PubMed Health⌕ Search

Biomedical subjects

S Lindquist

Publications and source records attributed to S Lindquist.

At least 73 records · Page 4Linked to original sources
PubMed1995

Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase.

Hsp90 is a protein chaperone whose functions are focused on a specific set of target proteins. The nature of Hsp90's interactions with these proteins is poorly understood. To provide tools for examining these interactions, we have isolated eight broadly distributed temperature-sensitive (ts) point mutations in the Hsp90 gene (HSP82) of Saccharomyces cerevisiae. The mutants fall into two distinct classes. One has a classic ts phenotype, with nearly wild-type activity at 25 degrees C and a precipitous loss of function at 34 degrees C. The remaining seven mutants, in contrast, cause a general reduction in Hsp90 function and are ts because they do not provide the high level of function required for growth at high temperatures. The effects of these mutants on two target proteins, a transcription factor (glucocorticoid receptor) and a tyrosine kinase (pp60v-src), provided several insights on Hsp90 function. First, Hsp90 is not only required to help the glucocorticoid receptor achieve a hormone-activable state, it is continuously required to maintain that state. Second, Hsp90's function in the maturation of pp60v-src involves separable roles in protein accumulation and kinase activation. Thus, Hsp90 is an integral component of both the steroid receptor and kinase signaling pathways. Finally, all eight point mutants affect the activation of both the glucocorticoid receptor and pp60v-src, indicating that Hsp90 promotes the activity of these very different target proteins through common mechanisms.

DNA Mutational Analysis↗
PubMed1994

Protein disaggregation mediated by heat-shock protein Hsp104.

The heat-inducible members of the Hsp100 (or Clp) family of proteins share a common function in helping organisms to survive extreme stress, but the basic mechanism through which these proteins function is not understood. Hsp104 protects cells against a variety of stresses, under many physiological conditions, and its function has been evolutionarily conserved, at least from Saccharomyces cerevisiae to Arabidopsis thaliana. Homology with the Escherichia coli ClpA protein suggests that Hsp104 may provide stress tolerance by helping to rid the cell of heat-denatured proteins through proteolysis. But genetic analysis indicates that Hsp104 may function like Hsp70 as a molecular chaperone. Here we investigate the role of Hsp104 in vivo using a temperature-sensitive Vibrio harveyi luciferase-fusion protein as a test substrate. We find that Hsp104 does not protect luciferase from thermal denaturation, nor does it promote proteolysis of luciferase. Rather, Hsp104 functions in a manner not previously described for other heat-shock proteins: it mediates the resolubilization of heat-inactivated luciferase from insoluble aggregates.

HSP70 Heat-Shock Proteins↗
PubMed1994

Conservation of Hsp90 macromolecular complexes in Saccharomyces cerevisiae.

In higher eukaryotic cells, the Hsp90 chaperone protein is found in complexes with other proteins, in addition to the substrate protein. These other proteins include Hsp70, p60, and several peptidyl-prolyl cis-trans isomerases (immunophilins). We utilized affinity chromatography to investigate whether Hsp82, the Hsp90 of Saccharomyces cerevisiae, is found in similar complexes in that organism. Six histidine residues were fused to the N terminus of Hsp82 to yield a fusion protein (Hsp82FP) with affinity for a nickel-ion matrix. Hsp82FP was shown to have wild-type function. In addition, when mammalian substrates of Hsp90 (glucocorticoid receptor and p60v-src) were expressed in yeast cells, these proteins bound to the affinity matrix only when isolated from cells containing Hsp82FP. Yeast homologs of Hsp70 (in particular, members of the Ssa subfamily), p60 (Sti1), and a 45-kDa immunophilin homolog were also isolated in this manner, by virtue of their specific, stable association with Hsp82. Thus, Hsp90 functions as part of a highly conserved macromolecular complex in eukaryotes.

Amino Acid Sequence↗
PubMed1994

Structure of the human beta-casein encoding gene.

The entire human beta-casein-encoding gene, Bca, was cloned and sequenced. The gene consists of eight exons ranging from 21 to 531 nucleotides (nt) in length and extending over 10,466 nt. Exon-2 contains the translational start, the entire signal sequence and the codons for the two first amino acids of the mature protein. This corresponds to the organization found in other species. The translational stop is localized to exon-7. Exon/intron boundaries are in accordance with the AG/GT rule and conform to suggested consensus sequences. Splice junctions are located between coding triplets. In all other species analyzed, Bca has been found to consist of nine exons; however, within intron-2 of the human gene, a sequence omitted from human mRNA, but corresponding to exon-3 of other known Bca genes, was revealed.

Amino Acid Sequence↗
PubMed1994

Saccharomyces cerevisiae Hsp104 protein. Purification and characterization of ATP-induced structural changes.

Heat-shock proteins (hsps) function in a variety of ways to help cells and organisms cope with environmental changes. One class of hsps, the Hsp100 proteins, is especially important for tolerance to a variety of extremely stressful conditions (e.g. high temperatures or high concentrations of ethanol). To begin to characterize the mechanism of action of Hsp100 proteins, we have initiated an in vitro analysis of the Saccharomyces cerevisiae Hsp104 protein. Here, we report the purification and initial structural characterization of the wild-type protein and three variants carrying mutations in the two ATP-binding site consensus elements. As demonstrated by both gel filtration chromatography and by cross-linking studies with glutaraldehyde, Hsp104 forms a homohexameric particle. By electron microscopy, these particles are ring-shaped and reminiscent of proteins in the Hsp60 and TF55/TCP families. In contrast to these other proteins, Hsp104 forms single rings, each containing only six subunits. More strikingly, the assembly and maintenance of Hsp104 particles are dependent upon the presence of adenine nucleotides. Oligomerization appears to primarily depend upon the second of the two ATP-binding sites in the protein.

Adenosine Triphosphate↗
PubMed1994

An Arabidopsis heat shock protein complements a thermotolerance defect in yeast.

The heat shock protein Hsp104 of the yeast Saccharomyces cerevisiae plays a key role in promoting survival at extreme temperatures. We found that when diverse higher plant species are exposed to high temperatures they accumulate proteins that are antigenically related to Hsp104. We isolated a cDNA corresponding to one of these proteins from Arabidopsis. The protein, AtHSP101, is 43% identical to yeast Hsp104. DNA gel blot analysis indicated that AtHSP101 is encoded by a single- or low-copy number gene. AtHsp101 mRNA was undetectable in the absence of stress but accumulated to high levels during exposure to high temperatures. When AtHSP101 was expressed in yeast, it complemented the thermotolerance defect caused by a deletion of the HSP104 gene. The ability of AtHSP101 to protect yeast from severe heat stress strongly suggests that this HSP plays an important role in thermotolerance in higher plants.

Adaptation, Physiological↗
PubMed1994

Preferential deadenylation of Hsp70 mRNA plays a key role in regulating Hsp70 expression in Drosophila melanogaster.

Following a standard heat shock, approximately 40% of Hsp70 transcripts in Drosophila melanogaster lack a poly(A) tail. Since heat shock disrupts other aspects of RNA processing, this observation suggested that heat might disrupt polyadenylation as well. We find, however, that as the temperature is increased a larger fraction of Hsp70 RNA is polyadenylated. Poly(A)-deficient Hsp70 RNAs arise not from a failure in polyadenylation but from the rapid and selective removal of poly(A) from previously adenylated transcripts. Poly(A) removal is highly regulated: poly(A) is (i) removed much more rapidly from Hsp70 RNAs than from Hsp23 RNAs, (ii) removed more rapidly after mild heat shocks than after severe heat shocks, and (iii) removed more rapidly after a severe heat shock if cells have first been conditioned by a mild heat treatment. Poly(A) seems to be removed by simple deadenylation rather than by endonucleolytic cleavage 5' of the adenylation site. During recovery from heat shock, deadenylation is rapidly followed by degradation. In cells maintained at high temperatures, however, the two processes are uncoupled and Hsp70 RNAs are deadenylated without being degraded. These deadenylated mRNAs are translated with low efficiency. Deadenylation therefore allows Hsp70 synthesis to be repressed even when degradation of the mRNA is blocked. Poly(A) tail shortening appears to play a key role in regulating Hsp70 expression.

Animals↗
PubMed1994

Isolation of mRNA and genomic DNA from epithelial cells in human milk and amplification by PCR.

Studies on the regulation of human milk protein genes and suitable cell culture systems have been limited due to restricted availability of tissue samples from lactating women. Although mammary gland tissue has been available from mammary reduction surgery, studies on tissue-specific expression of milk protein genes require samples obtained during specific stages of expression in the tissue, i.e., lactation. We have therefore developed a technique to isolate mRNA and genomic DNA directly from epithelial cells isolated from human milk and used PCR methodology to specifically amplify the cDNA and genomic DNA for human beta-casein. When comparing fresh human milk with milk that has been stored for five hours at 37 degrees C or frozen and thawed, we found that the amount of mRNA isolated was considerably higher from fresh milk. However, in spite of this, we could still isolate intact mRNA from frozen milk. The described methodology should be useful in studies on milk protein gene expression during lactation and studies on variants of milk protein genes.

Base Sequence↗
PubMed1993

Heat-shock protein hsp90 governs the activity of pp60v-src kinase.

During or immediately after synthesis in vertebrate cells, the oncogenic protein-tyrosine kinase pp60v-src associates with the approximately 90-kDa heat-shock protein (hsp90). In this complex, pp60v-src is not functional as a kinase. When pp60v-src is subsequently found inserted into the plasma membrane, it is active as a kinase and is no longer associated with hsp90. We have taken advantage of genetic manipulations possible in Saccharomyces cerevisiae to investigate the function and specificity of the association between hsp90 and pp60v-src. Expression of pp60v-src is known to be toxic to S. cerevisiae cells. We find that this toxicity is due to a very specific effect on growth, arrest at a particular point in the cell cycle. In cells expressing v-src, a mutation that lowers the level of hsp90 expression (i) relieves cell cycle arrest and rescues growth, (ii) reduces the level of tyrosine phosphorylation mediated by pp60v-src, (iii) changes the pattern of tyrosine phosphorylation, and (iv) reduces the concentration of pp60v-src. We conclude that hsp90 does not simply suppress pp60v-src kinase activity during transit to the plasma membrane, as previously suggested, but also stabilizes the protein and affects both its activity and specificity. This function of hsp90 is highly selective for pp60v-src: the same hsp90 mutation has no effect on the activity or specificity of the exogenous pp160v-abl tyrosine kinase; similarly, it does not affect the specificity and has only a very small effect on the activity of the exogenous pp60c-src kinase.

Cell Cycle↗
PubMed1993

The role of heat-shock proteins in thermotolerance.

The role of heat-shock proteins (hsps) in thermotolerance was examined in the budding yeast Saccharomyces cerevisiae and in the fruit fly Drosophila melanogaster. In yeast cells, the major protein responsible for thermotolerance is hsp 100. In cells carrying mutations in the hsp 100 gene, HSP 104, growth is normal at both high and low temperatures, but the ability of cells to survive extreme temperatures is severely impaired. The loss of thermotolerance is apparently due to the absence of the hsp 104 protein itself because, with the exception of the hsp 104 protein, no differences in protein profiles were observed between mutant and wild-type cells. Aggregates found in mutant cells at high temperatures suggest that the cause of death may be the accumulation of denatured proteins. No differences in the rates of protein degradation were observed between mutant and wild-type cells. This, and genetic analysis of cells carrying multiple hsp 70 and hsp 104 mutations, suggests that the primary function of hsp 104 is to rescue proteins from denaturation rather than to degrade them once they have been denatured. Drosophila cells do not produce a protein in the hsp 100 class in response to high temperatures. In this organism, hsp 70 appears to be the primary protein involved in thermotolerance. Thus, the relative importance of different hsps in thermotolerance changes from organism to organism.

Amino Acid Sequence↗
PubMed1993

AmpG, a signal transducer in chromosomal beta-lactamase induction.

The chromosomal ampC beta-lactamase in Citrobacter freundii and Enterobacter cloacae is inducible by beta-lactam antibiotics. When an inducible ampC gene is introduced on a plasmid into Escherichia coli together with its transcriptional regulator ampR, the plasmid-borne beta-lactamase is still inducible. We have isolated mutants, containing alterations in a novel E. coli gene, ampG, in which a cloned C. freundii ampC gene is unable to respond to beta-lactam inducers. The ampG gene was cloned, sequenced and mapped to minute 9.6 on the E. coli chromosome. The deduced amino acid sequence predicted AmpG to be a 53 kDa, transmembrane protein, which we propose acts as a signal transducer or permease in the beta-lactamase induction system. Immediately upstream of ampG there is another 579-base-pair-long open reading frame (ORF) encoding a putative lipoprotein shown to be non-essential for beta-lactamase induction. We have found that ampG and this ORF form an operon, whose promoter is located in front of the ORF. Located closely upstream of the putative promoter is the morphogene bolA, which is transcribed in the opposite orientation. However, using transcription fusions, we have found that the ampG transcription is not regulated by bolA. In addition, we show that transcription is probably not regulated by either the starvation specific sigma factor RpoS, which controls bolA, or by AmpD the negative regulator for ampC transcription.

Amino Acid Sequence↗
PubMed1993

Sequences of wild-type and mutant ampD genes of Citrobacter freundii and Enterobacter cloacae.

The ampD gene product regulates the expression of AmpC beta-lactamase in gram-negative bacteria and is proposed to be involved in peptidoglycan metabolism. In this study, we sequenced the ampD wild type and three mutant genes of Enterobacter cloacae and Citrobacter freundii. They exhibited a high degree of homology with the corresponding gene of Escherichia coli except in the carboxy termini, where, in the wild-type genes of E. cloacae and C. freundii, four additional amino acids yielding the Ser-X-X-Lys motif were found. Evidence that this C-terminal region of the ampD gene product is necessary for activity was shown by constructing a deletion of the last 16 amino acids. The spontaneous mutation of ampD02 is an out-of-frame insertion and yields an inactive AmpD protein. The single-base-pair substitution of Gly for Asp-121 in ampD05 is responsible for a hyperinducible phenotype. These results demonstrate regions of the ampD gene and the corresponding protein which have functional importance for the induction of AmpC beta-lactamase in E. cloacae.

Amino Acid Sequence↗
PubMed1993

Genetic evidence for a functional relationship between Hsp104 and Hsp70.

The phenotypes of single Hsp104 and Hsp70 mutants of the budding yeast Saccharomyces cerevisiae provide no clue that these proteins are functionally related. Mutation of the HSP104 gene severely reduces the ability of cells to survive short exposures to extreme temperatures (thermotolerance) but has no effect on growth rates. On the other hand, mutations in the genes that encode Hsp70 proteins have significant effects on growth rates but do not reduce thermotolerance. The absence of a thermotolerance defect in S. cerevisiae Hsp70 mutants is puzzling, since the protein clearly plays an important role in thermotolerance in a variety of other organisms. In this report, examination of the phenotypes of combined Hsp104 and Hsp70 mutants uncovers similarities in the functions of Hsp104 and Hsp70 not previously apparent. In the absence of the Hsp104 protein, Hsp70 is very important for thermotolerance in S. cerevisiae, particularly at very early times after a temperature upshift. Similarly, Hsp104 plays a substantial role in vegetative growth under conditions of decreased Hsp70 protein levels. These results suggest a close functional relationship between Hsp104 and Hsp70.

DNA Mutational Analysis↗
PubMed1993

Hyperthermia protects mice against the lethal effects of endotoxin.

Recently, investigators have reported that heat shock proteins (HSPs) can protect isolated cells from cytotoxicity induced by two important mediators of sepsis: interleukin-1 and tumor necrosis factor. The present study was undertaken to examine the hypothesis that transient whole body hyperthermia could decrease mortality from subsequent challenge with gram-negative endotoxin. We demonstrate that heat pretreatments improved long-term survival fivefold in a mouse endotoxin model and this was correlated with the production of HSPs. There was a marked difference in individual organ expression of the inducible 72-kDa heat shock protein (HSP72). Heat treatments caused significant HSP72 formation in lung, liver, kidney, and small intestine, but much lesser formation in heart, brain, and abdominal wall muscle. Additional experiments demonstrated that the protective effect of hyperthermic treatments against an endotoxin challenge occurred early, i.e., 1 and 2 h after heating, was maximal at 12 h, and had significantly diminished by 48 h. The formation and decay of HSP72 demonstrated a time course that paralleled the survival curve from endotoxin challenge, thus suggesting a possible role for HSP72 in the protective effect. Surprisingly, and in contrast to studies reported in incubated cells, endotoxin alone did not cause significant formation of HSP72 in vivo.

Anesthesia↗
PubMed1992

Hsp104 is required for tolerance to many forms of stress.

Heat-shock proteins (hsps) are induced by many types of stress. In Saccharomyces cerevisiae, a mutation in the HSP104 gene, a member of the highly conserved hsp100 gene family, reduces the ability of log-phase fermenting cells to withstand high temperatures after mild, conditioning pretreatments. Here, we examine the expression of hsp104 and its importance for survival under many different conditions. Hsp104 is expressed at a higher level in respiring cells than in fermenting cells and is required for the unusually high basal thermotolerance of respiring cells. Its expression in stationary phase cells and spores is crucial for the naturally high thermotolerance of these cell types and for their long-term viability at low temperatures. The protein is of critical importance in tolerance to ethanol and of moderate importance in tolerance to sodium arsenite. Thus, the hsp104 mutation establishes the validity of a long-standing hypothesis in the heat-shock field, namely, that hsps have broadly protective functions. Further, that a single protein is responsible for tolerance to heat, ethanol, arsenite and long-term storage in the cold indicates that the underlying causes of lethality are similar in an extraordinary variety of circumstances. Finally, the protein is of little or no importance in tolerance to copper and cadmium, suggesting that the lethal lesions produced by these agents are fundamentally different from those produced by heat.

Cadmium↗