CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation.

Proper folding of proteins (either newly synthesized or damaged in response to a stressful event) occurs in a highly regulated fashion. Cytosolic chaperones such as Hsc/Hsp70 are assisted by cofactors that modulate the folding machinery in a positive or negative manner. CHIP (carboxyl terminus of Hsc70-interacting protein) is such a cofactor that interacts with Hsc70 and, in general, attenuates its most well characterized functions. In addition, CHIP accelerates ubiquitin-dependent degradation of chaperone substrates. Using an in vitro ubiquitylation assay with recombinant proteins, we demonstrate that CHIP possesses intrinsic E3 ubiquitin ligase activity and promotes ubiquitylation. This activity is dependent on the carboxyl-terminal U-box. CHIP interacts functionally and physically with the stress-responsive ubiquitin-conjugating enzyme family UBCH5. Surprisingly, a major target of the ubiquitin ligase activity of CHIP is Hsc70 itself. CHIP ubiquitylates Hsc70, primarily with short, noncanonical multiubiquitin chains but has no appreciable effect on steady-state levels or half-life of this protein. This effect may have heretofore unanticipated consequences with regard to the chaperoning activities of Hsc70 or its ability to deliver substrates to the proteasome. These studies demonstrate that CHIP is a bona fide ubiquitin ligase and indicate that U-box-containing proteins may comprise a new family of E3s.

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins.

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.

Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells.

The mechanisms by which reactive species (RS) participate in the development of atherosclerosis remain incompletely understood. The present study was designed to test the hypothesis that RS produced in the vascular environment cause mitochondrial damage and dysfunction in vitro and, thus, may contribute to the initiating events of atherogenesis. DNA damage was assessed in vascular cells exposed to superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite. In both vascular endothelial and smooth muscle cells, the mitochondrial DNA (mtDNA) was preferentially damaged relative to the transcriptionally inactive nuclear beta-globin gene. Similarly, a dose-dependent decrease in mtDNA-encoded mRNA transcripts was associated with RS treatment. Mitochondrial protein synthesis was also inhibited in a dose-dependent manner by ONOO(-), resulting in decreased cellular ATP levels and mitochondrial redox function. Overall, endothelial cells were more sensitive to RS-mediated damage than were smooth muscle cells. Together, these data link RS-mediated mtDNA damage, altered gene expression, and mitochondrial dysfunction in cell culture and reveal how RS may mediate vascular cell dysfunction in the setting of atherogenesis.

Flavopiridol inhibits smooth muscle cell proliferation in vitro and neointimal formation In vivo after carotid injury in the rat.

BACKGROUND: Smooth muscle cell (SMC) proliferation is a critical component of neointimal formation in many models of vascular injury and in human lesions as well. Cell-cycle inhibition by gene transfer techniques can block SMC proliferation and lesion formation in animal models, although these methods are not yet applicable to the treatment of human disease. Flavopiridol is a recently identified, potent, orally available cyclin-dependent kinase inhibitor. METHODS AND RESULTS: Using human aortic SMCs, we found that flavopiridol in concentrations as low as 75 nmol/L resulted in nearly complete inhibition of basic fibroblast growth factor-induced and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone H1 phosphorylation, but had no effect on mitogen-activated protein kinase activation. Induction of the cell cycle-related proteins cyclin D1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cellular viability. To test whether flavopiridol had a similar activity in vivo when administered orally, we examined neointimal formation in rat carotid arteries after balloon injury. Flavopiridol 5 mg/kg reduced neointimal area by 35% and 39% at 7 and 14 days, respectively, after injury. CONCLUSIONS: Flavopiridol inhibits SMC growth in vitro and in vivo. Its oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions.

Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo.

Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O-2 and H2O2 generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47(phox) and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47(phox) and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47(phox) might be similarly regulated in vivo in a rat aorta balloon injury model and found that p47(phox) protein was increased after injury. Immunocytochemistry localized expression of p47(phox) to the neointima and media of injured arteries. Our data demonstrate that generation of O-2 and H2O2 is required for thrombin-mediated mitogenesis in VSMC and that p47(phox) is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.

Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.

Changes in the localization of catalase during differentiation of neutrophilic granulocytes.

The nature of the compartmentalization of catalase in human myeloid cells is an unresolved issue. Using a rabbit polyclonal antibody specific for catalase, indirect immunocytofluorescence of immature leukemic promyelocytes (HL-60 cells) showed a pattern of small, sharp, punctate staining in the cytoplasm of all cells, while mature neutrophils showed a larger diffuse, flocculent pattern of cytoplasmic staining. Differential centrifugation of nitrogen cavitates of HL-60 cells indicated that the putative catalase-containing compartment was relatively fragile compared with the compartment(s) that contained myeloperoxidase (MPO), beta-hexosaminidase, beta-glucuronidase, and lysosomal alpha-mannosidase activities. Parallel studies using dimethylsulfoxide (DMSO)-induced HL-60 cells and mature neutrophils showed that, in the course of differentiation, there was an apparent shift in the localization of catalase from the granule fraction to the cytosolic fraction. Percoll-sucrose density gradient centrifugation of HL-60 cell cavitates showed a catalase-containing compartment with a mean peak density (1.05 g/mL) significantly lower than that of the major myeloperoxidase-containing compartment (1.08 g/mL); in mature neutrophils, catalase activity comigrated with lactate dehydrogenase (LDH) activity. Catalase in isolated fractions was protected from proteolysis in the absence, but not in the presence, of 0.1% Triton X-100. Digitonin titration experiments confirmed the compartmentalized nature of catalase in immature HL-60 cells and were consistent with a cytosolic localization in mature neutrophils. Ultrastructural localization of catalase by Protein A-gold immunocytochemistry demonstrated four to six catalase-containing compartments in all HL-60 cell profiles. In mature neutrophils, catalase was localized primarily in the cytoplasmic matrix, although in fewer than 2% of the cell profiles, one to two catalase-containing compartments were observed. The changes in catalase localization that occur during myeloid differentiation appear to be similar to the changes that occur during erythroid and megakaryocytic differentiation, and may have potential clinical significance in the classification of acute leukemia and in the development of drug resistance.