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      Dynamic disulfide exchange in a crystallin protein in the human eye lens promotes cataract-associated aggregation

      , , , ,

      Journal of Biological Chemistry

      American Society for Biochemistry & Molecular Biology (ASBMB)

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          Abstract

          <p class="first" id="d2779187e208">Increased light scattering in the eye lens due to aggregation of the long-lived lens proteins, crystallins, is the cause of cataract disease. Several mutations in the gene encoding human γD-crystallin (HγD) cause misfolding and aggregation. Cataract-associated substitutions at Trp <sup>42</sup> cause the protein to aggregate <i>in vitro</i> from a partially unfolded intermediate locked by an internal disulfide bridge, and proteomic evidence suggests a similar aggregation precursor is involved in age-onset cataract. Surprisingly, WT HγD can promote aggregation of the W42Q variant while itself remaining soluble. Here, a search for a biochemical mechanism for this interaction has revealed a previously unknown oxidoreductase activity in HγD. Using <i>in vitro</i> oxidation, mutational analysis, cysteine labeling, and MS, we have assigned this activity to a redox-active internal disulfide bond that is dynamically exchanged among HγD molecules. The W42Q variant acts as a disulfide sink, reducing oxidized WT and forming a distinct internal disulfide that kinetically traps the aggregation-prone intermediate. Our findings suggest a redox “hot potato” competition among WT and mutant or modified polypeptides wherein variants with the lowest kinetic stability are trapped in aggregation-prone intermediate states upon accepting disulfides from more stable variants. Such reactions may occur in other long-lived proteins that function in oxidizing environments. In these cases, aggregation may be forestalled by inhibiting disulfide flow toward mutant or damaged polypeptides. </p>

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          Most cited references 94

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          Is Open Access

          PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse

          PhosphoSitePlus (http://www.phosphosite.org) is an open, comprehensive, manually curated and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses 1 30 000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The interface is designed for clarity and ease of navigation. From the home page, users can launch simple or complex searches and browse high-throughput data sets by disease, tissue or cell line. Searches can be restricted by specific treatments, protein types, domains, cellular components, disease, cell types, cell lines, tissue and sequences or motifs. A few clicks of the mouse will take users to substrate pages or protein pages with sites, sequences, domain diagrams and molecular visualization of side-chains known to be modified; to site pages with information about how the modified site relates to the functions of specific proteins and cellular processes and to curated information pages summarizing the details from one record. PyMOL and Chimera scripts that colorize reactive groups on residues that are modified can be downloaded. Features designed to facilitate proteomic analyses include downloads of modification sites, kinase–substrate data sets, sequence logo generators, a Cytoscape plugin and BioPAX download to enable pathway visualization of the kinase–substrate interactions in PhosphoSitePlus®.
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            The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data

            Affinity purification coupled with mass spectrometry (AP-MS) is now a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (e.g. proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. While the standard approach is to identify nonspecific interactions using one or more negative controls, most small-scale AP-MS studies do not capture a complete, accurate background protein set. Fortunately, negative controls are largely bait-independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the Contaminant Repository for Affinity Purification (the CRAPome) and describe the use of this resource to score protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely available online at www.crapome.org.
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              Amyloid formation by globular proteins under native conditions.

              The conversion of proteins from their soluble states into well-organized fibrillar aggregates is associated with a wide range of pathological conditions, including neurodegenerative diseases and systemic amyloidoses. In this review, we discuss the mechanism of aggregation of globular proteins under conditions in which they are initially folded. Although a conformational change of the native state is generally necessary to initiate aggregation, we show that a transition across the major energy barrier for unfolding is not essential and that aggregation may well be initiated from locally unfolded states that become accessible, for example, via thermal fluctuations occurring under physiological conditions. We review recent evidence on this topic and discuss its significance for understanding the onset and potential inhibition of protein aggregation in the context of diseases.
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                Author and article information

                Journal
                Journal of Biological Chemistry
                J. Biol. Chem.
                American Society for Biochemistry & Molecular Biology (ASBMB)
                0021-9258
                1083-351X
                November 16 2018
                November 16 2018
                November 16 2018
                September 21 2018
                : 293
                : 46
                : 17997-18009
                Article
                10.1074/jbc.RA118.004551
                6240864
                30242128
                © 2018

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