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A M Silverstein

Publications and source records attributed to A M Silverstein.

At least 19 recordsLinked to original sources
PubMed1997

Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin.

Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506- or cyclosporin A-binding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315-32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormone-free cells. Approximately one-half of the GR.hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and approximately 35% contains PP5. Only a small (but undetermined) fraction of the native GR.hsp90 heterocomplexes contain the cyclosporin A-binding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [3H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol.

Animals↗
PubMed1997

Geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus.

When they are translated, steroid receptors are assembled into a multiprotein complex containing hsp90, p23, an immunophilin, and often some hsp70. Some of the receptors, such as that for progesterone, have nuclear localization signals that are functional in the absence of hormone, and they move into the nucleus where they exist in the same multiprotein heterocomplex with hsp90. Other receptors, such as the glucocorticoid receptor, are localized predominantly in the cytoplasm in the absence of hormone and move into the nucleus in a hormone-dependent fashion. We have previously proposed that hsp90 and the immunophilin play a role in receptor trafficking [Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458]. In this work, we show that treatment of L cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, impedes dexamethasone-dependent trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus. Because geldanamycin treatment of hormone-free cells causes a rapid loss of steroid binding activity, receptors were prebound with dexamethasone by incubating cells with hormone at 0 degrees C prior to shifting the temperature to 37 degrees C for 20 min to permit receptor transformation and translocation in the presence or absence of geldanamycin. Geldanamycin does not cause steroid to dissociate from prebound receptors, and it does not inhibit hormone-mediated receptor transformation assayed by conversion to the DNA-binding state. However, as reported previously for the progesterone receptor, geldanamycin blocks assembly of the glucocorticoid receptor-hsp90 heterocomplex at an intermediate state of assembly where the receptor is bound to hsp70 and p60, both of which are required components in the assembly mechanism. Our observations support the proposal that dynamic association of receptors with hsp90 is required for receptor translocation from the cytoplasm to the nucleus.

Animals↗
PubMed1997

The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase.

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras.Raf, Ras.Src, Raf.Mek, and Raf.Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf.hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.

Animals↗
PubMed1996

The tetratricopeptide repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant.

We previously identified a protein-serine phosphatase designated PP5, based on the binding of its tetratricopeptide repeat (TPR) domain to the atrial natriuretic peptide receptor (Chinkers, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11075-11079). We have now identified another protein complex to which PP5 is targeted through its TPR domain. A 90-kDa protein, identified as heat shock protein 90 (hsp90) by immunoblotting, specifically co-immunoprecipitated from COS-7 cell lysates with the FLAG-tagged TPR domain of PP5. hsp90 also co-immunoprecipitated with full-length FLAG-tagged PP5 overexpressed in COS-7 cells and with endogenous PP5 from untransfected COS-7 cells or rat brain. During gel filtration, PP5 and hsp90 comigrated in a high molecular weight complex. Since glucocorticoid receptors (GR) exist as large heterocomplexes containing hsp90 bound to TPR proteins, we hypothesized that PP5 might be associated with these complexes. Consistent with this hypothesis, PP5 specifically co-immunoprecipitated with GR from mouse L cell lysates. To test the functional importance of this TPR-mediated association in living cells, we used a dominant negative PP5 mutant consisting only of its TPR domain. The mutant inhibited GR-mediated transactivation by approximately 70% in transfected CV-1 cells. This is the first evidence that the TPR proteins in steroid receptor heterocomplexes may be required for signaling in vivo.

Animals↗
PubMed1996

Paul Ehrlich: the founding of pediatric immunology.

In 1892-93, Paul Ehrlich published a series of experiments on the passive transfer of maternal antibody to fetus and newborn that deserve to be better known. He pointed out the importance of mother's milk to neonatal well-being. He was the first to define the difference between active and passive immunity. He was the first to demonstrate immune elimination of antigen and to explain the significance of each phase of the kinetics of the anamnestic antibody response. We point out how elegant were these early experiments and how far in advance of their times.

Allergy and Immunology↗
PubMed1996

Use of the thiol-specific derivatizing agent N-iodoacetyl-3-[125I]iodotyrosine to demonstrate conformational differences between the unbound and hsp90-bound glucocorticoid receptor hormone binding domain.

The hormone binding domain (HBD) of the glucocorticoid receptor (GR) contains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket. The HBD also contains the contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation. Binding of hsp90 to the receptor through its HBD inactivates the DNA binding domain (DBD). The DBD contains a number of cysteines essential to its DNA binding activity. Here, we assess the effects of hsp90 binding on the accessibility of cysteine residues in both the HBD and DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untransformed GR.hsp90 complex in a manner that is prevented by the sulfhydryl reagents cysteine and dithiothreitol but is not reversed by them. The 125I-labeled IAT derivative N-iodoacetyl-3-[125I]iodotyrosine ([125I]IAIT) covalently labels the immunopurified, hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90 leads to an approximately 2-fold increase in [125I]IAIT labeling of the full-length, 100-kDa GR. The increase in thiol labeling is related to the presence of hsp90 because it is blocked by molybdate, which prevents hsp90 dissociation. Cleavage of the [125I]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled fragment containing all of the cysteines in the HBD and the contact region for hsp90. Dissociation of hsp90 from the GR results in a 2.3-fold increase in [125I]IAIT labeling of the 15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment. These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conformational change in the HBD such that some of the thiols that are exposed in the GR*hsp90 complex become buried and are no longer accessible to the [125I]IAIT probe. In contrast, binding of the GR to hsp90 restricts access of cysteines in the DBD to this small thiol-derivatizing agent, a restriction that is relieved as a result of unmasking or conformational change accompanying hsp90 dissociation.

Animals↗
PubMed1995

MRI of lumbar intervertebral discs. Diurnal variations in signal intensities.

STUDY DESIGN: Eight volunteers underwent magnetic resonance imaging scans of their lumbar spines to determine diurnal variations in the scan results. OBJECTIVES: Magnetic resonance imaging of the lumbar spine typically is obtained under the assumption that results are not influenced by the time of day the scan is performed. To validate this assumption, asymptomatic volunteers had magnetic resonance images made of their lumbar spines, twice in one day. SUMMARY OF BACKGROUND DATA: No reports in the literature have indicated visible diurnal variations in magnetic resonance imaging of the spine. METHODS: Two magnetic resonance imaging scans were obtained of each volunteer's lumbar spine, the first 1 to 1.5 hours after waking and the second 8 to 10 hours later. The signal intensity was measured from each disc and mathematical standardization was performed against a saline phantom. Changes in disc height and bulge also were measured. Two blinded neuroradiologists reviewed each scan. RESULTS: Thirty-nine lumbar discs were studied. No visible changes could be detected between scans by blinded observers. However, the calculated signal intensity change was an average loss of 13.5%. An increase in disc bulge was measured in 48.6% of the discs. CONCLUSIONS: In this group of asymptomatic volunteers, the time of day that a magnetic resonance imaging scan was obtained did not influence the visual interpretation.

Adult↗
PubMed1995

From the forehead of Zeus: the ontogeny of the immune response.

Many of the most important developments that result in a fully functioning vertebrate immune system take place in the developing fetus. From a variety of gene segments there is assembled in B cells a congeries of antibody combining sites, one to a cell, which form the greater part of the large repertoire of immunological specificities that characterise the system. This capability is further expanded later by somatic mutations. Just as immunoglobulin isotypes are produced sequentially (IgM, IgD, IgG, IgE, IgA) as they are read along the chromosome, so does the fetus and neonate manifest immunological competence sequentially to different antigens by employing variable region germline genes as they appear along the chromosome. The generation of T cell receptor diversity is accomplished by a similar mechanism of gene segment translocations. Each stage in the lineage of T and B cells is associated with the appearance of unique combinations of surface molecular markers, which in T cells characterise also the specialised functions of different subsets. If the immune system does not spring forth quite fully formed from the evolved vertebrate genome, as Athena did from the forehead of Zeus, ontogenetic mechanisms have made it very nearly complete.

Antibody Diversity↗
PubMed1994

The structure and dynamics of immunology, 1951-1972: a prosopographical study of international meetings.

Immunology underwent a major change in direction during the 1950s and 1960s, from immunochemistry to immunobiology. A quantitative analysis of the participants at a large number of immunological meetings held during this critical period reveals much about the inner dynamics of the discipline. This new approach enables us to define the scientific leadership of the discipline and of its emerging subdisciplines, the interests of "mainstream" immunology, and when fields such as transplantation and allergy begin to join the mainstream. It is also suggested that some of these meetings, in addition to reflecting changes within the field, may have contributed substantially to these changes. Further, many of these meetings served to promote the institutionalization of the discipline and of its subdisciplines.

Allergy and Immunology↗
PubMed1991

The dynamics of conceptual change in twentieth century immunology.

I have attempted here to define three distinct eras in the 110-year history of the discipline of immunology. The first, extending from 1880 to about the First World War, centered around the new bacteriology and infectious diseases, and had a distinctly medical orientation. Several of the components of the original research program in immunology failed to maintain their original momentum or to fulfill their initial high promise, and went into decline. These include the development of new vaccines, serotherapeutic approaches, the study of cellular immunity, and the study of diseases that might be mediated by cytotoxic antibodies. Two other subprograms followed a somewhat different course; the study of anaphylaxis and related diseases passed primarily into the hands of clinical allergists, while the development and adaptation of serodiagnostic techniques passed into the hands of the new discipline of serology, both fields out of the mainstream of post-World War I immunology. As interest in the components of the old program was falling away, there developed a new area of interest in immunology. Leadership in the field devolved upon a new group of individuals with a predominantly chemical orientation to the study of antigens and antibodies, who pursued a research program and developed a theoretical base that reflected this orientation well. It may be interesting to examine more closely the forces responsible for this shift in emphasis. When interest in the old areas waned, the medically oriented practitioners did not switch to more immunochemical lines, but went in other directions. Karl Landsteiner was the only prominent "old-timer" who contributed significantly to the newer immunology, and it was his work that set the tone and attracted the new generation of immunochemists who became the reigning Denkkollektiv. A science does not change its precepts and approaches spontaneously; it is moved to the new position by those who explore fertile new areas. This is not to say, however, that there was no longer interesting and important work to be done along the old lines--it was just that such work was no longer "fashionable", as the reception of the work of Dienes, of Rich, of Rivers, and of the early Medawar illustrates. Whereas the earlier immunological program had interacted extensively with many different fields of biology and medicine, the immunochemical era was characterized by a relative introversion, as compared with the broad influence exerted by the earlier immunological program (92). We can date this second era from about the First World War until the late 1950s and early 1960s.(ABSTRACT TRUNCATED AT 400 WORDS)

Allergy and Immunology↗
PubMed1990

MR imaging of the brain in five members of a family with Pelizaeus-Merzbacher disease.

MR imaging was performed in five members of a family afflicted with Pelizaeus-Merzbacher disease. The individuals imaged included a male proband, his mother, and three maternal uncles. Clinically affected members showed generalized white matter signal aberration consistent with dys- and demyelination, basal ganglia, and thalamic signal aberration suggestive of pathologic iron storage and diffuse brain atrophy. These findings are similar to those seen in other leukodystrophies. The proband's mother was normal by neurologic examination but showed a suspicious but not definitely abnormal similar pattern of basal ganglionic and white matter signal aberration. In our limited patient sample, MR appears to be able to: (1) demonstrate a pattern of imaging abnormalities characteristic of Pelizaeus-Merzbacher disease (we do not know if this pattern is specific); (2) potentially detect the obligate carrier state; and (3) detect the facultative carrier state.

Adult↗
PubMed1987

Technetium-99m red blood cell venography in upper extremity deep venous thrombosis.

The efficacy of Tc-99m RBC venography has been demonstrated with respect to the study of lower extremity deep venous thrombosis. A case is presented where Tc-99m RBC venography was used to study the upper as well as lower extremities in a patient with upper extremity deep venous thrombosis (DVT) who was found to have pulmonary embolism.

Adult↗