances in our understanding of the possible functional contribution of PDIs to neurodegeneration, depicting a complex and poorly understood scenario. Possible therapeutic benefits of targeting PDIs in a disease context and their use as biomarkers are discussed.     

RNAi mechanisms and applications

Within the past two decades we have become increasingly aware of the roles that RNAs play in regulation of gene expression. The RNA world was given a booster shot with the discovery of RNA interference (RNAi), a compendium of mechanisms involving small RNAs (less than 30 bases long) that regulate the expression of genes in a variety of eukaryotic organisms. Rapid progress in our understanding of RNAi-based mechanisms has led to applications of this powerful process in studies of gene function as well as in therapeutic applications for the treatment of disease. RNAi-based therapies involve two-dimensional drug designs using only identification of good Watson-Crick base pairing between the RNAi guide strand and the target, thereby resulting in rapid design and testing of RNAi triggers. To date there are several clinical trials using RNAi, and we should expect the list of new applications to grow at a phenomenal rate. This article summarizes our current knowledge about the mechanisms and applications of RNAi.     

RNA structure and the mechanisms of alternative splicing

Alternative splicing is a widespread means of increasing protein diversity and regulating gene expression in eukaryotes. Much progress has been made in understanding the proteins involved in regulating alternative splicing, the sequences they bind to, and how these interactions lead to changes in splicing patterns. However, several recent studies have identified other players involved in regulating alternative splicing. A major theme emerging from these studies is that RNA secondary structures play an under appreciated role in the regulation of alternative splicing. This review provides an overview of the basic aspects of splicing regulation and highlights recent progress in understanding the role of RNA secondary structure in this process.     

哺乳动物细胞mRNA剪接调控的分子机制

mRNA的可变剪接是指一个单一的mRNA前体(pre-mRNA)经过不同的剪接加工方式生成多种mRNA变异体(variants)的过程,这些变异体最终可以编码合成具有不同结构和功能的蛋白质。在过去的10多年中,大量数据表明,可变剪接是增加转录组和蛋白质组多样性的重要资源,也是调控哺乳动物细胞基因表达的重要步骤。可变剪接具有高度的组织与发育阶段特异性,并受到外界信号的控制。剪接调控的紊乱与疾病的发生发展密切相关。该文将对哺乳动物细胞mRNA剪接调控的分子机制进行阐述。     

Protein microarrays and their applications

In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional protein analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes the basic technologies that are needed in the fabrication of protein arrays and their recent applications.     

HMM sampling and applications to gene finding and alternative splicing

The standard method of applying hidden Markov models to biological problems is to find a Viterbi (maximal weight) path through the HMM graph. The Viterbi algorithm reduces the problem of finding the most likely hidden state sequence that explains given observations, to a dynamic programming problem for corresponding directed acyclic graphs. For example, in the gene finding application, the HMM is used to find the most likely underlying gene structure given a DNA sequence. In this note we discuss the applications of sampling methods for HMMs. The standard sampling algorithm for HMMs is a variant of the common forward-backward and backtrack algorithms, and has already been applied in the context of Gibbs sampling methods. Nevetheless, the practice of sampling state paths from HMMs does not seem to have been widely adopted, and important applications have been overlooked. We show how sampling can be used for finding alternative splicings for genes, including alternative splicings that are conserved between genes from related organisms. We also show how sampling from the posterior distribution is a natural way to compute probabilities for predicted exons and gene structures being correct under the assumed model. Finally, we describe a new memory efficient sampling algorithm for certain classes of HMMs which provides a practical sampling alternative to the Hirschberg algorithm for optimal alignment. The ideas presented have applications not only to gene finding and HMMs but more generally to stochastic context free grammars and RNA structure prediction.