Quantitation and Intracellular Localization of the 85K Heat Shock Protein by Using Monoclonal and Polyclonal Antibodies

Abstract

Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by one-dimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.

MEK Inhibition Potentiates the Activity of Hsp90 Inhibitor 17-AAG against Pancreatic Cancer Cells

“…Hsp90 is a highly abundant molecular chaperone in eukaryotic cells . It simultaneously regulates the conformation, activation, maturation, and stability of a wide range of client proteins, many of which are mutated and/or overexpressed signaling proteins in cancers. − Hsp90 inhibition results in ubiquitination and proteasomal degradation of client proteins, thus rendering potent anticancer activity .…”

Design, Synthesis, and Biological Evaluation of Hydroquinone Derivatives of 17-Amino-17-demethoxygeldanamycin as Potent, Water-Soluble Inhibitors of Hsp90

“…6 Hsp90 is ubiquitously expressed and is one of the most abundant proteins inside the cell, constituting up to ∼2% of total protein. 7 Frequent overexpression of Hsp90 in solid and hematologic tumors suggests a role for the chaperone in oncogenesis. [8][9][10][11][12][13][14][15][16] Many of Hsp90 client proteins, including steroid hormone receptors, signaling kinases, transcription factors, and telomerase, appear to be involved in all the hallmarks of cancer.…”

“…Hsp90 68 is increasingly recognized as an important target for molecular cancer therapy due to its role in regulating key proteins in cell growth, survival, and differentiation pathways. − Together with co-chaperone proteins (e.g., Hsp70, Hip, Hop, Hsp40, Cdc37, p23), Hsp90 assists the folding, maturation, stability, and trafficking of a specific group of proteins called client proteins . Hsp90 is ubiquitously expressed and is one of the most abundant proteins inside the cell, constituting up to ∼2% of total protein . Frequent overexpression of Hsp90 in solid and hematologic tumors suggests a role for the chaperone in oncogenesis. − Many of Hsp90 client proteins, including steroid hormone receptors, signaling kinases, transcription factors, and telomerase, appear to be involved in all the hallmarks of cancer .…”

Hydrodynamic Properties and Quaternary Structure of the 90 kDa Heat-Shock Protein: Effects of Divalent Cations

“…In the presence of divalent cations, Hsp90 thermal oligomerization occurred at lower temperatures than in their absence (i.e., 40-45 °C). This process could result from the sum of two phenomena: (1) an oligomerization implicating the C-terminal domain and leading to oligomeric structures as observed in this study and (2) a marked elevation of Hsp90 chaperone and peptide binding activity, leading to N-terminal homotopic interactions that concomitantly increase the degree of oligomerization. The presence of oligomers with an apparent molecular mass of ∼500 kDa even at high temperatures for Hsp90 alone could be due to N-terminal intramolecular interactions.…”

“…Heat-shock protein 90 (Hsp90) 1 is one of the most abundant cytosolic proteins in eukaryotic cells, amounting to 1-2% of soluble proteins (1,2). This level further increases when cells are exposed to stresses such as heat shock, amino acid analogues, and heavy metals (3).…”