Alterations in the mouse and human proteome caused by Huntington's disease. (1/314)

Huntington's disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In our effort to identify proteins involved in processes upstream or downstream of the disease-causing huntingtin, we studied the proteome of a well established mouse model by large gel two-dimensional electrophoresis. We could demonstrate for the first time at the protein level that alpha1-antitrypsin and alphaB-crystalline both decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver and testes in mice. Reduced expression of the serine protease inhibitors alpha1-antitrypsin and contraspin was found in liver, heart, and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. In three brain regions obtained post-mortem from Huntington's disease patients, alpha1-antitrypsin expression was also altered. Reduced expression of the major urinary proteins not found in the brain was seen in the liver of affected mice, demonstrating that the disease exerts its influence outside the brain of transgenic mice at the protein level. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington's disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.  (+info)

The small heat shock protein alpha B-crystallin negatively regulates apoptosis during myogenic differentiation by inhibiting caspase-3 activation. (2/314)

Myoblasts respond to growth factor deprivation either by differentiating into multinucleated myotubes or by undergoing apoptosis; hence, the acquisition of apoptosis resistance by myogenic precursors is essential for their development. Here we demonstrate that the expression of the small heat shock protein alpha B-crystallin is selectively induced in C2C12 myoblasts that are resistant to differentiation-induced apoptosis, and we show that this induction occurs at an early stage in their differentiation in vitro. In contrast, the expression of several known anti-apoptotic proteins (FLIP, XIAP, Bcl-x(L)) was not altered during myogenesis. We also demonstrate that ectopic expression of alpha B-crystallin, but not the closely related small heat shock protein Hsp27, renders C2C12 myoblasts resistant to differentiation-induced apoptosis. Furthermore, we show that the myopathy-causing R120G alpha B-crystallin mutant is partly impaired in its cytoprotective function, whereas a pseudophosphorylation alpha B-crystallin mutant that mimics stress-induced phosphorylation is completely devoid of anti-apoptotic activity. Finally, we demonstrate that alpha B-crystallin negatively regulates apoptosis during myogenesis by inhibiting the proteolytic activation of caspase-3, whereas the R120G and pseudophosphorylation mutants are defective in this function. Taken together, our findings indicate that alpha B-crystallin is a novel negative regulator of myogenic apoptosis that directly links the differentiation program to apoptosis resistance.  (+info)

Orientation-dependent influence of an intergenic enhancer on the promoter activity of the divergently transcribed mouse Shsp/alpha B-crystallin and Mkbp/HspB2 genes. (3/314)

The mouse Shsp/alphaB-crystallin and Mkbp/HspB2 genes are closely linked and divergently transcribed. In this study, we have analyzed the contribution of the intergenic enhancer to Shsp/alphaB-crystallin and Mkbp/HspB2 promoter activity using dual-reporter vectors in transient transfection and transgenic mouse experiments. Deletion of the enhancer reduced Shsp/alphaB-crystallin promoter activity by 30- and 93-fold and Mkbp/HspB2 promoter activity by 6- and 10-fold in transiently transfected mouse lens alpha-TN4 and myoblast C2C12 cells, respectively. Surprisingly, inversion of the enhancer reduced Shsp/alphaB-crystallin promoter activity by 17-fold, but did not affect Mkbp/HspB2 promoter activity in the transfected cells. In contrast, enhancer activity was orientation-independent in combination with a heterologous promoter in transfected cells. Transgenic mouse experiments established the orientation dependence and Shsp/alphaB-crystallin promoter preference of the intergenic enhancer in its native context. The orientation dependence and preferential effect of the Shsp/alphaB-crystallin enhancer on the Shsp/alphaB-crystallin promoter provide an example of adaptive changes in gene regulation accompanying the functional diversification of duplicated genes during evolution.  (+info)

Retinal pigment epithelium is protected against apoptosis by alphaB-crystallin. (4/314)

PURPOSE: The degeneration of retinal pigment epithelial (RPE) cells is considered to be a crucial event in the pathophysiology of age-related macular degeneration (AMD). Cumulative oxidative damage has been implicated in the development of the changes seen in AMD. The present study was undertaken to evaluate the expression of the small heat shock protein alphaB-crystallin in the RPE in response to oxidative stress and to explore whether alphaB-crystallin expression confers an antiapoptotic cytoprotective effect on RPE cells. METHODS: Native human RPE cells from the macula and retinal periphery were analyzed by RT-PCR and Western blot analysis for expression of alphaB-crystallin. Monolayer cultures of human RPE cells were stressed by heat shock (42 degrees C for 20 minutes) or oxidant-mediated injury (50-300 micro M H(2)O(2) for 1 hour). Induction of alphaB-crystallin and the corresponding mRNA was assessed by Western and Northern blot analyses. To study the cytoprotective effect of alphaB-crystallin, human RPE cells were transfected with either a neomycin-selectable expression vector containing alphaB-crystallin cDNA or a control vector without alphaB-crystallin cDNA. Caspase-3 activity was determined by observing the cleavage of a colorimetric peptide substrate. Cell viability was quantified by combined propidium iodide and Hoechst 33342 staining. RESULTS: alphaB-crystallin is constitutively expressed in RPE under in vivo and in vitro conditions. Western blot analysis of freshly isolated RPE showed greater baseline expression levels in RPE derived from the macular area than in that from the more peripheral regions. Heat shock treatment and oxidative stress caused a significant increase in alphaB-crystallin mRNA and protein. Oxidant-mediated injury in RPE cells with baseline expression levels of alphaB-crystallin resulted in apoptotic cell death, as measured by caspase-3 activity, whereas RPE cells that had been stably transfected with alphaB-crystallin were more resistant to H(2)O(2)-induced cellular injury. CONCLUSIONS: alphaB-crystallin may function as a stress-inducible antiapoptotic protein in human RPE and is inducible by oxidative stress, a condition implicated in the pathogenesis of AMD. Overexpression of alphaB-crystallin may be an important mechanism for the RPE to prevent apoptotic cell death in response to cellular stress.  (+info)

The small heat-shock protein alpha B-crystallin promotes FBX4-dependent ubiquitination. (5/314)

AlphaB-crystallin is a small heat-shock protein in which three serine residues (positions 19, 45, and 59) can be phosphorylated under various conditions. We describe here the interaction of alphaB-crystallin with FBX4, an F-box-containing protein that is a component of the ubiquitin-protein isopeptide ligase SCF (SKP1/CUL1/F-box). The interaction with FBX4 was enhanced by mimicking phosphorylation of alphaB-crystallin at both Ser-19 and Ser-45 (S19D/S45D), but not at other combinations. Ser-19 and Ser-45 are preferentially phosphorylated during the mitotic phase of the cell cycle. Also alphaB-crystallin R120G, a mutant found to co-segregate with a desmin-related myopathy, displayed increased interaction with FBX4. Both alphaB-crystallin S19D/S45D and R120G specifically translocated FBX4 to the detergent-insoluble fraction and stimulated the ubiquitination of one or a few yet unknown proteins. These findings implicate the involvement of alphaB-crystallin in the ubiquitin/proteasome pathway in a phosphorylation- and cell cycle-dependent manner and may provide new insights into the alphaB-crystallin-induced aggregation in desmin-related myopathy.  (+info)

Mechanism of chaperone function in small heat-shock proteins. Phosphorylation-induced activation of two-mode binding in alphaB-crystallin. (6/314)

The consequences of alphaB-crystallin phosphorylation on its chaperone activity were investigated using a detailed analysis of the recognition and binding of destabilized T4 lysozyme (T4L) mutants by alphaB-crystallin phosphorylation mimics containing combinations of serine to aspartate substitutions. The T4L site-directed mutants were selected to constitute an energetic ladder of progressively destabilized proteins having similar structures in the folded state. alphaB-crystallin and its variants differentially recognize the T4L mutants, binding the more destabilized ones to a larger extent. Furthermore, the aspartate substitutions result in an increase in the extent of binding to the same T4L mutant and in the appearance of biphasic binding isotherms. The latter indicates the presence of two modes of binding characterized by different affinities and different numbers of binding sites. The transition to two-mode binding can also be induced by temperature or pH activation of the second mode. The similarity between the phosphorylation, pH, and temperature effects suggests a common structural origin. The location of the phosphorylation sites in the N-terminal domain and the hypothesized burial of this domain in the core of the oligomeric structure are consistent with a critical role for the destabilization of the quaternary structure in the process of recognition and binding by small heat-shock proteins.  (+info)

Morphological characterization of the Alpha A- and Alpha B-crystallin double knockout mouse lens. (7/314)

BACKGROUND: One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alpha A and alpha B are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alpha A- and alpha B-crystallin genes (alpha A/BKO). METHODS: Lenses from 129SvEvTac mice and alpha A/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. RESULTS: Equatorial and axial (sagittal) dimensions of lenses for alpha A/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alpha A/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alpha A/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alpha A/BKO mice as compared to wild mice. CONCLUSION: These results indicated that both alpha A- and alpha B-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.  (+info)

Mimicking phosphorylation of alphaB-crystallin on serine-59 is necessary and sufficient to provide maximal protection of cardiac myocytes from apoptosis. (8/314)

AlphaB-crystallin (alphaBC), a small heat shock protein expressed in high levels in the heart, is phosphorylated on Ser-19, 45, and 59 after stress. However, it is not known whether alphaBC phosphorylation directly affects cell survival. In the present study, constructs were prepared that encode forms of alphaBC harboring Ser to Ala (blocks phosphorylation) or Ser to Glu (mimics phosphorylation) mutations at positions 19, 45, and 59. The effects of each form on apoptosis of cultured cardiac myocytes after hyperosmotic or hypoxic stress were assessed. Compared with controls, cells that expressed alphaBC with Ser to Ala substitutions at all three positions, alphaBC(AAA), exhibited more stress-induced apoptosis. Cells expressing either alphaBC(AAE) or (EEE) exhibited 3-fold less apoptosis than cells expressing alphaBC(AAA), indicating that phosphorylation of Ser-59 confers protection. alphaBC is known to bind to procaspase-3 and to decrease caspase-3 activation. Compared with cells expressing alphaBC(AAA), the activation of caspase-3 was decreased by 3-fold in cells expressing alphaBC(AAE). These results demonstrate that mimicking the phosphorylation of alphaBC on Ser-59 is necessary and sufficient to confer caspase-3 inhibition and protection of cardiac myocytes against hyperosmotic or hypoxic stress. These findings provide direct evidence that alphaBC(S59P) contributes to the cardioprotection observed after physiologically relevant stresses, such as transient hypoxia. Identifying the targets of alphaBC(S59P) will reveal important details about the mechanism underlying the cytoprotective effects of this small heat shock protein.  (+info)

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