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      The interaction of unfolding α-lactalbumin and malate dehydrogenase with the molecular chaperone αB-crystallin: a light and X-ray scattering investigation

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          Abstract

          Purpose

          The molecular chaperone αB-crystallin is found in high concentrations in the lens and is present in all major body tissues. Its structure and the mechanism by which it protects its target protein from aggregating and precipitating are not known.

          Methods

          Dynamic light scattering and X-ray solution scattering techniques were used to investigate structural features of the αB-crystallin oligomer when complexed with target proteins under mild stress conditions, i.e., reduction of α-lactalbumin at 37 °C and malate dehydrogenase when heated at 42 °C. In this investigation, the size, shape and particle distribution of the complexes were determined in real-time following the induction of stress.

          Results

          Overall, it is observed that the mass distribution, hydrodynamic radius, and spherical shape of the αB-crystallin oligomer do not alter significantly when it complexes with its target protein.

          Conclusions

          The data are consistent with the target protein being located in the outer protein shell of the αB-crystallin oligomer where it is readily accessible for possible refolding via the action of other molecular chaperones.

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          Most cited references54

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          Alpha-crystallin can function as a molecular chaperone.

          J. Horwitz (1992)
          The alpha-crystallins (alpha A and alpha B) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially alpha B) are found in many cells and organs outside the lens, and alpha B is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that alpha-crystallin can function as a molecular chaperone. Stoichiometric amounts of alpha A and alpha B suppress thermally induced aggregation of various enzymes. In particular, alpha-crystallin is very efficient in suppressing the thermally induced aggregation of beta- and gamma-crystallins, the two other major mammalian structural lens proteins. alpha-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured gamma-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that alpha-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells alpha-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.
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            Crystal structure of a small heat-shock protein.

            The principal heat-shock proteins that have chaperone activity (that is, they protect newly made proteins from misfolding) belong to five conserved classes: HSP100, HSP90, HSP70, HSP60 and the small heat-shock proteins (sHSPs). The sHSPs can form large multimeric structures and have a wide range of cellular functions, including endowing cells with thermotolerance in vivo and being able to act as molecular chaperones in vitro; sHSPs do this by forming stable complexes with folding intermediates of their protein substrates. However, there is little information available about these structures or the mechanism by which substrates are protected from thermal denaturation by sHSPs. Here we report the crystal structure of a small heat-shock protein from Methanococcus jannaschii, a hyperthermophilic archaeon. The monomeric folding unit is a composite beta-sandwich in which one of the beta-strands comes from a neighbouring molecule. Twenty-four monomers form a hollow spherical complex of octahedral symmetry, with eight trigonal and six square 'windows'. The sphere has an outer diameter of 120 A and an inner diameter of 65 A.
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              Protective and therapeutic role for alphaB-crystallin in autoimmune demyelination.

              alphaB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease.
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                Author and article information

                Journal
                Mol Vis
                MV
                Molecular Vision
                Molecular Vision
                1090-0535
                2010
                18 November 2010
                : 16
                : 2446-2456
                Affiliations
                [1 ]School of Optometry and Vision Sciences, Cardiff University, Cardiff, UK
                [2 ]School of Chemistry & Physics, The University of Adelaide, Adelaide, SA, Australia
                [3 ]School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia
                [4 ]Department of Chemistry, University of Cambridge, Cambridge, UK
                [5 ]School of Pharmacy and Medical Sciences, The University of South Australia, Adelaide, SA, Australia
                Author notes
                Correspondence to: Justyn W. Regini, School of Optometry and Vision Sciences, Cardiff University, Maindy Rd, Cardiff CF24 4LU, UK; Phone: + 44 (0) 920870061; FAX: +44 (0) 2920874859; email: reginijw@ 123456cf.ac.uk

                The first two authors contributed equally to this work

                Article
                262 2010MOLVIS0326
                2998715
                21152271
                34feb8bf-5885-469f-9ab5-409e5e992674
                Copyright © 2010 Molecular Vision.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 29 July 2010
                : 12 November 2010
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                Vision sciences
                Vision sciences

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