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1.
Int J Mol Sci ; 24(13)2023 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-37446010

RESUMEN

The yeast Sup35 protein misfolds into the infectious [PSI+] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [PSI+] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [PSI+] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104.


Asunto(s)
Priones , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Priones/metabolismo , Solubilidad
2.
PLoS Genet ; 15(6): e1008219, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31242183

RESUMEN

Fes1 is a conserved armadillo repeat-containing Hsp70 nucleotide exchange factor important for growth at high temperature, proteasomal protein degradation and prion propagation. Depleting or mutating Fes1 induces a stress response and causes defects in these processes that are ascribed solely to disruption of Fes1 regulation of Hsp70. Here, we find Fes1 was essential for degradation of gluconeogenic enzymes by the vacuole import and degradation (Vid) pathway and for cell wall integrity (CWI), which is crucial for growth at high temperature. Unexpectedly, Fes1 mutants defective in physical or functional interaction with Hsp70 retained activities that support Vid and CWI. Fes1 and the Fes1 mutants bound to the Vid substrate Fbp1 in vitro and captured Slt2, a signaling kinase that regulates CWI, from cell lysates. Our data show that the armadillo domain of Fes1 binds proteins other than Hsp70, that Fes1 has important Hsp70-independent roles in the cell, and that major growth defects caused by depleting Fes1 are due to loss of these functions rather than to loss of Hsp70 regulation. We uncovered diverse functions of Fes1 beyond its defined role in regulating Hsp70, which points to possible multi-functionality among its conserved counterparts in other organisms or organelles.


Asunto(s)
Pared Celular/genética , Factores de Intercambio de Guanina Nucleótido/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas de Saccharomyces cerevisiae/genética , Citosol/metabolismo , Gluconeogénesis/genética , Glucosa/genética , Glucosa/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/genética , Vacuolas/genética
3.
Mol Cell ; 49(3): 464-73, 2013 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-23260660

RESUMEN

The heat shock protein 90 (Hsp90) family of heat shock proteins is an abundantly expressed and highly conserved family of ATP-dependent molecular chaperones. Hsp90 facilitates remodeling and activation of hundreds of proteins. In this study, we developed a screen to identify Hsp90-defective mutants in E. coli. The mutations obtained define a region incorporating residues from the middle and C-terminal domains of E. coli Hsp90. The mutant proteins are defective in chaperone activity and client binding in vitro. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Escherichia coli/citología , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/citología , Relación Estructura-Actividad
4.
Proc Natl Acad Sci U S A ; 115(10): E2210-E2219, 2018 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-29463764

RESUMEN

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that is essential in eukaryotes. It is required for the activation and stabilization of more than 200 client proteins, including many kinases and steroid hormone receptors involved in cell-signaling pathways. Hsp90 chaperone activity requires collaboration with a subset of the many Hsp90 cochaperones, including the Hsp70 chaperone. In higher eukaryotes, the collaboration between Hsp90 and Hsp70 is indirect and involves Hop, a cochaperone that interacts with both Hsp90 and Hsp70. Here we show that yeast Hsp90 (Hsp82) and yeast Hsp70 (Ssa1), directly interact in vitro in the absence of the yeast Hop homolog (Sti1), and identify a region in the middle domain of yeast Hsp90 that is required for the interaction. In vivo results using Hsp90 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Hsp70 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
5.
Proc Natl Acad Sci U S A ; 114(21): E4193-E4202, 2017 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-28484020

RESUMEN

Overproduction or deficiency of many chaperones and other cellular components cure the yeast prions [PSI+] (formed by Sup35p) or [URE3] (based on Ure2p). However, at normal expression levels, Btn2p and Cur1p eliminate most newly arising [URE3] variants but do not cure [PSI+], even after overexpression. Deficiency or overproduction of Hsp104 cures the [PSI+] prion. Hsp104 deficiency curing is a result of failure to cleave the Sup35p amyloid filaments to make new seeds, whereas Hsp104 overproduction curing occurs by a different mechanism. Hsp104(T160M) can propagate [PSI+], but cannot cure it by overproduction, thus separating filament cleavage from curing activities. Here we show that most [PSI+] variants arising spontaneously in an hsp104(T160M) strain are cured by restoration of just normal levels of the WT Hsp104. Both strong and weak [PSI+] variants are among those cured by this process. This normal-level Hsp104 curing is promoted by Sti1p, Hsp90, and Sis1p, proteins previously implicated in the Hsp104 overproduction curing of [PSI+]. The [PSI+] prion arises in hsp104(T160M) cells at more than 10-fold the frequency in WT cells. The curing activity of Hsp104 thus constitutes an antiprion system, culling many variants of the [PSI+] prion at normal Hsp104 levels.


Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/metabolismo , Priones/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
J Biol Chem ; 293(9): 3104-3117, 2018 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-29330300

RESUMEN

The [URE3] yeast prion is the self-propagating amyloid form of the Ure2 protein. [URE3] is cured by overexpression of several yeast proteins, including Ydj1, Btn2, Cur1, Hsp42, and human DnaJB6. To better understand [URE3] curing, we used real-time imaging with a yeast strain expressing a GFP-labeled full-length Ure2 construct to monitor the curing of [URE3] over time. [URE3] yeast cells exhibited numerous fluorescent foci, and expression of the GFP-labeled Ure2 affected neither mitotic stability of [URE3] nor the rate of [URE3] curing by the curing proteins. Using guanidine to cure [URE3] via Hsp104 inactivation, we found that the fluorescent foci are progressively lost as the cells divide until they are cured; the fraction of cells that retained the foci was equivalent to the [URE3] cell fraction measured by a plating assay, indicating that the foci were the prion seeds. During the curing of [URE3] by Btn2, Cur1, Hsp42, or Ydj1 overexpression, the foci formed aggregates, many of which were 0.5 µm or greater in size, and [URE3] was cured by asymmetric segregation of the aggregated seeds. In contrast, DnaJB6 overexpression first caused a loss of detectable foci in cells that were still [URE3] before there was complete dissolution of the seeds, and the cells were cured. We conclude that GFP labeling of full-length Ure2 enables differentiation among the different [URE3]-curing mechanisms, including inhibition of severing followed by seed dilution, seed clumping followed by asymmetric segregation between mother and daughter cells, and seed dissolution.


Asunto(s)
Proteínas Fúngicas/metabolismo , Imagen Molecular , Priones/metabolismo , Levaduras/citología , Factores de Tiempo , Levaduras/metabolismo
7.
J Biol Chem ; 291(8): 4035-47, 2016 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-26702057

RESUMEN

Human chaperone DnaJB6, an Hsp70 co-chaperone whose defects cause myopathies, protects cells from polyglutamine toxicity and prevents purified polyglutamine and Aß peptides from forming amyloid. Yeast prions [URE3] and [PSI(+)] propagate as amyloid forms of Ure2 and Sup35 proteins, respectively. Here we find DnaJB6-protected yeast cells from polyglutamine toxicity and cured yeast of both [URE3] prions and weak variants of [PSI(+)] prions but not strong [PSI(+)] prions. Weak and strong variants of [PSI(+)] differ only in the structural conformation of their amyloid cores. In line with its anti-prion effects, DnaJB6 prevented purified Sup35NM from forming amyloids at 37 °C, which produce predominantly weak [PSI(+)] variants when used to infect yeast, but not at 4 °C, which produces mostly strong [PSI(+)] variants. Thus, structurally distinct amyloids composed of the same protein were differentially sensitive to the anti-amyloid activity of DnaJB6 both in vitro and in vivo. These findings have important implications for strategies using DnaJB6 as a target for therapy in amyloid disorders.


Asunto(s)
Amiloide/metabolismo , Glutatión Peroxidasa/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Amiloide/genética , Glutatión Peroxidasa/genética , Proteínas del Choque Térmico HSP40/genética , Calor , Humanos , Chaperonas Moleculares/genética , Proteínas del Tejido Nervioso/genética , Factores de Terminación de Péptidos/genética , Priones/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
8.
PLoS Genet ; 10(10): e1004720, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25329162

RESUMEN

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.


Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Endopeptidasa Clp , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética
9.
Eukaryot Cell ; 13(5): 635-47, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24632242

RESUMEN

The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.


Asunto(s)
Proteínas de Choque Térmico/genética , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Expresión Génica , Proteínas de Choque Térmico/metabolismo , Factores de Terminación de Péptidos/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Solubilidad
10.
Eukaryot Cell ; 12(5): 739-45, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23504563

RESUMEN

Hsp100 chaperones protect microorganisms and plants from environmental stress by cooperating with Hsp70 and its nucleotide exchange factor (NEF) and Hsp40 cochaperones to resolubilize proteins from aggregates. The Saccharomyces cerevisiae Hsp104 (Sc-Hsp104)-based disaggregation machinery also is essential for replication of amyloid-based prions. Escherichia coli ClpB can substitute for Hsp104 to propagate [PSI(+)] prions in yeast, but only if E. coli DnaK and GrpE (Hsp70 and NEF) are coexpressed. Here, we tested if the reported inability of Schizosaccharomyces pombe Hsp104 (Sp-Hsp104) to support [PSI(+)] propagation was due to similar species-specific chaperone requirements and find that Sp-Hsp104 alone supported propagation of three different yeast prions. Sp-Hsp70 and Sp-Fes1p (NEF) likewise functioned in place of their Sa. cerevisiae counterparts. Thus, chaperones of these long-diverged species possess conserved activities that function in processes essential for both cell growth and prion propagation, suggesting Sc. pombe can propagate its own prions. We show that curing by Hsp104 overexpression and inactivation can be distinguished and confirm the observation that, unlike Sc-Hsp104, Sp-Hsp104 cannot cure yeast of [PSI(+)] when it is overexpressed. These results are consistent with a view that mechanisms underlying prion replication and elimination are distinct.


Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Choque Térmico/genética , Priones/metabolismo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Adenosina Trifosfatasas/biosíntesis , Prueba de Complementación Genética , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
11.
Proc Natl Acad Sci U S A ; 108(33): 13665-70, 2011 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-21808014

RESUMEN

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.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Priones/biosíntesis , Unión Proteica/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Proteínas HSP70 de Choque Térmico/genética , Metilación , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
12.
Proc Natl Acad Sci U S A ; 108(17): 6915-20, 2011 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-21474779

RESUMEN

Yeast Hsp104 and its bacterial homolog, ClpB, are Clp/Hsp100 molecular chaperones and AAA+ ATPases. Hsp104 and ClpB collaborate with the Hsp70 and DnaK chaperone systems, respectively, to retrieve and reactivate stress-denatured proteins from aggregates. The action of Hsp104 and ClpB in promoting cell survival following heat stress is species-specific: Hsp104 cannot function in bacteria and ClpB cannot act in yeast. To determine the regions of Hsp104 and ClpB necessary for this specificity, we tested chimeras of Hsp104 and ClpB in vivo and in vitro. We show that the Hsp104 and ClpB middle domains dictate the species-specificity of Hsp104 and ClpB for cell survival at high temperature. In protein reactivation assays in vitro, chimeras containing the Hsp104 middle domain collaborate with Hsp70 and those with the ClpB middle domain function with DnaK. The region responsible for the specificity is within helix 2 and helix 3 of the middle domain. Additionally, several mutants containing amino acid substitutions in helix 2 of the ClpB middle domain are defective in protein disaggregation in collaboration with DnaK. In a bacterial two-hybrid assay, DnaK interacts with ClpB and with chimeras that have the ClpB middle domain, implying that species-specificity is due to an interaction between DnaK and the middle domain of ClpB. Our results suggest that the interaction between Hsp70/DnaK and helix 2 of the middle domain of Hsp104/ClpB determines the specificity required for protein disaggregation both in vivo and in vitro, as well as for cellular thermotolerance.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas HSP70 de Choque Térmico/química , Proteínas de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae/química , Endopeptidasa Clp , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Nat Commun ; 14(1): 2489, 2023 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-37120429

RESUMEN

Hsp90 is an essential eukaryotic chaperone that regulates the activity of many client proteins. Current models of Hsp90 function, which include many conformational rearrangements, specify a requirement of ATP hydrolysis. Here we confirm earlier findings that the Hsp82-E33A mutant, which binds ATP but does not hydrolyze it, supports viability of S. cerevisiae, although it displays conditional phenotypes. We find binding of ATP to Hsp82-E33A induces the conformational dynamics needed for Hsp90 function. Hsp90 orthologs with the analogous EA mutation from several eukaryotic species, including humans and disease organisms, support viability of both S. cerevisiae and Sz. pombe. We identify second-site suppressors of EA that rescue its conditional defects and allow EA versions of all Hsp90 orthologs tested to support nearly normal growth of both organisms, without restoring ATP hydrolysis. Thus, the requirement of ATP for Hsp90 to maintain viability of evolutionarily distant eukaryotic organisms does not appear to depend on energy from ATP hydrolysis. Our findings support earlier suggestions that exchange of ATP for ADP is critical for Hsp90 function. ATP hydrolysis is not necessary for this exchange but provides an important control point in the cycle responsive to regulation by co-chaperones.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Nucleótidos/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Mol Cell Biol ; 43(4): 157-173, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37099734

RESUMEN

Hsp104 propagates the yeast prion [PSI+], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [PSI+] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation. Investigating this question, we now find, first, that mutating this site prevents both the curing of [PSI+] by Hsp104 overexpression and the trimming activity of Hsp104. Second, we find that depending on the specific member of the Hsp70 family binding to the N-terminal domain of Hsp104, both trimming and the curing caused by Hsp104 overexpression are either increased or decreased in parallel. Therefore, the binding of Hsp70 to the N-terminal domain of Hsp104 regulates both the rate of [PSI+] trimming by Hsp104 and the rate of [PSI+] curing by Hsp104 overexpression.


Asunto(s)
Priones , Proteínas de Saccharomyces cerevisiae , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Priones/genética , Priones/metabolismo , Factores de Terminación de Péptidos/química
15.
Biomolecules ; 13(9)2023 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-37759770

RESUMEN

The Sup35 prion protein of budding yeast has been reported to undergo phase separation to form liquid droplets both at low pH in vitro and when energy depletion decreases the intracellular pH in vivo. It also has been shown using purified proteins that this phase separation is driven by the prion domain of Sup35 and does not re-quire its C-terminal domain. In contrast, we now find that a Sup35 fragment consisting of only the N-terminal prion domain and the M-domain does not phase separate in vivo; this phase separation of Sup35 requires the C-terminal domain, which binds Sup45 to form the translation termination complex. The phase-separated Sup35 not only colocalizes with Sup45 but also with Pub1, a stress granule marker protein. In addition, like stress granules, phase separation of Sup35 appears to require mRNA since cycloheximide treatment, which inhibits mRNA release from ribosomes, prevents phase separation of Sup35. Finally, unlike Sup35 in vitro, Sup35 condensates do not disassemble in vivo when the intracellular pH is increased. These results suggest that, in energy-depleted cells, Sup35 forms supramolecular assemblies that differ from the Sup35 liquid droplets that form in vitro.

16.
Biology (Basel) ; 11(9)2022 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-36138771

RESUMEN

The accumulation of misfolded proteins as amyloids is associated with pathology in dozens of debilitating human disorders, including diabetes, Alzheimer's, Parkinson's, and Huntington's diseases. Expressing human amyloid-forming proteins in yeast is toxic, and yeast prions that propagate as infectious amyloid forms of cellular proteins are also harmful. The yeast system, which has been useful for studying amyloids and their toxic effects, has provided much insight into how amyloids affect cells and how cells respond to them. Given that an amyloid is a protein folding problem, it is unsurprising that the factors found to counteract the propagation or toxicity of amyloids in yeast involve protein quality control. Here, we discuss such factors with an emphasis on J-domain proteins (JDPs), which are the most highly abundant and diverse regulators of Hsp70 chaperones. The anti-amyloid effects of JDPs can be direct or require interaction with Hsp70.

17.
Biology (Basel) ; 11(12)2022 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-36552355

RESUMEN

Human J-domain protein (JDP) DnaJB6 has a broad and potent activity that prevents formation of amyloid by polypeptides such as polyglutamine, A-beta, and alpha-synuclein, related to Huntington's, Alzheimer's, and Parkinson's diseases, respectively. In yeast, amyloid-based [PSI+] prions, which rely on the related JDP Sis1 for replication, have a latent toxicity that is exposed by reducing Sis1 function. Anti-amyloid activity of DnaJB6 is very effective against weak [PSI+] prions and the Sup35 amyloid that composes them, but ineffective against strong [PSI+] prions composed of structurally different amyloid of the same Sup35. This difference reveals limitations of DnaJB6 that have implications regarding its therapeutic use for amyloid disease. Here, we find that when Sis1 function is reduced, DnaJB6 represses toxicity of strong [PSI+] prions and inhibits their propagation. Both Sis1 and DnaJB6, which are regulators of protein chaperone Hsp70, counteract the toxicity by reducing excessive incorporation of the essential Sup35 into prion aggregates. However, while Sis1 apparently requires interaction with Hsp70 to detoxify [PSI+], DnaJB6 counteracts prion toxicity by a different, Hsp70-independent mechanism.

18.
Proteins ; 79(1): 282-93, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21064130

RESUMEN

Interleukin-13 is a Th2-associated cytokine responsible for many pathological responses in allergic asthma including mucus production, inflammation, and extracellular matrix remodeling. In addition, IL-13 is required for immunity to many helminth infections. IL-13 signals via the type-II IL-4 receptor, a heterodimeric receptor of IL-13Rα1 and IL-4Rα, which is also used by IL-4. IL-13 also binds to IL-13Rα2, but with much higher affinity than the type-II IL-4 receptor. Binding of IL-13 to IL-13Rα2 has been shown to attenuate IL-13 signaling through the type-II IL-4 receptor. However, molecular determinants that dictate the specificity and affinity of mouse IL-13 for the different receptors are largely unknown. Here, we used high-density overlapping peptide arrays, structural modeling, and molecular docking methods to map IL-13 binding sequences on its receptors. Predicted binding sequences on mouse IL-13Rα1 and IL-13Rα2 were in agreement with the reported human IL-13 receptor complex structures and site-directed mutational analysis. Novel structural differences were identified between IL-13 receptors, particularly at the IL-13 binding interface. Notably, additional binding sites were observed for IL-13 on IL-13Rα2. In addition, the identification of peptide sequences that are unique to IL-13Rα1 allowed us to generate a monoclonal antibody that selectively binds IL-13Rα1. Thus, high-density peptide arrays combined with molecular docking studies provide a novel, rapid, and reliable method to map cytokine-receptor interactions that may be used to generate signaling and decoy receptor-specific antagonists.


Asunto(s)
Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Interleucina-13/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Humanos , Subunidad alfa1 del Receptor de Interleucina-13/química , Subunidad alfa2 del Receptor de Interleucina-13/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Análisis por Matrices de Proteínas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Homología Estructural de Proteína
19.
Biochem Biophys Res Commun ; 406(2): 177-82, 2011 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-21295540

RESUMEN

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% ß-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K(A)) between yeast AZ and yeast ODC is 6×10(7) (M(-1)). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.


Asunto(s)
Inhibidores de la Ornitina Descarboxilasa , Ornitina Descarboxilasa/química , Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Dicroismo Circular , Escherichia coli/genética , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/aislamiento & purificación , Estructura Secundaria de Proteína , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación
20.
Genetics ; 219(2)2021 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-34849884

RESUMEN

[PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI+] prions.


Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Factores de Terminación de Péptidos/genética , Dominios Proteicos , Proteostasis , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
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