No abstract

␣B-crystallin (␣B) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of ␣B has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that ␣B is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of ␣B in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize ␣B in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100 -200 nm, were characterized by the presence of flotillin-1, ␣-enolase, and Hsp70, the same proteins that associate with detergentresistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of ␣B is predominantly apical. Using OptiPrep gradients we demonstrate that ␣B resides in the DRM fraction. The secretion of ␣B is inhibited by the cholesterol-depleting drug, methyl ␤-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.The small heat shock protein, ␣B-crystallin (␣B) 2 is a developmentally regulated gene product whose association with multiple pathologies of varied antecedents such as neurodegeneration, oncogenesis, and cataracts suggests a vital function for this protein (1-3). Elevated levels of ␣B have been reported in Alexander, Alzheimer, and Parkinson diseases. It is expressed in astrocytes (4) and has been implicated in peripheral nerve myelination (5). It is known to be one of the main antigens involved in multiple sclerosis (6). Its expression in a subset of basal-like breast carcinomas has led to its characterization as a novel oncoprotein (7). It is also a potential tissue biomarker for renal cell carcinoma (8). Interestingly, ␣B has also been shown to activate T cells (9) and inhibit platelet aggregation (10).In the eye, apart from its predominant presence in the ocular lens, ␣B was initially reported in primary cultures of human retinal pigment epithelium (RPE) (11) and has since been shown to be expressed in the retina (1) and during early development of the rat eye in the embryonic RPE (12). It is highly expressed in rod outer segments as well as in the rat RPE, following intense light exposures that lead to photoreceptor cell degeneration (13). In age-related macular degeneration, high concentrations of ␣B transcripts are found in microdissected retinal tissue juxtaposed with subretinal lipoprotein deposits, known as ...

alphaA-Crystallin is highly upregulated in the retina during early EAU. This upregulation is localized primarily in the photoreceptor inner segments, the site of mitochondrial oxidative stress. Further, in early EAU, the photoreceptors preferentially use alphaA-crystallin to suppress mitochondrial oxidative stress-mediated apoptosis.

Far from being a physical entity, assembled of inanimate structural proteins, the ocular lens epitomizes the biological ingenuity that sustains an essential and near-perfect physical system of immaculate optics. Crystallins (a, 13, and y) provide transparency by dint of their high concentration, but it is debatable whether proteins that provide transparency are any different, biologically or structurally, from those that are present in non-transparent structures or tissues. It is becoming increasingly clear that crystallins may have a plethora of metabolic and regulatory functions, both within the lens as well as outside of it. a-Crystallins are members of a small heat shock family of proteins and l3/y-crystallins belong to the family of epidermis-specific differentiation proteins. Crystallin gene expression has been studied from the perspective of the lens specificity of their promoters. Mutations in a-, 13-, and ycrystallins are linked with the phenotype of the loss of transparency. Understanding catalytiC, non-structural properties of crystallins may be critical for understanding the malfunction in molecular cascades that lead to cataractogenesis and its eventual therapeutic amelioration.Crystallins, genes and cataract

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...