RESUMO
The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90's function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs.
Assuntos
Trifosfato de Adenosina/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Estrutura Terciária de Proteína , SumoilaçãoRESUMO
The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998-1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co-chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90-mediated folding of kinase and non-kinase clients-including Tsc2-thereby preventing their ubiquitination and proteasomal degradation.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Humanos , Fosforilação , Fosfotransferases/metabolismo , Complexo de Endopeptidases do Proteassoma , Dobramento de Proteína , Proteólise , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , UbiquitinaçãoRESUMO
Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in vitro and in vivo. This α helix participates in a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Treonina/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/química , Chaperoninas/genética , Chaperoninas/metabolismo , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Chaperonas Moleculares/genética , Fosforilação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Saccharomyces WEE1 (Swe1), the only "true" tyrosine kinase in budding yeast, is an Hsp90 client protein. Here we show that Swe1(Wee1) phosphorylates a conserved tyrosine residue (Y24 in yeast Hsp90 and Y38 in human Hsp90alpha) in the N domain of Hsp90. Phosphorylation is cell-cycle associated and modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases. Nonphosphorylatable mutants have normal ATPase activity, support yeast viability, and productively chaperone the Hsp90 client glucocorticoid receptor. Deletion of SWE1 in yeast increases Hsp90 binding to its inhibitor geldanamycin, and pharmacologic inhibition/silencing of Wee1 sensitizes cancer cells to Hsp90 inhibitor-induced apoptosis. These findings demonstrate that Hsp90 chaperoning of distinct client proteins is differentially regulated by specific posttranslational modification of a unique subcellular pool of the chaperone, and they provide a strategy to increase the cellular potency of Hsp90 inhibitors.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Tirosina/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Dimerização , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Interferência de RNA , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaçãoRESUMO
Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Quinases/metabolismo , Especificidade de Anticorpos , Quinase 4 Dependente de Ciclina/metabolismo , Ativação Enzimática , Células HCT116 , Humanos , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Saccharomyces cerevisiae , Especificidade por SubstratoRESUMO
Hsp90 is an essential and highly conserved modular molecular chaperone whose N and middle domains are separated by a disordered region termed the charged linker. Although its importance has been previously disregarded, because a minimal linker length is sufficient for Hsp90 activity, the evolutionary persistence of extensive charged linkers of divergent sequence in Hsp90 proteins of most eukaryotes remains unexplained. To examine this question further, we introduced human and plasmodium native and length-matched artificial linkers into yeast Hsp90. After evaluating ATPase activity and biophysical characteristics in vitro, and chaperone function in vivo, we conclude that linker sequence affects Hsp90 function, cochaperone interaction, and conformation. We propose that the charged linker, in addition to providing the flexibility necessary for Hsp90 domain rearrangements--likely its original purpose--has evolved in eukaryotes to serve as a rheostat for the Hsp90 chaperone machine.
Assuntos
Células Eucarióticas/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Deutério/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Solventes , Relação Estrutura-AtividadeRESUMO
Cellular homeostasis relies on both the chaperoning of proteins and the intracellular degradation system that delivers cytoplasmic constituents to the lysosome, a process known as autophagy. The crosstalk between these processes and their underlying regulatory mechanisms is poorly understood. Here, we show that the molecular chaperone heat shock protein 90 (Hsp90) forms a complex with the autophagy-initiating kinase Atg1 (yeast)/Ulk1 (mammalian), which suppresses its kinase activity. Conversely, environmental cues lead to Atg1/Ulk1-mediated phosphorylation of a conserved serine in the amino domain of Hsp90, inhibiting its ATPase activity and altering the chaperone dynamics. These events impact a conformotypic peptide adjacent to the activation and catalytic loop of Atg1/Ulk1. Finally, Atg1/Ulk1-mediated phosphorylation of Hsp90 leads to dissociation of the Hsp90:Atg1/Ulk1 complex and activation of Atg1/Ulk1, which is essential for initiation of autophagy. Our work indicates a reciprocal regulatory mechanism between the chaperone Hsp90 and the autophagy kinase Atg1/Ulk1 and consequent maintenance of cellular proteostasis.
Assuntos
Autofagia , Proteínas de Choque Térmico HSP90 , Animais , Fosforilação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Mamíferos/metabolismoRESUMO
12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used.
Assuntos
Anti-Infecciosos , Artroplastia de Quadril , Staphylococcus aureus Resistente à Meticilina , Mariposas , Animais , Feminino , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , DNARESUMO
Sgt1 is an adaptor protein implicated in a variety of processes, including formation of the kinetochore complex in yeast, and regulation of innate immunity systems in plants and animals. Sgt1 has been found to associate with SCF E3 ubiquitin ligases, the CBF3 kinetochore complex, plant R proteins and related animal Nod-like receptors, and with the Hsp90 molecular chaperone. We have determined the crystal structure of the core Hsp90-Sgt1 complex, revealing a distinct site of interaction on the Hsp90 N-terminal domain. Using the structure, we developed mutations in Sgt1 interfacial residues, which specifically abrogate interaction with Hsp90, and disrupt Sgt1-dependent functions in vivo, in plants and yeast. We show that Sgt1 bridges the Hsp90 molecular chaperone system to the substrate-specific arm of SCF ubiquitin ligase complexes, suggesting a role in SCF assembly and regulation, and providing multiple complementary routes for ubiquitination of Hsp90 client proteins.
Assuntos
Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Modelos Biológicos , Chaperonas Moleculares/química , Conformação Molecular , Plasmídeos/metabolismo , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie , Nicotiana/genética , Ubiquitina/química , Ubiquitina-Proteína Ligases/químicaRESUMO
Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.
Assuntos
Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Nucleotídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Proteínas de Choque Térmico HSP90/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Nucleotídeos/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Elucidating the mechanism of action of an antifungal or cytotoxic compound is a time-consuming process. Yeast chemogenomic profiling provides a compelling solution to the problem but is experimentally complex. Here, we demonstrate the use of a highly simplified yeast chemical genetic assay comprising just 89 yeast deletion strains, each diagnostic for a specific mechanism of action. We use the assay to investigate the mechanism of action of two antifungal chalcone compounds, trans-chalcone and 4'-hydroxychalcone, and narrow down the mechanism to transcriptional stress. Crucially, the assay eliminates mechanisms of action such as topoisomerase I inhibition and membrane disruption that have been suggested for related chalcone compounds. We propose this simplified assay as a useful tool to rapidly identify common off-target mechanisms.
Assuntos
Chalcona , Chalconas , Chalcona/farmacologia , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Chalconas/farmacologiaRESUMO
Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8-/- mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.
Assuntos
Proteínas ADAM/genética , Antígenos CD/genética , Regulação da Expressão Gênica , Proteínas de Membrana/genética , RNA/genética , Síndrome do Desconforto Respiratório/genética , Proteínas ADAM/biossíntese , Animais , Antígenos CD/biossíntese , Células Cultivadas , Modelos Animais de Doenças , Humanos , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologiaRESUMO
There are six essential genes in the Saccharomyces cerevisiae genome which encode proteins bearing the tetratricopeptide repeat (TPR) domain that mediates protein-protein interaction. Thus far, the function of one of them, YNL313c, remains unknown. Our conditional mutants of YNL313c display osmoremedial temperature sensitivity, hypersensitivity to both Calcofluor White and low concentrations of SDS, and osmoremedial caffeine sensitivity. These are hallmarks of mutants that display cell wall defects. Accordingly we rename the gene as EMW1 (essential for maintenance of the cell wall). Loss of Emw1p function is not associated with abrogation of the cell wall integrity (CWI) MAP kinase cascade. Instead, emw1(ts) mutants activate this cascade even at permissive temperature, indicating that loss of Emw1p function does not cause a defect in sensors and effectors of cell wall signalling, but leads to a cell wall defect directly. Constitutive activation of the CWI cascade is reflected by the overproduction of chitin by emw1(ts) mutants, a compensatory response frequently displayed by cell wall mutants. Growth is restored to emw1(ts) mutants incubated at otherwise non-permissive temperature when GFA1 is overexpressed. GFA1 encodes the hexosephosphate aminotransferase that catalyses the rate-limiting step in the pathway that synthesizes the chitin precursor UDP-GlcNAc. The possibility that Emw1p is required for function of Gfa1p was ruled out, because the emw1(ts) phenotype persists when the requirement for Gfa1p is bypassed. Furthermore, if loss of Emw1p function leads to loss of function of Gfa1p, then chitin synthesis would be diminished. Instead, a stimulation of the synthesis of this polymer is detected. Consequently, the defect associated with emw1(ts) mutants may be associated with compromise in one of the remaining processes that depend on UDP-GlcNAc, namely N-glycosylation or glycosylphosphatidylinositol (GPI)-anchor synthesis.
Assuntos
Parede Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Antifúngicos/toxicidade , Benzenossulfonatos/toxicidade , Cafeína/toxicidade , Parede Celular/metabolismo , Quitina/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Dodecilsulfato de Sódio/toxicidade , TemperaturaRESUMO
OBJECTIVES: The substrate specificity of wild-type human phenylalanine monooxygenase (wt-hPAH) has been investigated with respect to the mucoactive drug, S-carboxymethyl-L-cysteine and its thioether metabolites. The ability of wt-hPAH to metabolise other S-substituted cysteines was also examined. METHODS: Direct assays of PAH activity were by HPLC with fluorescence detection; indirect assays involved following disappearance of the cofactor by UV spectroscopy. KEY FINDINGS: wt-hPAH catalysed the S-oxygenation of S-carboxymethyl-L-cysteine, its decarboxylated metabolite, S-methyl-L-cysteine, and both their corresponding N-acetylated forms. However, thiodiglycolic acid was not a substrate. The enzyme profiles for both phenylalanine and S-carboxymethyl-L-cysteine showed allosteric kinetics at low substrate concentrations, with Hill constants of 2.0 and 1.9, respectively, for the substrate-activated wt-hPAH. At higher concentrations, both compounds followed Michaelis-Menten kinetics, with non-competitive substrate inhibition profiles. The thioether compounds, S-ethyl-L-cysteine, S-propyl-L-cysteine and S-butyl-L-cysteine were all found to be substrates for phenylalanine monooxygenase. CONCLUSIONS: Phenylalanine monooxygenase may play a wider role outside intermediary metabolism in the biotransformation of dietary-derived substituted cysteines and other exogenous thioether compounds.
Assuntos
Carbocisteína/metabolismo , Fenilalanina Hidroxilase/metabolismo , Sulfetos/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cisteína/análogos & derivados , Cisteína/metabolismo , Ativação Enzimática , Fluorescência , Humanos , Cinética , Lisofosfatidilcolinas/metabolismo , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , Especificidade por Substrato , Sulfetos/química , Xenobióticos/química , Xenobióticos/metabolismoRESUMO
Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including remodelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 interacts physically with the plasma-membrane-associated phosphoinositide 4-kinase, Stt4. In the present study, we demonstrate that phenotypes of ypp1 and stt4 conditional mutants are identical, namely osmoremedial temperature sensitivity, hypersensitivity to cell wall destabilizers and defective organization of actin. We go on to show that overexpression of STT4 suppresses the temperature-sensitive growth defect of ypp1 mutants. In contrast, overexpression of genes encoding the other two phosphoinositide 4-kinases in yeast, Pik1 and Lsb6, do not suppress this phenotype. This implies a role for Ypp1 in Stt4-dependent events at the plasma membrane, as opposed to a general role in overall metabolism of phosphatidylinositol 4-phosphate. Use of a pleckstrin homology domain sensor reveals that there are substantially fewer plasma-membrane-associated 4-phosphorylated phosphoinositides in ypp1 mutants in comparison with wild-type cells. Furthermore, in vivo labelling with [(3)H]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in ypp1 mutants. This is the principal cause of lethality under non-permissive conditions in ypp1 mutants, as limiting the activity of the Sac1 phosphoinositide 4-phosphate phosphatase leads to restoration of viability. Additionally, the endocytic defect associated with elevated levels of PtdIns4P in sac1Delta cells is restored in combination with a ypp1 mutant, consistent with the opposing effects that these two mutations have on levels of this phosphoinositide.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Alelos , Endocitose , Sistema de Sinalização das MAP Quinases , Mutação , Fenótipo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.
Assuntos
Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Calorimetria , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Humanos , Cinética , Ligantes , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , UltracentrifugaçãoRESUMO
Heat shock protein 90 (Hsp90) is a molecular chaperone required for the activity of many of the most important regulatory proteins of eukaryotic cells (the Hsp90 'clients'). Vertebrates have two isoforms of cytosolic Hsp90, Hsp90alpha and Hsp90beta. Hsp90beta is expressed constitutively to a high level in most tissues and is generally more abundant than Hsp90alpha, whereas Hsp90alpha is stress-inducible and overexpressed in many cancerous cells. Expressed as the sole Hsp90 of yeast, human Hsp90alpha and Hsp90beta are both able to provide essential Hsp90 functions. Activations of certain Hsp90 clients (heat shock transcription factor, v-src) were more efficient with Hsp90alpha, rather than Hsp90beta, present in the yeast. In contrast, activation of certain other clients (glucocorticoid receptor; extracellular signal-regulated kinase-5 mitogen-activated protein kinase) was less affected by the human Hsp90 isoform present in these cells. Remarkably, whereas expression of Hsp90beta as the sole Hsp90 of yeast rendered cells highly sensitive to the Hsp90 inhibitor radicicol, comparable expression of Hsp90alpha did not. This raises the distinct possibility that, also for mammalian systems, alterations to the Hsp90alpha/Hsp90beta ratio (as with heat shock) might be a significant factor affecting cellular susceptibility to Hsp90 inhibitors.
Assuntos
Proteínas de Choque Térmico HSP90/fisiologia , Macrolídeos/farmacologia , Isoformas de Proteínas/fisiologia , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Western Blotting , Primers do DNA , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Garlic contains the organosulfur compound allicin which exhibits potent antifungal activity. Here we demonstrate the use of a highly simplified yeast chemical genetic screen to characterize its mode of action. By screening 24 validated yeast gene deletion "signature" strains for which hypersensitivity is characteristic for common antifungal modes of action, yeast lacking the high affinity Cu2+ transporter Ctr1 was found to be hypersensitive to allicin. Focusing on transition metal related genes identified two more hypersensitive strains lacking the Cu2+ and Zn2+ transcription factors Mac1 and Zap1. Hypersensitivity in these strains was reversed by the addition of Cu2+ and Zn2+ ions, respectively. The results suggest the antifungal activity of allicin is mediated through restricted Cu2+ and Zn2+ uptake or inhibition of Cu2+ and Zn2+ metalloproteins. As certain antimicrobial modes of action are much more common than others, the approach taken here provides a useful way to identify them early on.
Assuntos
Antifúngicos/farmacologia , Cobre/metabolismo , Alho/química , Ensaios de Triagem em Larga Escala/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Zinco/metabolismo , Dissulfetos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The plasma membrane is dynamic, with both its lipid and protein composition changing to facilitate adaptation to the ambient conditions. Biochemical activities to pre-existing proteins will also change. To monitor these variations, the cell membrane must be isolated. Moreover, the preparations must be free of contamination from the variety of other membranes in the cell, principally those associated with the golgi, endoplasmic reticulum (ER), the nucleus, and the vacuole. We describe a method for isolating plasma membranes that avoids incubation with enzymes that degrade the cell wall, thereby avoiding physiological changes that may lead to alteration in profile and activity of membrane proteins as well as avoiding changes that may alter lipid composition. We have used this method to show that, in response to heat shock, the plasma membrane acquires a novel heat-shock protein (HSP) and displays a decline in the levels of the abundant H+ translocating ATPase.