Alteration of protein subcellular location and domain formation by alternative translational initiation

Alternative translation is an important cellular mechanism contributing to the generation of proteins and the diversity of protein functions. Instead of studying individual cases, we systematically analyzed the alteration of protein subcellular location and domain formation by alternative translational initiation in eukaryotes. The results revealed that 85.7% of alternative translation events generated biological diversity, attributed to different subcellular localizations and distinct domain contents in alternative isoforms. Analysis of isoelectric point values revealed that most N-terminal truncated isoforms significantly lowered their isoelectric point values targeted at different subcellular localizations, whereas they had conserved domain contents the same as the full-length isoforms. Furthermore, Fisher's exact test indicated that the two ways-targeting at different cellular compartments and changing domain contents-were negatively associated. The N-term truncated isoforms should have only one way to diversify their functions distinct from the full-length ones. The peculiar consequence of subcellular relocation as well as change of domain contents reflected the very high level of biological complexity as alternative usage of initiation codons.     

A Novel,Universally Active C-terminal Protein Degradation Signal Generated by Alternative Splicing

Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.     

Bioinformatics meets proteomics--bridging the gap between mass spectrometry data analysis and cell biology

Proteomics research programs typically comprise the identification of protein content of any given cell, their isoforms, splice variants, post-translational modifications, interacting partners and higher-order complexes under different conditions. These studies present significant analytical challenges owing to the high proteome complexity and the low abundance of the corresponding proteins, which often requires highly sensitive and resolving techniques. Mass spectrometry plays an important role in proteomics and has become an indispensable tool for molecular and cellular biology. However, the analysis of mass spectrometry data can be a daunting task in view of the complexity of the information to decipher, the accuracy and dynamic range of quantitative analysis, the availability of appropriate bioinformatics software and the overwhelming size of data files. The past ten years have witnessed significant technological advances in mass spectrometry-based proteomics and synergy with bioinformatics is vital to fulfill the expectations of biological discovery programs. We present here the technological capabilities of mass spectrometry and bioinformatics for mining the cellular proteome in the context of discovery programs aimed at trace-level protein identification and expression from microgram amounts of protein extracts from human tissues.     

Proteomics in radiation research: present status and future perspectives

Rapidly developing postgenome research has made proteins an attractive target for biological analysis. The well-established term of proteome is defined as the complete set of proteins expressed in a given cell, tissue or organism. Unlike the genome, a proteome is rapidly changing as it tends to adapt to microenvironmental signals. The systematic analysis of the proteome at a given time and state is referred to as proteomics. This technique provides information on the molecular and cellular mechanisms that regulate physiology and pathophysiology of the cell. Applications of proteome profiling in radiation research are increasing. However, the large-scale proteomics data sets generated need to be integrated into other fields of radiation biology to facilitate the interpretation of radiation-induced cellular and tissue effects. The aim of this review is to introduce the most recent developments in the field of radiation proteomics.     

Blood coagulation and alternative pre-mRNA splicing: an overview

Throughout the 20th century, great advances were made in understanding of how blood coagulation occurs, what physiological and biochemical mechanisms are responsible for its regulation, and what genes and their protein products comprise the essential components of the hemostatic network. Recently, complete sequencing of the human genome revealed that the structural diversity of higher eukaryotes cannot be solely attributed to the number of protein-encoding genes, whereas tools of molecular biology helped establish that pre-mRNAs produced by most protein-encoding genes undergo alternative splicing, a mechanism that enables production of multiple protein isoforms by a single gene. Research in the field of thrombosis and hemostasis revealed that the genes encoding several critical proteins at various junctures of the coagulation cascade produce alternatively spliced protein isoforms with distinct structural and biochemical characteristics, revealing a principally novel dimension in the regulation of blood clotting and, possibly, a few novel therapeutic approaches to treatment of abnormal hemostasis. This review summarizes recently published data pertaining to biosynthesis of the alternatively spliced isoforms of tissue factor (TF, or coagulation factor III), tissue factor pathway inhibitor (TFPI), and coagulation factor XI (FXI), and discusses future directions of this continuously evolving area of biomedical research, with an emphasis on molecular mechanics responsible for regulation of constitutive as well as alternative pre-mRNA splicing.     

High-throughput expression,purification, and characterization of recombinant Caenorhabditis elegans proteins

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.     

New insights into viral structure and virus–cell interactions through proteomics

Although genomics techniques such as DNA microarrays have been widely used in virology, much more limited use has been made of proteomics. Although difficult, proteomics can greatly contribute to an understanding of virus–cell interactions, including the ternary structure of viral receptors at the cell surface, post-translational modifications and isoforms of critical viral and cellular proteins and even to the structure of viruses. Proteomics techniques also offer the potential for discovering markers for diagnostic and prognostic tests of viral infections in vivo. This review describes the use of several proteomic approaches for the analysis of HIV–cellular receptor interactions, the molecular mechanisms of transport of herpes simplex virus within neurons, and the structure of the tegument of herpes simplex virus.     

Comparative analysis of protein interaction networks

Recent advances in proteomics and computational biology have lead to a flood of protein interaction data and resulting interaction networks (e.g. (Gavin et al., 2002)). Here I first analyse the status and quality of parts lists (genes and proteins), then comparatively assess large-scale protein interaction data (von Mering et al., 2002) and finally try to identify biological meaningful units (e.g. pathways, cellular processes) within interaction networks that are derived from the conservation of gene neighborhood (Snel et al., 2002). Possible extensions of gene neighborhood analysis to eukaryotes (von Mering and Bork, 2002) will be discussed.     

Proteomics in clinical trials and practice: present uses and future promise

The study of clinical proteomics is a promising new field that has the potential to have many applications, including the identification of biomarkers and monitoring of disease, especially in the field of oncology. Expression proteomics evaluates the cellular production of proteins encoded by a particular gene and exploits the differential expression and post-translational modifications of proteins between healthy and diseased states. These biomarkers may be applied towards early diagnosis, prognosis, and prediction of response to therapy. Functional proteomics seeks to decipher protein-protein interactions and biochemical pathways involved in disease biology and targeted by newer molecular therapeutics. Advanced spectrometry technologies and new protein array formats have improved these analyses and are now being applied prospectively in clinical trials. Further advancement of proteomics technology could usher in an era of personalized molecular medicine, where diseases are diagnosed at earlier stages and where therapies are more effective because they are tailored to the protein expression of a patient's malignancy.     

Prioritizing targets for structural biology through the lens of proteomics: The archaeal protein TGAM_1934 from Thermococcus gammatolerans

ORFans are hypothetical proteins lacking any significant sequence similarity with other proteins. Here, we highlighted by quantitative proteomics the TGAM_1934 ORFan from the hyperradioresistant Thermococcus gammatolerans archaeon as one of the most abundant hypothetical proteins. This protein has been selected as a priority target for structure determination on the basis of its abundance in three cellular conditions. Its solution structure has been determined using multidimensional heteronuclear NMR spectroscopy. TGAM_1934 displays an original fold, although sharing some similarities with the 3D structure of the bacterial ortholog of frataxin, CyaY, a protein conserved in bacteria and eukaryotes and involved in iron–sulfur cluster biogenesis. These results highlight the potential of structural proteomics in prioritizing ORFan targets for structure determination based on quantitative proteomics data. The proteomic data and structure coordinates have been deposited to the ProteomeXchange with identifier PXD000402 ( http://proteomecentral.proteomexchange.org/dataset/PXD000402 ) and Protein Data Bank under the accession number 2mcf, respectively.     

Proteomic tools for cell biology

Acquisition of large bodies of genomic sequence is facilitating the use of global techniques to assay cellular function. DNA microarrays have enabled the measurement of global mRNA levels and are able to detect changes in gene expression between different cellular states. Since much of the regulation of physiolgical processes happens post-translationally, measuring only the mRNA levels gives an incomplete picture. Strategies to assay global expression, localization, or interaction of proteins fall into the emerging field of proteomics, with various combinations of techniques being utilized to separate and identify proteins. In this review, we will present a general overview of the currently available proteomic tools and then give examples of how these tools are being utilized to answer questions in cell biology.