Ubiquitination of protein kinase C-alpha and degradation by the proteasome

Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.     

Constitutive expression of heat shock protein 90 (HSP90) in neurons of the rat brain

Immunoblot analysis, immunocytochemistry and immuno-electron microscopy were employed to study the expression of HSP90 protein in the adult rat brain, using a specific polyclonal antiserum. Immunoblot analysis demonstrated equal levels of HSP90 in microdissected extracts from hippocampus, cortex, striatum and cerebellum. Immunocytochemistry and immuno-electron microscopy provided evidence that HSP90 is markedly expressed throughout all neuronal subpopulations of the CNS but not in non-neuronal cells except ependyma and choroid plexus. At the ultrastructural level, HSP90 immunoreactivity was predominantly found in perikarya but to a lesser extent also in dendrites and nuclei. The constitutive expression of HSP90 in widespread neuronal cell populations suggests a functional role in the physiological molecular program of CNS neurons.     

The degradation of apolipoprotein B100 is mediated by the ubiquitin-proteasome pathway and involves heat shock protein 70

Apolipoprotein B (apoB) is the major protein component of atherogenic lipoproteins of hepatic origin. In HepG2 cells, the standard cell culture model of human hepatic lipoprotein metabolism, there is a limited availability of core lipids in the endoplasmic reticulum for association with nascent apoB. Under these conditions, apoB is partially translocated, interacts with cytosolic Hsp70, and undergoes rapid degradation. We show that increasing the expression of Hsp70 in HepG2 cells promotes apoB degradation. In addition, apoB is polyubiquitinated and its degradation both normally and after Hsp70 induction is blocked by inhibitors of the proteasome. The apoB that accumulates after proteasome inhibition is endoplasmic reticulum-associated and can be assembled into lipoproteins and secreted if new lipid synthesis is stimulated. Thus, apoB is the first example of a wild-type mammalian protein whose secretion is regulated by degradation in the cytosol via the ubiquitin-proteasome pathway. Furthermore, targeting of this secretory protein to the proteasome is regulated by the molecular chaperone Hsp70 and the availability of apoB's lipid-ligands.     

Involvement of valosin-containing protein, an ATPase Co-purified with IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IkappaBalpha

The inactivation of the prototype NF-kappaB inhibitor, IkappaBalpha, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IkappaBalpha immune complexes. The ubiquitinated IkappaBalpha conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-kappaB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IkappaBalpha complexes sediment in the 19 S fractions, while the unmodified IkappaBalpha sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IkappaBalpha are critical for VCP binding, which in turn is necessary but not sufficient for IkappaBalpha degradation; while the N-terminal domain of IkappaBalpha is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between IkappaBalpha and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IkappaBalpha.     

Intestinal expression of human heat shock protein 90 in patients with Crohn's disease and ulcerative colitis

Heat shock proteins are induced by several stress factors and are potential antigens in autoimmune disorders. Expression of heat shock protein 90 (HSP 90) was investigated in patients with inflammatory bowel disease and normal controls. We combined western blot analysis with laser densitometry for quantitation. Localization of HSP 90 was investigated by immunohistochemistry. Western blots showed a significant mucosal expression of HSP 90, which was comparable in patients and controls. There was also no difference between normal and inflamed mucosa in inflammatory bowel disease. In immunohistochemical staining studies, HSP 90 was detected in epithelial cells, mononuclear cells, giant cells, nerve cells, and endothelial cells of small vessels. There was no difference in the intensity of staining or localization in patients with inflammatory bowel disease compared to controls. These findings render a potential protective or immunogenic function of HSP 90 in inflammatory bowel disease unlikely.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.     

Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells

The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells, In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization.     

Detection of autoantibodies to the 90 kDa heat shock protein in systemic lupus erythematosus and other autoimmune diseases

Expression of the highly conserved 90 kDa heat shock protein (Hsp90) is elevated in the peripheral blood mononuclear cells of approximately 25% of patients with SLE. Conflicting data have been published about the frequency of antibodies to Hsp90 with the previous methodology using a complex Western blot system. We now describe an ELISA to measure autoantibodies to Hsp90 and Hsp70 in SLE patients, healthy controls and patients with a variety of autoimmune rheumatic diseases. IgG and IgM antibodies were elevated in 26 and 35% of SLE patients, respectively. These results show autoantibodies to Hsp90 (but not Hsp70) are elevated in a significant proportion of patients with SLE (P < 0.025) compared to healthy controls; and that those with raised antibody levels were more likely to have renal disease and a low C3 level (P < 0.02).     

Effect of stages of lactation on the concentration of a 90-kilodalton heat shock protein in bovine mammary tissue

From the normal mammary tissue of a Holstein cow in late lactation, a heat shock protein (90 kDa) was purified by ammonium sulfate fractionation and five-step column chromatography. From 70 g of tissue, 9.5 mg of this heat shock protein were obtained; samples had 98% purity and 19% recovery. The molecular mass of the 90-kDa heat shock protein was estimated to be 86 kDa by SDS-PAGE. Analysis of the amino-terminal amino acid sequence suggested that the protein had been purified as a mixture of two isoforms. The contents of the heat shock protein in cytoplasmic fractions of mammary tissues from Holstein heifers and cows at lactation and involution were measured by quantitative immunoblot analysis using rabbit antiserum raised against the purified heat shock proteins. The contents of the heat shock protein were higher in tissues from lactating cows than in those from heifers and involuting cows. The elevated concentrations of cytoplasmic 90-kDa heat shock protein in lactating tissue suggested that this protein is involved in mammary differentiation and lactation.     

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated. 2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii. 3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h. 4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.     

Autoimmune diabetes induced by the beta-cell toxin STZ. Immunity to the 60-kDa heat shock protein and to insulin

Administered at a suitably low dose, the toxin streptozotocin (STZ) can trigger an autoimmune process leading to destruction of the beta-cells of the pancreatic islets. In this study, we examined specific immunological reactions in mice before and during the development of STZ-induced autoimmune diabetes. We now report that the development of spontaneous autoantibodies to insulin can serve as a marker of susceptibility to a low dose of STZ. Susceptible male mice of the C57BL/KsJ strain manifested such anti-insulin antibodies, and resistant female mice did not. Administration of a low dose of STZ (five daily doses each of 30 mg/kg) induced transient hyperglycemia approximately 20-30 days later, which temporarily remitted but was followed by intractable diabetes approximately 2.5 months later. The diabetogenic process triggered by the low dose of STZ was associated with an increase in the level of anti-insulin antibodies bearing the Dana and Micha (DM) idiotype, later followed by the appearance of anti-idiotypic antibodies that peaked before the onset of diabetes. Antibodies and T-cells reactive to hsp60 (heat shock protein) were triggered by the low-dose STZ administration and persisted throughout the period that preceded clinical diabetes. T-cells reactive to the p277 peptide of hsp60 were also observed. Finally, active immunization to hsp60 caused transient hyperglycemia by itself and also aggravated the hyperglycemia induced by low-dose STZ. Thus, autoantibodies to insulin can indicate susceptibility to a toxic trigger of diabetes, and a low dose of a toxin can activate the insulin and hsp60 autoimmunity that has been detected previously in the spontaneous autoimmune diabetes of NOD strain mice.     

A small influence of HSP90 levels on the trehalose and heat shock element inductions of the yeast heat shock response

The T cell receptor (TCR) V beta repertoire in peripheral blood lymphocytes (PBL) of a large number of healthy individuals was analysed by quantifying V beta-specific mRNA using the method of anchored multiprimer DNA amplification and a reverse dot blot assay. Among 16 V beta gene families examined, particular V beta genes were noted to be unequally expressed in the PBL of 70 healthy donors. The frequently used genes belong to the V beta 4, 5, 6, 8 and 13 (12) families, while V beta 1, 9 and 15 were the least frequently used gene families. This bias in gene usage was observed in all individuals. Marked deviation from the mean percentage usage was noted for some V beta genes in individuals when their PBL were examined serially, but the common pattern of biased usage was not grossly distorted. When the TCR repertoire of different ethnic groups was examined, a lower mean frequency of V beta 3.2 was seen in the repertoire of 19 Caucasians compared with 25 age-matched Samoans (P < 0.003). Conversely, the expression of V beta 5.1 and V beta 5.3 was higher in Caucasians than in 51 age-matched Polynesians (Maoris and Samoans, P < 0.003). Considering the 20% co-efficient of variation in the estimate of V beta gene usage, our data from 70 unrelated individuals suggest that in PBL, individual variations in the TCR repertoire were superimposed upon a common biased usage of V beta genes in the general population.     

Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery

Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90- to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions.     

Expression of heat shock protein 27 in chromomycosis

We report on a 58-year-old woman with long-lasting (36 years) chromomycosis on the foot and secondary self-inoculation from foot to hand 4 years ago. Mycological classification was performed after culture on Sabouraud glucose agar. We used haematoxylin and eosin and Giemsa staining and an antibody to heat shock protein (HSP) 27 (Stress Gen, Clone G3.1) on paraffin-embedded and cryostat specimens of chromomycosis. The mycological culture revealed the fungus Fonsecaea pedosoi. Histopathology revealed dermal fibrosis with persistent fungi (Medlar bodies), numerous mast cells and pseudoepitheliomatous hyperplasia. Immunohistochemically, HSP 27 was positively identified in F. pedrosoi. Moreover, in differentiating keratinocytes in the pseudoepitheliomatous lesions of chromomycosis, HSP 27 was increasingly expressed from basal layers to stratum spinosum in the epidermis but not in keratinocytes directly bordering Medlar bodies. In chromomycosis, HSP 27 is expressed, in accordance with its role as a marker of differentiation and proliferation, in keratinocytes and also in F. pedrosoi. It remains unknown if these results might explain the therapeutic efficacy of hyperthermic treatment.     

Cytosolic degradation of T-cell receptor alpha chains by the proteasome

The T-cell antigen receptor (TCR) is an hetero-oligomeric membrane complex composed of at least seven transmembrane polypeptide chains that has served as a model for the assembly and degradation of integral membrane proteins in the endoplasmic reticulum (ER). Unassembled TCRalpha chains fail to mature to the Golgi apparatus and are rapidly degraded by a non-lysosomal "ER degradation" pathway that has been proposed to be autonomous to the ER. In these studies we show that the degradation of core-glycosylated TCRalpha is blocked by N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and lactacystin, implicating the proteasome in ER degradation. Either acute or chronic treatment of TCRalpha-transfected cells with proteasome inhibitors cause the core-glycosylated TCRalpha chains to progressively shift to an approximately 28-kDa form that lacks N-linked oligosaccharides and the N-terminal signal peptide. The susceptibility of this 28-kDa species to extravesicular protease indicates that it is not protected by the ER membrane and, hence, cytoplasmic. These data suggest a model in which TCRalpha chains that are translocated across the membrane, core-glycosylated, but fail to assemble are dislocated back to the cytoplasm for degradation by cytoplasmic proteasomes. Our data also suggest that covalent modification of TCRalpha with ubiquitin is not required for its degradation.