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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.

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The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.

The MM281 strain of Salmonella typhimurium which possesses mutations in each its three known Mg 2؉ transport systems and requires 100 mM Mg 2؉ for growth was used to screen a genomic library from the gram-positive alkaliphilic bacterium Bacillus firmus OF4 for clones that could restore the ability to grow without Mg 2؉ supplementation. . Mutant strains lacking a functional CorA transporter demonstrate a significant reduction in the ability to take up Mg 2ϩ as well as increased resistance to the cytotoxic effects of Co 2ϩ in the culture medium. This phenotype is not unique to S. typhimurium and E. coli. Mg 2ϩ transport mutants of Bacillus subtilis and Rhodobacter capsulatus that also exhibit resistance to Co 2ϩ have been described (11,13,19,20). This phenotype implies the presence of a CorA-like system in these organisms and has prompted the hypothesis that CorA is widely distributed throughout prokaryotes (22). As part of studies to examine the structure and distribution of CorA-like systems, an Mg 2ϩ transport-deficient strain of S. typhimurium was used to screen a plasmid library from the gram-positive alkaliphile Bacillus firmus OF4 (9, 10) for clones that could complement the corA mutation. In this study, we describe the isolation and characterization of the MgtE transport system, which complements the Mg 2ϩ uptake mutations in S. typhimurium and restores sensitivity to Co 2ϩ . Sequence analysis, however, indicates that the MgtE locus encodes a 34-kDa protein quite unlike CorA and appears to represent a new class of Mg 2ϩ transport system. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains used in this study are listed in Table 1 with their sources. Plasmids were maintained in either E. coli DH5␣ or S. typhimurium JR501 (3). Plasmid complementation assays employed S. typhimurium MM281 as the host (7,8,24).Culture media and reagents. Luria-Bertani (LB) broth was routinely used as the complex growth medium. N-minimal broth was used as the minimal growth medium (16). Antibiotics were added to complex and minimal culture media, respectively, at the following concentrations (in micrograms per liter): sodium ampicillin, 100 and 30; tetracycline hydrochloride, 20 and 10; kanamycin sulfate, 50 and 100; chloramphenicol, 25 and 10. Growth of MM281 requires MgSO 4 supplementation at a final concentration of 100 mM in both complex and minimal culture media. Restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase, Klenow fragment of DNA polymerase I, S1 nuclease, and T4 polynucleotide kinase were obtained from Life Technologies, Inc. (Gibco-BRL, Gaithersburg, Md.). Sequencing enzymes and associated biochemicals were obtained from U. S. Biochemicals Inc. (Cleveland, Ohio).57 Co 2ϩ was obtained from Amersham (Arlington, Ill.). Synthetic oligodeoxynucleotide primers were obtained from Oligo's Etc. (Wilsonville, Oreg.). Additional chemicals were obtained from standard suppliers.DNA manipulation. Plasmid DNA was prepared from 500-ml cultures with the Wizard Maxiprep DNA isolation...