Connexins: synthesis, post-translational modifications, and trafficking in health and disease

Connexins are tetraspan transmembrane proteins that form gap junctions and facilitate direct intercellular communication, a critical feature for the development, function, and homeostasis of tissues and organs. In addition, a growing number of gap junction-independent functions are being ascribed to these proteins. The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional level, and undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function. Here, we summarize these key regulatory events, with emphasis on how these affect connexin multifunctionality in health and disease

Recent advances in malaria genomics and epigenomics

Malaria continues to impose a significant disease burden on low- and middle-income countries in the tropics. However, revolutionary progress over the last 3 years in nucleic acid sequencing, reverse genetics, and post-genome analyses has generated step changes in our understanding of malaria parasite (Plasmodium spp.) biology and its interactions with its host and vector. Driven by the availability of vast amounts of genome sequence data from Plasmodium species strains, relevant human populations of different ethnicities, and mosquito vectors, researchers can consider any biological component of the malarial process in isolation or in the interactive setting that is infection. In particular, considerable progress has been made in the area of population genomics, with Plasmodium falciparum serving as a highly relevant model. Such studies have demonstrated that genome evolution under strong selective pressure can be detected. These data, combined with reverse genetics, have enabled the identification of the region of the P. falciparum genome that is under selective pressure and the confirmation of the functionality of the mutations in the kelch13 gene that accompany resistance to the major frontline antimalarial, artemisinin. Furthermore, the central role of epigenetic regulation of gene expression and antigenic variation and developmental fate in P. falciparum is becoming ever clearer. This review summarizes recent exciting discoveries that genome technologies have enabled in malaria research and highlights some of their applications to healthcare. The knowledge gained will help to develop surveillance approaches for the emergence or spread of drug resistance and to identify new targets for the development of antimalarial drugs and perhaps vaccines

Small heat-shock proteins: important players in regulating cellular proteostasis

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease.SM was supported by a Royal Society Dorothy Hodgkin Fellowship, HE is supported by an Australian Research Council Future Fellowship (FT110100586) and JC is supported by a National Health and Medical Research Council Project Grant (#1068087)

Structural characterization of intrinsically disordered proteins by NMR spectroscopy.

Recent advances in NMR methodology and techniques allow the structural investigation of biomolecules of increasing size with atomic resolution. NMR spectroscopy is especially well-suited for the study of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) which are in general highly flexible and do not have a well-defined secondary or tertiary structure under functional conditions. In the last decade, the important role of IDPs in many essential cellular processes has become more evident as the lack of a stable tertiary structure of many protagonists in signal transduction, transcription regulation and cell-cycle regulation has been discovered. The growing demand for structural data of IDPs required the development and adaption of methods such as 13C-direct detected experiments, paramagnetic relaxation enhancements (PREs) or residual dipolar couplings (RDCs) for the study of 'unstructured' molecules in vitro and in-cell. The information obtained by NMR can be processed with novel computational tools to generate conformational ensembles that visualize the conformations IDPs sample under functional conditions. Here, we address NMR experiments and strategies that enable the generation of detailed structural models of IDPs

Mass Spectrometry in the Elucidation of the Glycoproteome of Bacterial Pathogens

Presently some three hundred post-translational modifications are known to occur in bacteria in vivo. Many of these modifications play critical roles in the regulation of proteins and control key biological processes. One of the most predominant modifications, N- and O-glycosylations are now known to be present in bacteria (and archaea) although they were long believed to be limited to eukaryotes. In a number of human pathogens these glycans have been found attached to the surfaces of pilin, flagellin and other surface and secreted proteins where it has been demonstrated that they play a role in the virulence of these bacteria. Mass spectrometry characterization of these glycosylation events has been the enabling key technology for these findings. This review will look at the use of mass spectrometry as a key technology for the detection and mapping of these modifications within microorganisms, with particular reference to the human pathogens, Campylobacter jejuni and Mycobacterium tuberculosis. The overall aim of this review will be to give a basic understanding of the current ‘state-of-the-art’ of the key techniques, principles and technologies, including bioinformatics tools, involved in the analysis of the glycosylation modifications

Deconvoluting the biology and druggability of protein lipidation using chemical proteomics

Lipids are indispensable cellular building blocks, and their post-translational attachment to proteins makes them important regulators of many biological processes. Dysfunction of protein lipidation is also implicated in many pathological states, yet its systematic analysis presents significant challenges. Thanks to innovations in chemical proteomics, lipidation can now be readily studied by metabolic tagging using functionalized lipid analogs, enabling global profiling of lipidated substrates using mass spectrometry. This has spearheaded the first deconvolution of their full scope in a range of contexts, from cells to pathogens and multicellular organisms. Protein N-myristoylation, S-acylation, and S-prenylation are the most well-studied lipid post-translational modifications because of their extensive contribution to the regulation of diverse cellular processes. In this review, we focus on recent advances in the study of these post-translational modifications, with an emphasis on how novel mass spectrometry methods have elucidated their roles in fundamental biological processes

Delineating the role of βIV-Tubulins in pancreatic cancer: βIVb-tubulin inhibition sensitizes pancreatic cancer cells to vinca alkaloids

Pancreatic cancer (PC) is a lethal disease which is characterized by chemoresistance. Components of the cell cytoskeleton are therapeutic targets in cancer. βIV-tubulin is one such component that has two isotypes—βIVa and βIVb. βIVa and βIVb isotypes only differ in two amino acids at their C-terminus. Studies have implicated βIVa-tubulin or βIVb-tubulin expression with chemoresistance in prostate, breast, ovarian and lung cancer. However, no studies have examined the role of βIV-tubulin in PC or attempted to identify isotype specific roles in regulating cancer cell growth and chemosensitivity. We aimed to determine the role of βIVa- or βIVb-tubulin on PC growth and chemosensitivity. PC cells (MiaPaCa-2, HPAF-II, AsPC1) were treated with siRNA (control, βIVa-tubulin or βIVb-tubulin). The ability of PC cells to form colonies in the presence or absence of chemotherapy was measured by clonogenic assays. Inhibition of βIVa-tubulin in PC cells had no effect chemosensitivity. In contrast, inhibition of βIVb-tubulin in PC cells sensitized to vinca alkaloids (Vincristine, Vinorelbine and Vinblastine), which was accompanied by increased apoptosis and enhanced cell cycle arrest. We show for the first time that βIVb-tubulin, but not βIVa-tubulin, plays a role in regulating vinca alkaloid chemosensitivity in PC cells. The results from this study suggest βIVb-tubulin may be a novel therapeutic target and predictor of vinca alkaloid sensitivity for PC and warrants further investigation

Deriving a mutation index of carcinogenicity using protein structure and protein interfaces

With the advent of Next Generation Sequencing the identification of mutations in the genomes of healthy and diseased tissues has become commonplace. While much progress has been made to elucidate the aetiology of disease processes in cancer, the contributions to disease that many individual mutations make remain to be characterised and their downstream consequences on cancer phenotypes remain to be understood. Missense mutations commonly occur in cancers and their consequences remain challenging to predict. However, this knowledge is becoming more vital, for both assessing disease progression and for stratifying drug treatment regimes. Coupled with structural data, comprehensive genomic databases of mutations such as the 1000 Genomes project and COSMIC give an opportunity to investigate general principles of how cancer mutations disrupt proteins and their interactions at the molecular and network level. We describe a comprehensive comparison of cancer and neutral missense mutations; by combining features derived from structural and interface properties we have developed a carcinogenicity predictor, InCa (Index of Carcinogenicity). Upon comparison with other methods, we observe that InCa can predict mutations that might not be detected by other methods. We also discuss general limitations shared by all predictors that attempt to predict driver mutations and discuss how this could impact high-throughput predictions. A web interface to a server implementation is publicly available at http://inca.icr.ac.uk/

the contribution of mass spectrometry based proteomics to understanding epigenetics

Chromatin is a macromolecular complex composed of DNA and histones that regulate gene expression and nuclear architecture. The concerted action of DNA methylation, histone post-translational modifications and chromatin-associated proteins control the epigenetic regulation of the genome, ultimately determining cell fate and the transcriptional outputs of differentiated cells. Deregulation of this complex machinery leads to disease states, and exploiting epigenetic drugs is becoming increasingly attractive for therapeutic intervention. Mass spectrometry (MS)-based proteomics emerged as a powerful tool complementary to genomic approaches for epigenetic research, allowing the unbiased and comprehensive analysis of histone post-translational modifications and the characterization of chromatin constituents and chromatin-associated proteins. Furthermore, MS holds great promise for epigenetic biomarker discovery and represents a useful tool for deconvolution of epigenetic drug targets. Here, we will provide an ov..