Importance of low-oligomeric-weight species for prion propagation in the yeast prion system Sup35/Hsp104

The [PSI+] determinant of Saccharomyces cerevisiae, consisting of the cytosolic translation termination factor Sup35, is a prion-type genetic element that induces an inheritable conformational change and converts the Sup35 protein into amyloid fibers. The molecular chaperone Hsp104 is required to maintain self-replication of [PSI+]. We observe in vitro that addition of catalytic amounts of Hsp104 to the prion-determining region of the NM domain of Sup35, Sup355-26, results in the dissociation of oligomeric Sup35 into monomeric species. Several intermediates of Sup355-26 could be detected during this process. Strong interactions are found between Hsp104 and hexameric/tetrameric Sup355-26, whereas the intermediate and monomeric "release" forms show a decreased affinity with respect to Hsp104, as monitored by saturation transfer difference and diffusion-ordered NMR spectroscopic experiments. Interactions are mediated mostly by the side chains of Gln, Asn, and Tyr residues in Sup355-26. No interaction can be detected between Hsp104 and higher oligomeric states (>/=8) of Sup355-26. Taking into account the fact that Hsp104 is required for maintenance of [PSI+], we suggest that low-oligomeric-weight species of Sup35 are important for prion propagation in yeast.     

Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division

Propagation of the yeast prion [PSI+], a self-replicating aggregated form of Sup35p, requires Hsp104. One model to explain this phenomenon proposes that, in the absence of Hsp104, Sup35p aggregates enlarge but fail to replicate thus becoming diluted out as the yeast divide. To test this model, we used live imaging of Sup35p-GFP to follow the changes that occur in [PSI+] cells after the addition of guanidine to inactivate Hsp104. After guanidine addition there was initially an increase in aggregation of Sup35p-GFP; but then, before the yeast divided, the aggregates began to dissolve, and after approximately 6 h the Sup35-GFP looked identical to the Sup35-GFP in [psi+] cells. Although plating studies showed that the yeast were still [PSI+], this reduction in aggregation suggested that curing of [PSI+] by inactivation of Hsp104 might be independent of cell division. This was tested by measuring the rate of curing of [PSI+] cells in both dividing and nondividing cells. Cell division was inhibited by adding either alpha factor or farnesol. Remarkably, with both of these methods, we found that the rate of curing was not significantly affected by cell division. Thus, cell division is not a determining factor for curing [PSI+] by inactivating Hsp104 with guanidine. Rather, curing apparently occurs because Sup35-GFP polymers slowly depolymerize in the absence of Hsp104 activity. Hsp104 then counteracts this curing possibly by catalyzing formation of new polymers.     

Interactions of the chaperone Hsp104 with yeast Sup35 and mammalian PrP

[PSI+] is a genetic element in yeast for which a heritable change in phenotype appears to be caused by a heritable change in the conformational state of the Sup35 protein. The inheritance of [PSI+] and the physical state of Sup35 in vivo depend on the protein chaperone Hsp104 (heat shock protein 104). Although these observations provide a strong genetic argument in support of the “protein-only” or “prion” hypothesis for [PSI+], there is, as yet, no direct evidence of an interaction between the two proteins. We report that when purified Sup35 and Hsp104 are mixed, the circular dichroism (CD) spectrum differs from that predicted by the addition of the proteins’ individual spectra, and the ATPase activity of Hsp104 is inhibited. Similar results are obtained with two other amyloidogenic substrates, mammalian PrP and β-amyloid 1-42 peptide, but not with several control proteins. With a group of peptides that span the PrP protein sequence, those that produced the largest changes in CD spectra also caused the strongest inhibition of ATPase activity in Hsp104. Our observations suggest that (i) previously described genetic interactions between Hsp104 and [PSI+] are caused by direct interaction between Hsp104 and Sup35; (ii) Sup35 and PrP, the determinants of the yeast and mammalian prions, respectively, share structural features that lead to a specific interaction with Hsp104; and (iii) these interactions couple a change in structure to the ATPase activity of Hsp104.     

Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride

Guanidine hydrochloride (Gdn.HCl) blocks the propagation of yeast prions by inhibiting Hsp104, a molecular chaperone that is absolutely required for yeast prion propagation. We had previously proposed that ongoing cell division is required for Gdn.HCl-induced loss of the [PSI+] prion. Subsequently, Wu et al.[Wu Y, Greene LE, Masison DC, Eisenberg E (2005) Proc Natl Acad Sci USA 102:12789-12794] claimed to show that Gdn.HCl can eliminate the [PSI+] prion from alpha-factor-arrested cells leading them to propose that in Gdn.HCl-treated cells the prion aggregates are degraded by an Hsp104-independent mechanism. Here we demonstrate that the results of Wu et al. can be explained by an unusually high rate of alpha-factor-induced cell death in the [PSI+] strain (780-1D) used in their studies. What appeared to be no growth in their experiments was actually no increase in total cell number in a dividing culture through a counterbalancing level of cell death. Using media-exchange experiments, we provide further support for our original proposal that elimination of the [PSI+] prion by Gdn.HCl requires ongoing cell division and that prions are not destroyed during or after the evident curing phase.     

羟基脲抑制盐酸胍对于酵母朊病毒[PSI+]的治愈

摘 要：目的 研究细胞分裂与盐酸胍作用下酵母朊病毒[PSI+]聚集体解聚的关系。方法 本研究借助重组表达Sup35p-GFP的菌株,采用表型分析方法与半变性琼脂糖凝胶电泳（SDD-AGE）结合蛋白质免疫印迹技术,分析了羟基脲抑制细胞分裂情况下对于盐酸胍解聚酵母朊病毒聚集体的影响。结果 羟基脲抑制细胞分裂的情况下,表型分析的数据显示盐酸胍不能治愈酵母朊病毒[PSI+],蛋白水平的实验数据证实了这一结果并发现,羟基脲的存在使得盐酸胍解聚酵母朊病毒聚集体的能力明显下降。结论 这暗示着盐酸胍治愈朊病毒[PSI+]是需要细胞进行分裂的。     

V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors

The Raf family includes three members, of which B-Raf is frequently mutated in melanoma and other tumors. We show that Raf-1 and A-Raf require Hsp90 for stability, whereas B-Raf does not. In contrast, mutated, activated B-Raf binds to an Hsp90-cdc37 complex, which is required for its stability and function. Exposure of melanoma cells and tumors to the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in the degradation of mutant B-Raf, inhibition of mitogen-activated protein kinase activation and cell proliferation, induction of apoptosis, and antitumor activity. These data suggest that activated mutated B-Raf proteins are incompetent for folding in the absence of Hsp90, thus suggesting that the chaperone is required for the clonal evolution of melanomas and other tumors that depend on this mutation. Hsp90 inhibition represents a therapeutic strategy for the treatment of melanoma.     

A novel role for DYX1C1, a chaperone protein for both Hsp70 and Hsp90, in breast cancer

Aims  With three consecutive tetratricopeptide repeat (TPR) motifs at its C-terminus essential for neuronal migration, and a p23 domain at its N-terminus, DYX1C1 was the first gene proposed to have a role in developmental dyslexia. In this study, we attempted to identify the potential interaction of DYX1C1 and heat shock protein, and the role of DYX1C1 in breast cancer. Main methods  GST pull-down, a yeast two-hybrid system, RT-PCR, site-directed mutagenesis approach. Key findings  Our study initially confirmed DYX1C1, a dyslexia related protein, could interact with Hsp70 and Hsp90 via GST pull-down and a yeast two-hybrid system. And we verified that EEVD, the C-terminal residues of DYX1C1, is responsible for the identified association. Further, DYX1C1 mRNA was significantly overexpressed in malignant breast tumor, linking with the up-regulated expression of Hsp70 and Hsp90. Significance  These results suggest that DYX1C1 is a novel Hsp70 and Hsp90-interacting co-chaperone protein and its expression is associated with malignancy. Yuxin Chen, Muzi Zhao, Saiqun Wang contributed equally to this work.     

Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p

Organisms encode multiple homologous heat shock protein (Hsp)-70s, which are essential protein chaperones that play the major role in cellular protein "quality control." Although Hsp70s are functionally redundant and highly homologous, many possess distinct functions. A regulatory motif underlying such distinctions, however, is unknown. The 98% identical cytoplasmic Hsp70s Ssa1p and Ssa2p function differently with regard to propagation of yeast [URE3] prions and in the vacuolar-mediated degradation of gluconeogenesis enzymes, such as FBPase. Here, we show that the Hsp70 nucleotide binding domain (NBD) regulates these functional specificities. We find little difference in ATPase, protein refolding, and amyloid inhibiting activities of purified Ssa1p and Ssa2p, but show that interchanging NBD residue alanine 83 (Ssa1p) and glycine 83 (Ssa2p) switched functions of Ssa1p and Ssa2p in [URE3] propagation and FBPase degradation. Disrupting the degradation pathway did not affect prion propagation, however, indicating these are two distinct processes where Ssa1/2p chaperones function differently. Our results suggest that the primary evolutionary pressure for Hsp70 functional distinctions is not to specify interactions of Hsp70 with substrate, but to specify the regulation of this activity. Our data suggest a rationale for maintaining multiple Hsp70s and suggest that subtle differences among Hsp70s evolved to provide functional specificity without affecting overall enzymatic activity.     

CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation

Hsp104 is a ring-forming AAA+ machine that recognizes both aggregated proteins and prion-fibrils as substrates and, together with the Hsp70 system, remodels substrates in an ATP-dependent manner. Whereas the ability to disaggregate proteins is dependent on the Hsp104 M-domain, the location of the M-domain is controversial and its exact function remains unknown. Here we present cryoEM structures of two Hsp104 variants in both crosslinked and noncrosslinked form, in addition to the structure of a functional Hsp104 chimera harboring T4 lysozyme within the M-domain helix L2. Unexpectedly, we found that our Hsp104 chimera has gained function and can solubilize heat-aggregated β-galactosidase (β-gal) in the absence of the Hsp70 system. Our fitted structures confirm that the subunit arrangement of Hsp104 is similar to other AAA+ machines, and place the M-domains on the Hsp104 exterior, where they can potentially interact with large, aggregated proteins.     

Amino acid sequence, active-site residue, and effect of suicide inhibitors on cephalosporinase of Citrobacter freundii GN346

The structural gene for a cephalosporinase of Citrobacter freundii GN346 was sequenced. From the nucleotide sequence, the entire amino acid sequence of the mature enzyme with 361 amino acids and a molecular weight of 39,867 was determined. The active-site serine was directly confirmed to be serine 64 by studies in which the enzyme was labeled with dansylpenicillin. In investigations comparing the inhibitory effect of sulbactam (penicillanic acid sulfone) and cloxacillin sulfone on the cephalosporinase and on TEM-2-type penicillinase, sulbactam was found to be an effective progressive inhibitor but a poor competitive inhibitor for the cephalosporinase. The cephalosporinase and the inhibitor formed a long-lived complex with a half-life of 550 minutes. Cloxacillin sulfone could not inactivate the cephalosporinase progressively but irreversibly inactivated the penicillinase.     

Amino acid residue at position 13 in HLA-DR beta chain plays a critical role in the development of Kaposi's sarcoma in AIDS patients

BACKGROUND: In 1994 human herpesvirus 8 (HHV-8) was identified as the causative agent of Kaposi's sarcoma (KS). Moreover, the crucial role of HLA molecules in determining susceptibility to several infections was recognized. OBJECTIVES: To evaluate the influence of HLA-DRB1 polymorphism in KS susceptibility among HHV-8 infected AIDS patients. DESIGN: A matched case-control study was designed to identify possible biological and environmental risk factors for HIV associated KS. Cases were defined as any AIDS patient with a clinical diagnosis of KS and controls as any AIDS patient with an indicative disease other than KS or with CD4 cells counts < 200 x 10 cells/l, diagnosed at +/- 4 months after case diagnosis. Each case was matched with two controls by sex, age and transmission category. METHODS: HHV-8 serostatus was determined by immunofluorescence assay for the latency associated antigen encoded by Orf73, ELISA for Orf73 and ELISA for the lytic antigen Orf65. DRB1 typing was carried out with a commercially available PCR-sequence specific primer assay. RESULTS: Comparison of marker frequencies in HHV-8 infected AIDS patients with or without KS showed a positive association between KS and HLA-DRB1 alleles containing phenylalanine at position 13 [odds ratio (OR), 2.24; P = 0.016]. A negative association was observed when the residue at the same position was glycine (OR, 0.16; P = 0.009). CONCLUSION: These observations suggest a possible role for HLA-DRB1 in the development of KS in HHV-8 infected individuals with HIV co-infection. Progression to KS in HHV-8 infected AIDS patients may also depend on host factors controlling the immune response.     

Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12.

The 21,000-dalton protein (p21) encoded by the ras oncogene of Harvey murine sarcoma virus (v-Ha-ras) becomes phosphorylated (pp21) in vivo and in vitro on threonine residue 59. p21 molecules encoded by cellular ras genes (c-Ha-ras-1) contain an alanine at position 59, and thus these p21 molecules are not phosphorylated. In this investigation, recombinant ras genes have been constructed between the 5' p21 coding region of normal (EC) or oncogenically activated (EJ) human c-Ha-ras-1 and the 3' p21 coding region of v-Ha-ras to generate p21 molecules containing a threonine phosphoacceptor site at position 59 and a glycine (EC/v-Ha) or valine (EJ/v-Ha) at residue 12. In transformed NIH 3T3 mouse fibroblast cells labeled with [35S]methionine, the ratio of pp21 to p21 was strikingly modulated by the amino acid at residue 12. v-Ha-ras p21 has an arginine at position 12, and 24% of total p21 was in the phosphorylated form. A glycine at residue 12 decreased the amount of pp21 to 14% of total p21, and a valine at residue 12 dramatically increased this value to 50%. In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM). These results indicate that a biochemical activity of p21 distinguishes between previously observed biological differences of normal and activated human ras genes.     

Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.

The murine BP-1 antigen (also called 6C3) is a homodimeric, phosphorylated cell surface glycoprotein that is expressed on immature B-lineage cells, bone marrow stromal cell lines, thymic cortical epithelial cells, endothelial cells, enterocytes, and renal proximal tubular cells. The amino acid sequence deduced from a BP-1 cDNA predicted a type II integral membrane protein with a zinc-binding motif (His-Glu-Xaa-Xaa-His) found in zinc-dependent metallopeptidases, and functional analysis suggested that BP-1 is aminopeptidase A [APA; L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7]. Here we constructed an expression vector in which the BP-1 cDNA was placed downstream from the SR alpha promoter and used this construct to transfect COS-7 and Ltk- cells. Both transfectants expressed the BP-1 antigen on the cell surface and APA activity. The enzymatic activity of recombinant APA was increased by Ca2+ and inhibited by Zn2+, consistent with previous reports with purified APA. Point mutation of one of the histidine residues in the zinc-binding motif to phenylalanine completely abolished APA enzymatic activity, suggesting the structure of the zinc-binding motif of APA is critical for catalytic activity. Both wild-type and mutant BP-1 were glycosylated, transported to the cell surface, and possessed molecular weights similar to native BP-1 molecules on the murine 18.81 pre-B-cell line. The successful expression of both wild-type and mutant APA should allow more precise analysis of the diverse physiological roles of this ectoenzyme.     

Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization.

We have studied the intracellular compartmentalization in yeast of Escherichia coli beta-galactosidase bearing heterologous amino acid sequences at its amino terminus. Chimeras containing as few as 74 NH2-terminal amino acids of GAL4, a yeast positive regulatory protein, at the amino terminus accumulate in the cell nucleus. This and other results are consistent with the proposal that the GAL4 gene product mediates positive control by binding to DNA and that the information for nuclear localization resides in its amino terminus. The amino acid sequence of the GAL4 amino terminus does not agree with the previously proposed consensus sequences responsible for nuclear localization. The beta-galactosidase activity in cells bearing the non-nuclear chimeric proteins is 10-fold greater than in cells bearing chimeric proteins that specifically concentrate in the nucleus.     

Activity of the Hsp70 chaperone complex--DnaK, DnaJ, and GrpE--in initiating phage lambda DNA replication by sequestering and releasing lambda P protein.

Initiation of DNA replication by phage lambda requires the ordered assembly and disassembly of a specialized nucleoprotein structure at the origin of replication. In the disassembly pathway, a set of Escherichia coli heat shock proteins termed the Hsp70 complex--DnaK, DnaJ, and GrpE--act with ATP to release lambda P protein from the nucleo-protein complex, freeing the DnaB helicase for its DNA-unwinding reaction. To investigate the mechanism of the release reaction, we have examined the interaction between P and the three heat shock proteins by glycerol gradient sedimentation and gel electrophoresis. We have discovered an ATP-dependent ternary interaction between P, DnaK, and DnaJ; this P.DnaK.DnaJ complex is dissociated by GrpE. We have concluded that the function of the Hsp70 complex in sequestering and releasing P protein provides for the critical step in the disassembly pathway. Based on our data and other work on protein folding, the formation of the P.DnaK.DnaJ complex might involve a conformational shift to a folding intermediate of P.     

Histidine residue at position 226 is critical for iodide uptake activity of human sodium/iodide symporter

The sodium/iodide symporter (SLC5A5; also known as NIS), a transmembrane glycoprotein principally in the thyroid gland, is responsible for the accumulation of iodide necessary for thyroid hormones. Our previous study indicated that a novel exon 6 deletion (residues 233-280) in SLC5A5 loses the iodide uptake activity. Herein we characterized the role of His-226 in iodide transport of SLC5A5. His-226, a highly conserved extracellular residue among SLC5A5 homologs, was replaced with alanine, aspartic acid, glutamic acid, or lysine. All the SLC5A5 mutants were expressed normally in the cells and targeted correctly to the plasma membrane. However, all of the mutants displayed severe defects in iodide uptake, suggesting that His-226 was critical for iodide uptake. Kinetic analysis further showed that mutation at His-226 led to a dramatic decrease in V(max). These findings suggested that the decreased levels of iodide uptake activity of SLC5A5 mutants resulted from lower catalytic rates. In conclusion, our data first identified the involvement of extracellular charged amino acid residue in the iodide uptake ability of SLC5A5.