Use of proteomics to define targets of T-cell immunity

The mammalian immune system has evolved to display peptides derived from microbial antigens to immune effector cells. Liberated from the intact antigens through distinct proteolytic mechanisms, these peptides are subsequently transported to the cell surface while bound to chaperone-like receptors known as major histocompatibility complex molecules. These complexes are then scrutinized by T-cells that express receptors with specificity for specific major histocompatibility complex–peptide complexes. In normal uninfected cells, this process of antigen processing and presentation occurs continuously, with the resultant array of self-antigen-derived peptides displayed on the surface of these cells. Changes in this cellular peptide array alert the immune system to changes in the intracellular environment that may be associated with infection, oncogenesis or other abnormal cellular processes, resulting in a cascade of events that result in the elimination of the abnormal cell. Since peptides play such an essential role in informing the immune system of infection with viral or microbial pathogens and the transformation of cells in malignancy, the tools of proteomics, in particular mass spectrometry, are ideally suited to study these immune responses at a molecular level. Recent advances in studies of immune responses that have utilized mass spectrometry and associated technologies are reviewed. The authors gaze into the future and look at current challenges and where proteomics will impact in immunology over the next 5 years.     

Use of proteomics for the identification of novel drug targets in brain diseases

In spite of the rapid advances in the development of the new proteomic technologies, there are, to date, relatively fewer studies aiming to explore the neuronal proteome. One of the reasons is the complexity of the brain, which presents high cellular heterogeneity and a unique subcellular compartmentalization. Therefore, tissue fractionation of the brain to enrich proteins of interest will reduce the complexity of the proteomics approach leading to the production of manageable and meaningful results. In this review, general considerations and strategies of proteomics, the advantages and challenges to exploring the neuronal proteome are described and summarized. In addition, this article presents an overview of recent advances of proteomic technologies and shows that proteomics can serve as a valuable tool to globally explore the changes in brain proteome during various disease states. Understanding the molecular basis of brain function will be extremely useful in identifying novel targets for the treatment of brain diseases.     

Platelet proteomics applied to the search for novel antiplatelet therapeutic targets

Introduction: Unwanted platelet activation is associated with numerous diseases, mainly thrombosis-related. In this context, proteomics has emerged as a novel tool with potential for drug target discovery and to scrutinize the effects of antiplatelet drugs.

Areas covered: The present review presents the main findings of platelet proteomic studies to date in the context of drug target discovery and perspectives for the future ahead. It includes data and evidences obtained from literature searches on PubMed as well as commentaries derived from the authors’ experience and opinions.

Expert commentary: Platelet proteomics applied to drug target discovery is a young field. Recent studies have shown promising data, especially in the context of coronary artery disease. However, challenges remain such as establishing definitive guidelines for blood collection and platelet isolation, essential to guarantee data reproducibility. Recent advances in quantitative platelet proteomics should lead to novel studies with higher clinical impact in the near future.     

Bacterial proteomics and identification of potential vaccine targets

Currently, the greatest causes of human morbidity and mortality are infectious diseases. Vaccination remains the most effective measure to lessen this burden on the human population. Many vaccines presently in use were developed using techniques first proposed by Louis Pasteur, which involved the use of inactivated, attenuated live forms, or extracts of pathogenic organisms to immunize the host and provide protection from the disease. The advent of the genomic era has recently led to a new generation of rationally designed vaccines developed using a process termed reverse vaccinology. This approach uses genomic data in silico to identify proteins encoded by the pathogen as potential vaccine candidates. Proteomic technologies serve as an important complement to the reverse vaccinology approach to antigen discovery. Proteomic techniques are able to identify proteins that are expressed by the pathogen during infection of a host and the subset of proteins that reside on the surface of the pathogen. These two traits should be considered central factors to vaccine antigen selection as they assure that the host will be able to mount an effective immune response that leads to lasting protection from the pathogen.     

High-throughput screening of structural proteomics targets using NMR

We applied a high-throughput strategy for the screening of targets for structural proteomics of Xanthomonas axonopodis pv citri. This strategy is based on the rapid (1)H-(15)N HSQC NMR analysis of bacterial lysates containing selectively (15)N-labelled heterologous proteins. Our analysis permitted us to classify the 19 soluble candidates in terms of 'foldedness', that is, the extent to which they present a well-folded solution structure, as reflected by the quality of their NMR spectra. This classification allowed us to define a priority list to be used as a guide to select protein candidates for further structural studies.     

Use of proteomics to understand seed development in rice

Rice is an important cereal crop and has become a model monocot for research into crop biology. Rice seeds currently feed more than half of the world's population and the demand for rice seeds is rapidly increasing because of the fast‐growing world population. However, the molecular mechanisms underlying rice seed development is incompletely understood. Genetic and molecular studies have developed our understanding of substantial proteins related to rice seed development. Recent advancements in proteomics have revolutionized the research on seed development at the single gene or protein level. Proteomic studies in rice seeds have provided the molecular explanation for cellular and metabolic events as well as environmental stress responses that occur during embryo and endosperm development. They have also led to the new identification of a large number of proteins associated with regulating seed development such as those involved in stress tolerance and RNA metabolism. In the future, proteomics, combined with genetic, cytological, and molecular tools, will help to elucidate the molecular pathways underlying seed development control and help in the development of valuable and potential strategies for improving yield, quality, and stress tolerance in rice and other cereals. Here, we reviewed recent progress in understanding the mechanisms of seed development in rice with the use of proteomics.     

Presynaptic Calmodulin targets: lessons from structural proteomics

Introduction: Calmodulin (CaM) is a highly conserved Ca²⁺-binding protein that is exceptionally abundant in the brain. In the presynaptic compartment of neurons, CaM transduces changes in Ca²⁺ concentration into the regulation of synaptic transmission dynamics.

Areas covered: We review selected literature including published CaM interactor screens and outline established and candidate presynaptic CaM targets. We present a workflow of biochemical and structural proteomic methods that were used to identify and characterize the interactions between CaM and Munc13 proteins. Finally, we outline the potential of ion mobility-mass spectrometry (IM-MS) for conformational screening and of protein-protein cross-linking for the structural characterization of CaM complexes.

Expert commentary: Cross-linking/MS and native MS can be applied with considerable throughput to protein mixtures under near-physiological conditions, and thus effectively complement high-resolution structural biology techniques. Experimental distance constraints are applicable best when obtained by combining different cross-linking strategies, i.e. by using cross-linkers with different spacer length and reactivity, and by using the incorporation of unnatural photo-reactive amino acids. Insights from structural proteomics can be used to generate CaM-insensitive mutants of CaM targets for functional studies in vitro or ideally in vivo.     

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity

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Anti-tumor immunity in adult T-cell leukemia

Adult T-cell leukemia (ATL) occurs in a small population of human T-cell leukemia virus type I (HTLV-I)-infected individuals. It has been noted that ATL is incidentally associated with mother-to-child infection which occurs mainly through breast-feeding, elevated levels of proviral load, and insufficiency in HTLV-I-specific cytotoxic T lymphocyte (CTL) responses. Among these, anti-tumor potentials of HTLV-I-specific CTL have been shown in ex vivo analysis of human HTLV-I-infected individuals and also in vivo experiments by using rat models of HTLV-I-infected lymphomas. In another rat model of HTLV-I-infection, orally infected rats showed significantly higher HTLV-I proviral load but lower HTLV-I-specific cellular immune responses than in intraperitoneally infected rats. As a result, persistent viral load was inversely correlated with levels of virus-specific T-cell responses. HTLV-I-specific T-cell responses in orally infected rats recovered by re-immunization. Conversion of Tax-specific T-cell responses from low to high levels was also observed in an ATL patient who obtained complete remission after hematopoietic stem cell transplantation. These findings suggest that HTLV-I-specific immune unresponsiveness associated with oral HTLV-I infection may be a potential risk factor for development of ATL, allowing expansion of the infected cell reservoir in vivo, and that immunological strategies targeting Tax may potentially reduce the risk of ATL and induce therapeutic effects on ATL.     

Use of laboratory animals to define physiological functions and bioavailability of zinc

For the past 50 years laboratory animals have been used to ascertain the metabolic bases for signs of zinc deficiency such as sharply reduced food intake, severe dermatitis, slow wound healing, delayed sexual development and function, reduced immunocompetence, severe teratogenic abnormalities, and abnormal metabolism of carbohydrate, lipid, and protein. Current evidence indicates that many of these symptoms may be consequences of inhibition of early steps in nucleic acid metabolism that lead to problems with cellular replication and growth and also that zinc plays an important role in membrane structure and function. Bioavailability of zinc to experimental animals was early shown to be reduced by plant protein diets and to be further reduced by feeding excess calcium. Current evidence indicates phytic acid in plant proteins to be a major inhibitor of zinc absorption, although food-processing methods can either increase or decrease zinc bioavailability. The inhibitory effect of phytic acid is very dependent on dietary calcium in association with phytate and zinc. Usual calcium intakes by humans are much below those demonstrated in animals to cause phytate inhibition of dietary zinc availability.     

Use of proteomics to discover novel markers of cardiac allograft rejection

Endomyocardial biopsy remains the most reliable method of detecting rejection following cardiac transplantation. Despite numerous attempts to detect rejection using a blood assay, none have proved reliable enough to replace the biopsy. Here, we have investigated the hypothesis that proteomics has the potential to reveal many molecules which are upregulated in the heart during rejection, some of which may serve as novel blood markers of rejection. Initially, sequential cardiac biopsies (33 in total) from 4 patients were analysed by two-dimensional gel electrophoresis according to whether they showed rejection (n = 16) or no rejection (n = 17); over 100 proteins were found to be upregulated by between 2- and 50-fold during rejection. Of these, 13 were identified and were found to be cardiac specific or heat shock proteins. Two of these (alphaB-crystallin, tropomyosin) were measured by ELISA in the sera of 17 patients followed for 3 months after their transplants. Mean levels of alphaB-crystallin and tropomyosin were significantly higher in sera associated with biopsies showing 1A (p = 0.007) or all grades of rejection (p = 0.022) compared to no rejection. These studies demonstrate that proteomics is a powerful method that can be used to identify novel serum markers of human cardiac allograft rejection.     

Chemical proteomics identifies unanticipated targets of clinical kinase inhibitors.

Kinases represent one of the most important target classes of current drug discovery efforts. However, because the vast majority of potential small-molecule therapeutics is directed toward the highly conserved ATP-binding cleft, kinase inhibitors often exhibit significant unintended off-target effects. A recent report describes a chemical proteomics methodology that enables the simultaneous in vivo quantification of the on- and off-binding targets of kinase inhibitors across hundreds of nucleotide-dependent enzymes.     

Plant immunity: AvrPto targets the frontline

Bacterial pathogens must suppress host defences to cause disease. New research shows that the Pseudomonas effector protein AvrPto does so by directly targeting plant transmembrane receptor kinases involved in bacterial perception.