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.

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 ubiquitinconjugating 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.

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2-and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IB␣, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-B activation, SsnB effectively blunted the NF-B activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases. Toll-like receptors (TLRs)2 are key components of innate immunity (1) expressed by macrophages, dendritic cells, and many other cell types (2, 3). TLRs serve as the first line of defense against invading pathogens such as bacteria and viruses. Currently more than a dozen TLRs have been identified, with the first nine being well characterized. Some TLRs, including TLR1, -2, -4, -5, and -6, are mainly located on the plasma membrane and recognize bacterial, fungal, and protozoan pathogens, whereas others, including TLR3, -7, -8, and -9, are mainly located on endosomal/lysosomal membranes where they bind viral RNAs or DNAs (4 -6). All TLRs use leucine-rich repeats to sense the ligands and the Toll/IL-1 receptor homologue (TIR) domain to trigger downstream signaling by binding to adaptor proteins MyD88 (7-9), TIRAP/Mal (10, 11), or TRIF (12, 13). The signaling initiated by TLRs is a double-edged sword. On the one hand, it may lead to confining or eliminating the invading organisms (14, 15); on the other hand, a prolonged and exaggerated response can cause tissue and organ damage (16,17). Moreover, TLR signaling triggered by exogenous or endogenous ligands contributes to the pathogenesis of many chronic inflammatory diseases (18). For example, TLR2 and TLR4 are involved in atherosclerosis (19,20), autoimmune colitis (21), systemic lupus erythematosus (22, 23), diabetes (24, 25), and Alzheimer disease (26,27). Thus, blockade of excessive TLR signaling is a therapeutic approach being actively pursu...

Endothelial nitric-oxide synthase (eNOS), the enzyme responsible for production of endothelial NO, is under tight and complex regulation. Proper cellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. In addition, the molecular chaperone Hsp90 interacts with eNOS and positively regulates eNOS activity. Hsp90 is modulated by physical interaction with its co-chaperones. CHIP (carboxyl terminus of Hsp70-interacting protein) is such a co-chaperone that remodels the Hsp90 heterocomplex and causes protein degradation of some Hsp90 substrates through the ubiquitin-protein isopeptide ligase activity of CHIP. Here we show that CHIP incorporated into the eNOS⅐Hsp90 complex and specifically decreased soluble eNOS levels in transiently transfected COS cells. Surprisingly, in contrast to the effects of the Hsp90 inhibitor geldanamycin, which induces eNOS ubiquitylation and its subsequent protein degradation, CHIP did not target eNOS for ubiquitylation and proteasome-dependent degradation. Instead, CHIP partitioned soluble eNOS into an insoluble and inactive cellular compartment, presumably through its co-chaperone activity. This effect seems to be due to displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation. Consistent with observations from overexpression studies, eNOS localization to the membrane and activity were increased in mouse lung endothelial cells lacking CHIP. Taken together, these results demonstrate a novel co-chaperone-dependent mechanism through which eNOS trafficking is regulated and suggest a potentially generalized role for CHIP in protein trafficking through the Golgi compartment.The nitric-oxide synthases (NOSs) 1 are a family of mammalian enzymes that catalyze the oxidation of L-arginine to produce NO and L-citrulline. Three NOS isoforms exist in mammalian cells, neuronal (nNOS; NOS1), inducible (iNOS; NOS2), and endothelial (eNOS; NOS3), named after the cell types in which they were originally discovered. All NOS isoforms have similar primary structures, including an oxygenase domain at the N terminus, a reductase domain at the C terminus, and a hinge calmodulin domain in between. eNOS is unique among the NOS isoforms in that it is dually acylated by myristate and palmitate. Cysteine palmitoylation is necessary for targeting of eNOS to the specific plasmalemmal microdomain, caveolae (1), and both fatty acylations are required for specific targeting of eNOS to the Golgi (2). Correct subcellular trafficking and localization to the plasmalemma is necessary for eNOS function. eNOS produces NO (and/or other reactive nitrogen species) in vascular endothelial cells and cardiomyocytes in response to a variety of agonists and mechanical stimuli (i.e. shear) (3, 4). Mislocalization of the enzyme to either domain impairs agonist-stimulated eNOS activation and optimal NO release from cells, implying that the proper subcellular localization of eNOS is critical for optima...