βB1-Crystallin: Thermodynamic Profiles of Molecular Interactions

β-Crystallins are structural proteins maintaining eye lens transparency and opacification. Previous work demonstrated that dimerization of both βA3 and βB2 crystallins (βA3 and βB2) involves endothermic enthalpy of association (∼8 kcal/mol) mediated by hydrophobic interactions.Thermodynamic profiles of the associations of dimeric βA3 and βB1 and tetrameric βB1/βA3 were measured using sedimentation equilibrium. The homo- and heteromolecular associations of βB1 crystallin are dominated by exothermic enthalpy (-13.3 and -24.5 kcal/mol, respectively).Global thermodynamics of βB1 interactions suggest a role in the formation of stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those β-crystallins which associate by predominately hydrophobic forces participate in a weaker protein associations

Errors in macromolecular synthesis after stress. A study of the possible protective role of the small heat shock proteinsBiochemistry

The general goal of this thesis was to gain insight in what small heat shock proteins (sHsps) do with respect to macromolecular synthesis during a stressful situation in the cell. It is known that after a non-lethal heat shock, cells are better protected against a subsequent more severe heat shock, a phenomenon known as thermotolerance and attributed to the presence of the heat shock proteins. The question we asked first is whether the error rate in macromolecular synthesis (transcription, RNA processing, quality control mechanisms and translation) is increased during or after a heat shock in the cell. To know which of these processes might be failing in a cell during stress we designed different reporter systems and we studied the possible protective effect that the sHsps might have. We have shown that there is molecular misreading of sequence repeats and that this misreading is induced by stress. The sHsps did not prevent this. Concerning splicing, the sHsp Hsp27 provided a faster recovery of splicing after heat shock and a faster rephosphorylation of an inhibitor of splicing, dSRp38. Regarding mRNA quality control mechanisms, no defect in nonsense-mediated decay could be detected after heat shock. Finally we describe a relaxed stringency of AUG selection induced by heat shock. To determine whether the sHsps are able to help in cotranslational protein folding and/or heteromer formation, we examined the folding and assembly of beta-crystallins. In the lens, beta-crystallins are surrounded by a high concentration of the sHsp alpha-crystallin, so these are suitable model proteins to test if sHsps facilitate the folding of other crystallins in the lens, particularly that of heterodimeric crystallins. We described that the folding of betaA4-crystallin is helped by its heterodimeric partner, betaB2-crystallin, but not by sHsp

mRNA made during heat shock enters the first round of translation

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Influence of hormones and growth factors on lens protein composition: The effect of dexamethasone and PDGF-AA

Purpose: To investigate the effect of hormones and ocular growth factors on the expression of alpha-, beta-, and gamma-crystallins in rat lens epithelial and fiber cells. Methods: PDGF-AA, EGF, NGF, M-CSF, BMP-2, BMP-4, dexamethasone, and estrogen were tested for their ability to alter the spectrum of crystallins in explanted newborn rat lens epithelial cells or in vitro differentiating newborn rat lens fiber cells. The accumulation of alphaA-, aB-, betaA3/1-, betaB2-, and gamma-crystallin was measured by western blot and dot blot analysis. The morphology of the rat lens explants after culture was examined by hematoxylin-eosin staining, while crystallins were localized by immunofluoresence. Results: Only dexamethasone and PDGF-AA showed an effect on relative crystallin levels. In the presence of dexamethasone the amount of alphaB-crystallin was increased in lens epithelial cells, but dexamethasone did not affect the crystallin spectrum in fiber cells. In rat lens epithelial explants cultured with PDGF-AA an increase in beta- and gamma-crystallin expression was seen. The spectrum of beta- and gamma-crystallins synthesized differed from that present in lens fiber cells. The cells expressing beta- and gamma-crystallin after culture with PDGF-AA were scattered in the epithelial cell layer and retained an epithelial morphology. PDGF-AA did not change the spectrum of crystallins synthesized in lens fiber cells but did enhance the rate of fiber cell differentiation, in agreement with results of others. Conclusions: Both dexamethasone and PDGF-AA influence crystallin gene expression in cultured rat lens epithelial cells. Dexamethasone enhances the expression of alphaB-crystallin while culturing in the presence of PDGF-AA caused an increase in beta- as well as gamma-crystallin synthesis. Since at least the gamma-crystallin genes are known to be silenced in epithelial cells by DNA methylation, PDGF-AA may be able to induce one of the steps towards fiber cell differentiation in some epithelial cells

In vivo heteromer formation. Expression of soluble betaA4-crystallin requires ession of a heteromeric partner

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Hsp27 Enhances Recovery of Splicing as well as Rephosphorylation of SRp38 after Heat Shock

A heat stress causes a rapid inhibition of splicing. Exogenous expression of Hsp27 did not prevent that inhibition but enhanced the recovery of splicing afterward. Another small heat shock protein, αB-crystallin, had no effect. Hsp27, but not αB-crystallin, also hastened rephosphorylation of SRp38—dephosphorylated a potent inhibitor of splicing—after a heat shock, although it did not prevent dephosphorylation by a heat shock. The effect of Hsp27 on rephosphorylation of SRp38 required phosphorylatable Hsp27. A Hsp90 client protein was required for the effect of Hsp27 on recovery of spicing and on rephosphorylation of SRp38. Raising the Hsp70 level by either a pre-heat shock or by exogenous expression had no effect on either dephosphorylation of SRp38 during heat shock or rephosphorylation after heat shock. The phosphatase inhibitor calyculin A prevented dephosphorylation of SRp38 during a heat shock and caused complete rephosphorylation of SRp38 after a heat shock, indicating that cells recovering from a heat shock are not deficient in kinase activity. Together our data show that the activity of Hsp27 in restoring splicing is not due to a general thermoprotective effect of Hsp27, but that Hsp27 is an active participant in the (de)phosphorylation cascade controlling the activity of the splicing regulator SRp38

The effect of alphaB-crystallin and Hsp27 on the availability of translation tion factors in heat-shocked cells

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