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S Lindquist

Publications and source records attributed to S Lindquist.

At least 55 records · Page 3Linked to original sources
PubMed1997

Chaperone-supervised conversion of prion protein to its protease-resistant form.

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet. The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.

Adenosine Triphosphate↗
PubMed1997

In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone.

In the highly concentrated environment of the cell, polypeptide chains are prone to aggregation during synthesis (as nascent chains await the emergence of the remainder of their folding domain), translocation, assembly, and exposure to stresses that cause previously folded proteins to unfold. A large and diverse group of proteins, known as chaperones, transiently associate with such folding intermediates to prevent aggregation, but in many cases the specific functions of individual chaperones are still not clear. In vivo, Hsp90 (heat shock protein 90) plays a role in the maturation of components of signal transduction pathways but also exhibits chaperone activity with diverse proteins in vitro, suggesting a more general function. We used a unique temperature-sensitive mutant of Hsp90 in Saccharomyces cerevisiae, which rapidly and completely loses activity on shift to high temperatures, to examine the breadth of Hsp90 functions in vivo. The data suggest that Hsp90 is not required for the de novo folding of most proteins, but it is required for a specific subset of proteins that have greater difficulty reaching their native conformations. Under conditions of stress, Hsp90 does not generally protect proteins from thermal inactivation but does enhance the rate at which a heat-damaged protein is reactivated. Thus, although Hsp90 is one of the most abundant chaperones in the cell, its in vivo functions are highly restricted.

HSP90 Heat-Shock Proteins↗
PubMed1997

Cdc37 is a molecular chaperone with specific functions in signal transduction.

Cdc37 is required for cyclin-dependent kinase activation and is genetically linked with the activity of several other kinases, including oncogenic v-Src, casein kinase II, MPS-1 kinase, and sevenless. Strikingly, many pathways involving Cdc37 also involve the protein chaperone Hsp90. The identification of Cdc37 as the 50-kD protein in several Hsp90-kinase complexes, together with other data, led to the recent suggestion that Cdc37 is a kinase-targeting "subunit" of Hsp90. We directly examined the effect of Cdc37 on Hsp90 functions. Rather than simply acting as an accessory factor for Hsp90, Cdc37 is itself a protein chaperone with properties remarkably similar to those of Hsp90. In vitro, Cdc37 maintains denatured beta-galactosidase in an activation-competent state without reactivating it and stabilizes mature, but unstable, casein kinase II. In vivo, Cdc37 overexpression can compensate for decreased Hsp90 function, but the proteins are not interchangeable. Cdc37 can compensate for Hsp90 in maintaining the activity of v-Src kinase but does not maintain the activity of the glucocorticoid receptor. Thus, the very similar chaperone activities of the two proteins, uncovered through in vitro analysis, diverge in vivo in specific signal transduction pathways.

Cell Cycle Proteins↗
PubMed1997

Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae.

The [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of [PSI+].

Fungal Proteins↗
PubMed1997

Home health nurses: stress, self-esteem, social intimacy, and job satisfaction.

A survey of 253 home health care nurses' perceptions of work-related stress, self-esteem, social intimacy, and job satisfaction found that stress has a negative correlation with self-esteem, social intimacy, and job satisfaction. A positive correlation, however, was found between self-esteem and social intimacy and job satisfaction. Health system administrators, owners, and directors had significantly higher levels of self-esteem, nurses with 5 years or more in their home health nursing position had significantly higher levels of self-esteem. The survey found that nurses with less than a baccalaureate degree possessed significantly lower levels of sociability than those with a graduate or baccalaureate degree. Administrators and managers scored significantly higher on sociability than head nurses.

Adult↗
PubMed1997

In vivo analysis of the Hsp90 cochaperone Sti1 (p60).

Hsp90 interacts with Sti1 (p60) in lysates of yeast and vertebrate cells. Here we provide the first analysis of their interaction in vivo. Saccharomyces cerevisiae mutations that eliminate Sti1 or reduce intracellular concentrations of Hsp90 individually have little or no effect on growth at normal temperatures. However, when combined, the mutations greatly reduce or eliminate growth. Furthermore, overexpression of Sti1 has allele-specific effects on cells carrying various hsp90ts point mutations. These genetic interactions provide strong evidence that Hsp90 and Sti1 interact in vivo and that their functions are closely allied. Indeed, deletion of STI1 reduces the in vivo activity of the Hsp90 target protein, glucocorticoid receptor (GR). Mutations in GR that eliminate interaction with Hsp90 also eliminate the effects of the sti1 deletion. Examination of GR protein complexes in the sti1 deletion mutant reveals a selective increase in the concentration of GR-Ydj1 complexes, supporting previous hypotheses that Ydj1 functions at an early step in the maturation of GR and that Sti1 acts at an intermediate step. Deletion of STI1 also reduces the in vivo activity of another, unrelated Hsp90 target protein, v-Src. Our data indicate that Sti1 is a general factor in the maturation of Hsp90 target proteins and support earlier suggestions that Hsp90 matures even very different target proteins by a similar mechanism.

Amino Acid Isomerases↗
PubMed1997

Three variants of parathyroid hormone-related protein messenger RNA are expressed in human mammary gland.

PTH-related protein (PTHrP) is found in a variety of tissues; particularly high levels are present in human milk. The structure of the human PTHrP gene is complex, and alternative splicing allows expression of three different variants PTHrP139, PTHrP173, and PTHrP141, respectively. To determine which of the variants are expressed in human mammary gland a reverse transcriptase polymerase chain reaction (RT-PCR) method was elaborated, distinguishing the three variants. mRNA isolated from human milk cells, human mammary epithelial cells (HMEC) and human nonlactating mammary gland cells were analyzed. The RT-PCR experiments resulted in amplification of DNA fragments corresponding to all three variants for all three cell sources tested. The nucleotide sequences of the PCR fragments were determined and verified to be identical to the reported sequences. Hence, it is concluded that human mammary gland epithelial cells express three variants of PTHrP. Whether these have different physiologic effects in the mammary gland or in the breast fed infant remain to be explored.

Adult↗
PubMed1996

A cyclophilin function in Hsp90-dependent signal transduction.

Cpr6 and Cpr7, the Saccharomyces cerevisiae homologs of cyclophilin-40 (CyP-40), were shown to form complexes with Hsp90, a protein chaperone that functions in several signal transduction pathways. Deletion of CPR7 caused severe growth defects when combined with mutations that decrease the amount of Hsp90 or Sti1, another component of the Hsp90 chaperone machinery. The activities of two heterologous Hsp90-dependent signal transducers expressed in yeast, glucocorticoid receptor and pp60(v-src) kinase, were adversely affected by cpr7 null mutations. These results suggest that CyP-40 cyclophilins play a general role in Hsp90-dependent signal transduction pathways under normal growth conditions.

Amino Acid Isomerases↗
PubMed1996

Support for the prion hypothesis for inheritance of a phenotypic trait in yeast.

A cytoplasmically inherited genetic element in yeast, [PSI+], was confirmed to be a prionlike aggregate of the cellular protein Sup35 by differential centrifugation analysis and microscopic localization of a Sup35-green fluorescent protein fusion. Aggregation depended on the intracellular concentration and functional state of the chaperone protein Hsp104 in the same manner as did [PSI+] inheritance. The amino-terminal and carboxy-terminal domains of Sup35 contributed to the unusual behavior of [PSI+]. [PSI+] altered the conformational state of newly synthesized prion proteins, inducing them to aggregate as well, thus fulfilling a major tenet of the prion hypothesis.

Amino Acid Sequence↗
PubMed1996

Heat-shock protein 104 expression is sufficient for thermotolerance in yeast.

In all organisms, mild heat pretreatments induce tolerance to high temperatures. In the yeast Saccharomyces cerevisiae, such pretreatments strongly induce heat-shock protein (Hsp) 104, and hsp104 mutations greatly reduce high-temperature survival, indicating Hsp1O4 plays a critical role in induced thermotolerance. Surprisingly, however, a heat-shock transcription factor mutation (hsf1-m3) that blocks the induction of Hsps does not block induced thermotolerance. To resolve these apparent contradictions, we reexamined Hsp expression in hsf1-m3 cells. HsplO4 was expressed at a higher basal level in this strain than in other S. cerevisiae strains. Moreover, whereas the hsf1-m3 mutation completely blocked the induction of Hsp26 by heat, it did not block the induction of Hsp1O4. HSP104 could not be deleted in hsf1-m3 cells because the expression of heat-shock factor (and the viability of the strain) requires nonsense suppression mediated by the yeast prion [PSI+], which in turn depends upon Hsp1O4. To determine whether the level of Hsp1O4 expressed in hsf1-m3 cells is sufficient for thermotolerance, we used heterologous promoters to regulate Hsp1O4 expression in other strains. In the presence of other inducible factors (with a conditioning pretreatment), low levels of Hsp1O4 are sufficient to provide full thermotolerance. More remarkably, in the absence of other inducible factors (without a pretreatment), high levels of Hsp1O4 are sufficient. We conclude that Hsp1O4 plays a central role in ameliorating heat toxicity. Because Hsp1O4 is nontoxic and highly conserved, manipulating the expression of Hsp1OO proteins provides an excellent prospect for manipulating thermotolerance in other species.

Estradiol↗
PubMed1996

HSP100/Clp proteins: a common mechanism explains diverse functions.

The HSP100/Clp proteins are a newly discovered family with a great diversity of functions, such as increased tolerance to high temperatures, promotion of proteolysis of specific cellular substrates and regulation of transcription. HSP100/Clp proteins are also synthesized in a variety of specific patterns and, in eukaryotes, are localized to different subcellular compartments. Recent data suggest that a common ability to disassemble higher-order protein structures and aggregates unifies the molecular functions of this diverse family.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1996

Mad cows meet mad yeast: the prion hypothesis.

Obscure work on the inheritance of two peculiar genetic traits in yeast has recently collided with work on infectious, neurodegenerative diseases in mammals. The impact illuminates both fields. The yeast work reveals a new mechanism of genetic variation that is based on differences in protein conformation rather than differences in nucleic acid. In the light of this work the prion hypothesis for transmissible neurodegenerative disease no longer seems so bizarre. Indeed, the mammalian prion hypothesis is now placed within a larger framework that is likely to be of universal importance in biology. Yeast prions, as the genetic elements are called, can be cured, offering new insights into the molecular processes involved in protein-based inheritance and new hope for the treatment of neurodegenerative diseases in man.

Animals↗
PubMed1995

The basis for a heat-induced developmental defect: defining crucial lesions.

Because lethal heat shocks perturb a multitude of cellular processes, the primary lesions responsible for death from heat stress remain to be defined. In Drosophila, sublethal heat treatments produce developmental anomalies that frequently mimic the effects of known mutations and are hence referred to as phenocopies. Mutations subject to phenocopy mimicry provide signposts to those biological processes most sensitive to heat and most important for the function and survival of the organism as a whole. We have analyzed a particular developmental defect inducible in early embryos of Drosophila melanogaster. By molecular, phenotypic, and genetic criteria, we have found extensive parallels between this phenocopy and certain dominant mutations in the segmentation gene fushi tarazu (ftz). Our analysis of this phenocopy indicates that the crucial lesion is interference with proper turnover of ftz protein, resulting in ftz overexpression. Our results provide a novel explanation for a heat-induced developmental defect. Perturbations in relative amounts of important regulatory proteins may be a common mechanism by which heat-shock phenocopies arise.

Abdomen↗
PubMed1995

Role of the protein chaperone YDJ1 in establishing Hsp90-mediated signal transduction pathways.

The substrate-specific protein chaperone Hsp90 (heat shock protein 90) from Saccharomyces cerevisiae functions in diverse signal transduction pathways. A mutation in YDJ1, a member of the DnaJ chaperone family, was recovered in a synthetic-lethal screen with Hsp90 mutants. In an otherwise wild-type background, the ydj1 mutation exerted strong and specific effects on three Hsp90 substrates, derepressing two (the estrogen and glucocorticoid receptors) and reducing the function of the third (the tyrosine kinase p60v-src). Analysis of one of these substrates, the glucocorticoid receptor, indicated that Ydj1 exerts its effects through physical interaction with Hsp90 substrates.

Base Sequence↗
PubMed1995

Heat-shock proteins Hsp104 and Hsp70 reactivate mRNA splicing after heat inactivation.

BACKGROUND: The heat-shock protein Hsp104 plays a crucial role in the survival of cells exposed to high temperatures and other severe stresses, but its specific functions and the biological pathways on which it operates have been unclear. Indeed, very little is known about the specific cellular processes in which any of the heat-shock proteins acts to affect thermotolerance. One essential process that is particularly sensitive to heat in many organisms is the splicing of intervening sequences from mRNA precursors. RESULTS: We have examined the role of Hsp104 in the repair of splicing after disruption by heat shock. When splicing in the budding yeast Saccharomyces cerevisiae was disrupted by a brief heat shock, it recovered much more rapidly in wild-type strains than in strains containing hsp104 mutations. Constitutive expression of Hsp104 promoted the recovery of heat-damaged splicing in the absence of other protein synthesis, but did not protect splicing from the initial disruption, suggesting that Hsp104 functions to repair splicing after heat damage rather than to prevent the initial damage. A modest reduction in the recovery of splicing after heat shock in an hsp70 mutant suggested that Hsp70 may also function in the repair of splicing. The roles of Hsp104 and Hsp70 were confirmed by the ability of the purified proteins to restore splicing in extracts that had been heat-inactivated in vitro. Together, these two proteins were able to restore splicing to a greater degree than could be accomplished by an optimal concentration of either protein alone. CONCLUSIONS: Our findings provide the first demonstration of the roles of heat-shock proteins in a biological process that is known to be particularly sensitive to heat in vivo. The results support previous genetic arguments that the Hsp104 and Hsp70 proteins have different, but related, functions in protecting cells from the toxic effects of high temperatures. Because Hsp104 and Hsp70 are able to function in vitro, after the heat-damaged substrate or substrates have been generated, neither protein is required to bind to its target(s) during heating in order to effect repair.

Fungal Proteins↗