Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library of anti-HSP90 monoclonal antibodies (mAbs) and by limited proteolysis with trypsin and chymotrypsin. Thirty-three mAbs were obtained by immunization with bacterially expressed human HSP90␣ and HSP90␤ isoforms. Among them, ten and three mAbs reacted specifically with HSP90␣ and HSP90␤, respectively. Immunoblotting and enzyme-linked immunosorbent analyses revealed that major immunogenic domains were located at two restricted regions of HSP90␣, i.e. amino acids 227-310 (designated Region I) and 702-716 (Region II), corresponding to a highly charged region and a region near the C terminus, respectively. Taken together with the characteristics of the amino acid sequences, these two immunogenic regions appeared to be exposed at the outer surface of HSP90. We further investigated the domain structures of HSP90 by limited proteolysis in combination with N-terminal sequencing and immunoblotting analyses. Tryptic cleavages of HSP90␣ at low concentrations revealed the existence of major susceptible sites at Arg 400 -Glu 401 , Lys 615 -Ala 616 , and Arg 620 -Asp 621 . Proteolysis at higher trypsin concentrations caused successive cleavages only toward the N-terminal direction from these sites, and Region I was included in the region selectively deleted during this process, thereby further suggesting its surface location. From these results, we propose three domain structures of HSP90 consisting of amino acids 1-400, 401-615, and 621-732. Differences in the protease sensitivity and immunogenicity further suggest that every domain is composed of two subdomains. This is the first study describing the domain structures and the immunogenic regions of HSP90.The 90-kDa heat-shock protein (HSP90) 1 is one of the major stress proteins in eukaryotic cells. There are at least two HSP90 genes, and two HSP90 isoform proteins, ␣ and ␤, are HSP90 is believed to have a chaperone-like activity for particular molecules that are involved in signal transduction, such as steroid receptors (6), casein kinase II (7), pp60 v-src (8), elF2␣ kinase (9), and aryl hydrocarbon receptor (dioxin receptor) (10). HSP90 specifically binds to these proteins; and, in most cases, this interaction is essential for the function of the proteins (9,11,12). However, a variety of evidence, i.e. the abundance of HSP90 in cells even under nonstressed conditions, the conserved amino acid sequences from prokaryotic to eukaryotic cells (13), and the indispensability in yeast (3), strongly suggests that HSP90 is involved in more fundamental functions of cells. In fact, several studies have recently shown that HSP90 functions as a general chaperone. That is, it interacts with various proteins less specifically and modulates their conformation. For instance, the refolding of citrate synthase is significantly enhanced by the co-presence of HSP90 (14). The spontaneous refolding of denatured dihydrofolate reductase and irreversible denaturation of firefly luciferase are prevented b...

No abstract

Two isoforms of the 90-kDa heat shock protein, HSP90alpha and HSP90beta, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90alpha predominantly exists as a homodimer and that HSP90beta is present mainly as a monomer [Minami, Kawasaki, Miyata, Suzuki and Yahara (1991) J. Biol. Chem. 266, 10099-10103]. However, only the dimeric form has been observed under other analytical conditions such as gradient centrifugation. In this study, therefore, we investigated native forms of HSP90 by use of immunochemical techniques with isoform-specific monoclonal antibodies recently developed in our laboratory. Glycerol gradient centrifugation at the physiological salt concentration as well as native PAGE analysis of rat liver cytosol revealed oligomeric forms of HSP90alpha sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90beta oligomers sedimenting at 6-12S. All of the HSP90beta oligomers, however, migrated at 100-kDa monomer and 190-kDa dimer positions on native PAGE. A novel two-dimensional double native PAGE revealed that the entity was converted from the HSP90beta dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90alpha oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90alpha migrated as dimers, but a considerable amount did not penetrate into the gel under native PAGE conditions, indicating the existence of oligomeric forms. Electrophoretic studies of deletion mutants of HSP90 demonstrated that the C-terminal 200 amino acids were capable of forming oligomers. Taken together, we conclude that both of the HSP90 isoforms predominantly exist as oligomeric forms in the cytosol even under unstressed conditions but that they artificially dissociate into smaller forms when subjected to native PAGE.

No abstract

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.