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Engholm-Keller K.,University of Southern Denmark | Engholm-Keller K.,Childrens Medical Research Institute | Larsen M.R.,University of Southern Denmark
Proteomics | Year: 2013

Phosphorylation, the reversible addition of a phosphate group to amino acid side chains of proteins, is a fundamental regulator of protein activity, stability, and molecular interactions. Most cellular processes, such as inter- and intracellular signaling, protein synthesis, degradation, and apoptosis, rely on phosphorylation. This PTM is thus involved in many diseases, rendering localization and assessment of extent of phosphorylation of major scientific interest. MS-based phosphoproteomics, which aims at describing all phosphorylation sites in a specific type of cell, tissue, or organism, has become the main technique for discovery and characterization of phosphoproteins in a nonhypothesis driven fashion. In this review, we describe methods for state-of-the-art MS-based analysis of protein phosphorylation as well as the strategies employed in large-scale phosphoproteomic experiments with focus on the various challenges and limitations this field currently faces. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Liao W.W.P.,University of Sydney | Arthur J.W.,University of Sydney | Arthur J.W.,Childrens Medical Research Institute
Autoimmunity Reviews | Year: 2011

The Major Histocompatibility Complex (MHC) constitutes an important part of the human immune system. During infection, pathogenic proteins are processed into peptide fragments by the antigen processing machinery. These peptides bind to MHC molecules and the MHC-peptide complex is then transported to the cell membrane where it elicits an immune response via T-cell binding. Understanding the molecular mechanism of this process will greatly assist in determining the aetiology of various diseases and in the design of effective drugs. One of the most challenging aspects of this area of research is understanding the specificity and sensitivity of the binding process. An empirical approach to the problem is unfeasible as there are over 512 billion potential binding peptides for each MHC molecule. Computational approaches offer the promise of predicting peptide binding, thus dramatically reducing the number of peptides proceeding to experimental verification. Various bioinformatic approaches have been developed to predict whether or not a particular peptide will bind to a particular MHC allele. Currently, peptide binding prediction methods can be categorised into three major groups: motif- and scoring matrix-based methods, artificial intelligence- (AI-) based methods, and structure-based methods. The first two are sequence-based approaches and are generally based on common sequence motifs in peptides known to bind to MHC molecules. The structure-based approach concerns the structural features and the distribution of energy between the binding peptide and the MHC molecule. Although knowledge of the molecular structure of the MHC molecules is expected to lead to better predictions of peptide binding, the development of structure-based methods has been relatively slow compared to sequence-based methods. Comparisons of various methods showed that the best sequence-based methods significantly outperform structure-based methods. This may be improved by producing more structures and binding data desperately needed by many alleles, especially class II molecules. On the other hand, the large number of verification methods and indicators used by structure-based studies hinders critical evaluation of the methods. Adopting commonly used assessment procedures can demonstrate the relative performance of structure-based methods in a straightforward comparison with other methods. This review provides an overview of current methods for predicting peptide binding to the MHC, with a focus on structure-based methods, and explores the potential for future development in this area. © 2011 Elsevier B.V.

Speidel D.,Childrens Medical Research Institute
Trends in Cell Biology | Year: 2010

Apoptosis induced by p53 is firmly established as a central mechanism of tumour suppression. In addition to its complex functions as a nuclear transcription factor, p53 can act in the cytosol and mitochondria to promote apoptosis through transcription-independent mechanisms. Recent studies have shown that physical and functional interactions of p53 with various members of the Bcl-2 family provide the basis for this alternative route of p53-mediated cell death. However, different models of how these interactions promote apoptosis have been proposed. This review focuses on the mechanisms, regulation and physiological roles of transcription-independent p53 activities and highlights recent findings suggesting that the utilisation of these activities provides a promising alternative strategy for p53-based cancer therapy. © 2009 Elsevier Ltd. All rights reserved.

Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

Pera M.F.,University of Southern California | Tam P.P.L.,Childrens Medical Research Institute | Tam P.P.L.,University of Sydney
Nature | Year: 2010

During early mammalian development, as the pluripotent cells that give rise to all of the tissues of the body proliferate and expand in number, they pass through transition states marked by a stepwise restriction in developmental potential and by changes in the expression of key regulatory genes. Recent findings show that cultured stem-cell lines derived from different stages of mouse development can mimic these transition states. They further reveal that there is a high degree of heterogeneity and plasticity in pluripotent populations in vitro and that these properties are modulated by extrinsic signalling. Understanding the extrinsic control of plasticity will guide efforts to use human pluripotent stem cells in research and therapy. © 2010 Macmillan Publishers Limited. All rights reserved.

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