αB‐crystallin exists as an independent protein in the heart and in the lens

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The molecular cascade of stress response in higher eukaryotes commences in the cytoplasm with the trimerization of the heat shock factor 1 (HSF1), followed by its transport to the nucleus, where it binds to the heat shock element leading to the activation of transcription from the down-stream gene(s). This well-established paradigm has been mostly studied in cultured cells. The developmental and tissue-specific control of the heat shock transcription factors (HSFs) and their interactions with heat shock promoters remain unexplored. We report here that in the rat lens, among the three mammalian HSFs, expression of HSF1 and HSF2 is largely fetal, whereas the expression of HSF4 is predominantly postnatal. Similar pattern of expression of HSF1 and HSF4 is seen in fetal and adult human lenses. This stagespecific inverse relationship between the expression of HSF1/2 and HSF4 suggests tissue-specific management of stress depending on the presence or absence of specific HSF(s). In addition to real-time PCR and immunoblotting, gel mobility shift assays, coupled with specific antibodies and HSE probes, derived from three different heat shock promoters, establish that there is no HSF1 or HSF2 binding activity in the postnatal lens nuclear extracts. Using this unique, developmentally modulated in vivo system, we demonstrate 1) specific patterns of HSF4 binding to heat shock elements derived from ␣B-crystallin, Hsp70, and Hsp82 promoters and 2) that it is HSF4 and not HSF1 or HSF2 that interacts with the canonical heat shock element of the ␣B-crystallin gene.Induced transcription from heat shock promoters is mediated by the activation of transacting HSFs 1 (1, 2). There are four known HSFs (HSF1, HSF2, HSF3, and HSF4). HSF3 is an avian HSF (3, 4). Although yeast and Drosophila melanogaster have a single gene that encodes an HSF, higher eukaryotes, animals, and plants have multiple genes that code for HSFs (4 -6). HSF1 and HSF2 transcription factors have almost identical gene structures (4). The heat shock response starts with the cytoplasmic HSF and its trimerization and transport to the nucleus, where it binds to the heat shock element (HSE) in the heat shock promoter, activating transcription of the down stream heat shock gene(s) (1, 4). Both HSF1 and HSF2 contain three hydrophobic repeats, HR-A, -B, and -C. HR-A and -B are involved in trimerization upon reception of the stress signal. HR-C, located at the carboxyl terminus, has been suggested to inhibit trimerization in the uninduced state. HSF4, on the other hand, does not contain the HR-C domain; it therefore exists as a trimeric unit and binds to the DNA constitutively (for review, see Ref. 4).HSF1 is considered to be the universal HSF and mediates expression of heat shock genes upon reception of a stress signal such as high temperature, whereas HSF2 is associated with developmental control. Although it has not been experimentally established, the assumption in this generalization is that all tissues and cells contain HSF1 as a pre-existing HSF in the cytoplasm to enab...

Section: Methodsmentioning

confidence: 99%

Developmentally Dictated Expression of Heat Shock Factors: Exclusive Expression of HSF4 in the Postnatal Lens and Its Specific Interaction with αB-crystallin Heat Shock Promoter

Somasundaram¹,

Bhat

2004

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“…The results provide a base line for further studies of modification-related changes in protein interaction and conformation. In addition, ␣A-and ␣B-crystallins were recently detected in other tissues, such as heart, kidney, spleen, and retina (12)(13)(14)(15). These findings raise question about the role or function of ␣A-and ␣B-crystallins in these tissues.…”

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confidence: 99%

Conformational and Functional Differences between Recombinant Human Lens αA- and αB-Crystallin

Sun

Das

Liang

1997

158

136

Human and other mammalian lens proteins are composed of three major crystallins: ␣-, ␤-, and ␥-crystallin. ␣-Crystallin plays a prominent role in the supramolecular assembly required to maintain lens transparency. With age, the crystallins, especially ␣-crystallin, undergo posttranslational modifications that may disrupt the supramolecular assembly, and the lens becomes susceptible to other stresses resulting in cataract formation. Because these modifications occur even at a relatively young age, it is difficult to obtain pure, unmodified crystallins for in vitro experiments. ␣-Crystallin is composed of two subunits, ␣A and ␣B. Before the application of recombinant DNA technology, these two ␣-crystallin subunits were separated from calf lens in the denatured state and reconstituted by the removal of the denaturant, but they were not refolded properly. In the present studies, we applied the recombinant DNA technology to prepare native, unmodified ␣A-and ␣B-crystallins for conformational and functional studies. The expressed proteins from Escherichia coli are in the native state and can be studied directly. First, ␣A and ␣B cDNAs were isolated from a human lens epithelial cell cDNA library. The cDNAs were cloned into a pAED4 expression vector and then expressed in E. coli strain BL21(DE3). Pure recombinant ␣A-and ␣B-crystallins were obtained after purification by gel filtration and DEAE liquid chromatography. They were subjected to conformational studies involving various spectroscopic measurements and an assessment of chaperone-like activity. ␣A-and ␣B-crystallins have not only different secondary structure, but also tertiary structure. 1-Anilino-8-naphthalene sulfonate fluorescence indicates that ␣B-crystallin is more hydrophobic than ␣A-crystallin. The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for ␣B-than for ␣A-crystallin. The resulting data provide a base line for further studies of human lens ␣-crystallin.␣-Crystallin is the major lens structural protein responsible for the supramolecular assembly necessary to maintain lens transparency. It is isolated as a large water-soluble aggregate with a mass of 800 kDa and is composed of two homologous subunits, ␣A and ␣B, each with a molecular mass of 20 kDa. With aging and cataract formation, ␣-crystallins gradually become a high molecular weight aggregate and eventually an insoluble protein (1). This is manifested by the observation that the major crystallin component in the insoluble protein fraction of aged and cataractous lenses is ␣-crystallin (2). The aggregation and insolubilization of ␣-crystallin are believed to arise from posttranslational modifications, which accumulate significantly because of the low turnover rate of lens crystallins. Two-dimensional gel electrophoresis indicated that human lens crystallins were modified extensively even at a relatively young age (3). The significance of the modification is that crystallins become partially unfolded, and this partially unfolding exposes ...

“…It is a M r ϳ0.6 -1.0 ϫ 10 6 complex composed of two structurally related subunits, designated ␣A and ␣B, which are encoded by genes localized to chromosomes 21 and 11, respectively (4,5). While expression of the ␣A gene appears to be preferentially but not exclusively restricted to lens cells (6), the ␣B gene product has been described in a broad number of tissues and organs including the brain, skeletal muscle, heart, and kidney (7,8). Elevated expression of the ␣B gene has been correlated with several neurological diseases (9).…”

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confidence: 99%

Cloning, Expression, and Chaperone-like Activity of Human αA-Crystallin

Andley

Mathur

Griest

et al. 1996

152

136

One of the major protein components of the ocular lens, ␣-crystallin, is composed of ␣A and ␣B chain subunits that have structural homology to the family of mammalian small heat shock proteins. Like other small heat shock proteins, ␣-crystallin subunits associate to form large oligomeric aggregates that express chaperone-like activity, as defined by the ability to suppress nonspecific aggregation of proteins destabilized by treatment with a variety of denaturants including heat, UV irradiation, and chemical modification. It has been proposed that age-related loss of sequences at the C terminus of the ␣A chain subunit may be a factor in the pathogenesis of cataract due to diminished capacity of the truncated crystallin to protect against nonspecific aggregation of lens proteins. To evaluate the functional consequences of ␣-crystallin modification, two mutant forms of ␣A subunits were prepared by site-directed mutagenesis. Like wild type (WT), aggregates of ϳ540 kDa were formed from a tryptophan-free ␣A mutant (W9F). When added in stoichiometric amounts, both WT and W9F subunits completely suppressed the heat-induced aggregation of aldose reductase. In contrast, subunits encoded by a truncation mutant in which the Cterminal 17 residues were deleted (R157STOP), despite having spectroscopic properties similar to WT, formed much larger aggregates with a marked reduction in chaperone-like activity. Similar results were observed when the chaperone-like activity was assessed through inhibition of ␥-crystallin aggregation induced by singlet oxygen. These results demonstrate that the structurally conservative substitution of Phe for Trp-9 has a negligible effect on the functional interaction of ␣A subunits, and that deletion of C-terminal sequences from the ␣A subunit results in substantial loss of chaperone-like activity, despite overall preservation of secondary structure.The major components of the mammalian lens fiber cells are the ␣-, ␤-, and ␥-crystallins, which constitute an estimated 35% wet weight of the lens. The crystallins contribute to the transparency and refractive power of the lens by short range interactions among themselves and cytoskeletal elements in a highly concentrated matrix (1-3). ␣-Crystallin is one of the most abundant of the crystallins in mature lens fiber cells. It is a M r ϳ0.6 -1.0 ϫ 10 6 complex composed of two structurally related subunits, designated ␣A and ␣B, which are encoded by genes localized to chromosomes 21 and 11, respectively (4, 5).