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1.
J Biol Chem ; 300(8): 107541, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38992438

RESUMO

The amyloid precursor protein (APP) is a key protein in Alzheimer's disease synthesized in the endoplasmic reticulum (ER) and translocated to the plasma membrane where it undergoes proteolytic cleavages by several proteases. Conversely, to other known proteases, we previously elucidated rhomboid protease RHBDL4 as a novel APP processing enzyme where several cleavages likely occur already in the ER. Interestingly, the pattern of RHBDL4-derived large APP C-terminal fragments resembles those generated by the η-secretase or MT5-MMP, which was described to generate so-called Aη fragments. The similarity in large APP C-terminal fragments between both proteases raised the question of whether RHBDL4 may contribute to η-secretase activity and Aη-like fragments. Here, we identified two cleavage sites of RHBDL4 in APP by mass spectrometry, which, intriguingly, lie in close proximity to the MT5-MMP cleavage sites. Indeed, we observed that RHBDL4 generates Aη-like fragments in vitro without contributions of α-, ß-, or γ-secretases. Such Aη-like fragments are likely generated in the ER since RHBDL4-derived APP-C-terminal fragments do not reach the cell surface. Inherited, familial APP mutations appear to not affect this processing pathway. In RHBDL4 knockout mice, we observed increased cerebral full-length APP in comparison to wild type (WT) in support of RHBDL4 being a physiologically relevant protease for APP. Furthermore, we found secreted Aη fragments in dissociated mixed cortical cultures from WT mice, however significantly fewer Aη fragments in RHBDL4 knockout cultures. Our data underscores that RHBDL4 contributes to the η-secretease-like processing of APP and that RHBDL4 is a physiologically relevant protease for APP.


Assuntos
Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide , Animais , Humanos , Camundongos , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Knockout , Proteólise
2.
Biochemistry ; 62(6): 1209-1218, 2023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36857408

RESUMO

The physiological functions of the rhomboid-related protein 4 (RHBDL4) are emerging, but their molecular details remain unclear. Because increased expression of RHBDL4 has been clinically linked to poorer outcomes in cancer patients, this association urgently demands a better understanding of RHBDL4. To elucidate the molecular interactions and pathways that RHBDL4 may be involved in, we conducted proximity-dependent biotin identification (BioID) assays. Our analyses corroborated several of the expected protein interactors such as the transitional endoplasmic reticulum (ER) ATPase VCP/p97 (TERA), but they also described novel putative interactors including IRS4, PGAM5, and GORS2. Using proximity-ligation assays, we validated VCP/p97, COPB, and VRK2 as proteins that are in proximity to RHBDL4. Overall, our results support the emerging functions of RHBDL4 in ER quality control and also point toward putative RHBDL4 functions in protein membrane insertion and membrane organization and trafficking.


Assuntos
Proteínas de Membrana , Peptídeo Hidrolases , Humanos , Endopeptidases , Proteínas de Membrana/metabolismo
3.
Chembiochem ; 17(9): 843-51, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-26792008

RESUMO

The unfolded protein response (UPR) initiated by the transmembrane kinase/ribonuclease Ire1 has been implicated in a variety of diseases. Ire1, with its unique position in the UPR, is an ideal target for the development of therapies; however, the identification of specific kinase inhibitors is challenging. Recently, the development of covalent inhibitors has gained great momentum because of the irreversible deactivation of the target. We identified and determined the mechanism of action of the Ire1-inhibitory compound UPRM8. MS analysis revealed that UPRM8 inhibition occurs by covalent adduct formation at a conserved cysteine at the regulatory DFG+2 position in the Ire1 kinase activation loop. Mutational analysis of the target cysteine residue identified both UPRM8-resistant and catalytically inactive Ire1 mutants. We describe a novel covalent inhibition mechanism of UPRM8, which can serve as a lead for the rational design and optimization of inhibitors of human Ire1.


Assuntos
Cisteína/metabolismo , Endorribonucleases/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Pirimidinonas/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinonas/química , Pirimidinonas/farmacologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Resposta a Proteínas não Dobradas/efeitos dos fármacos
4.
Mol Cell Proteomics ; 11(9): 710-23, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22665516

RESUMO

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.


Assuntos
Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Mapas de Interação de Proteínas , Animais , Ciclofilinas/metabolismo , Células Epiteliais , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imunoglobulina G/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Peptidilprolil Isomerase/metabolismo , Ratos
5.
PLoS Pathog ; 6(2): e1000753, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-20140196

RESUMO

Candida albicans, the major fungal pathogen of humans, causes life-threatening infections in immunocompromised individuals. Due to limited available therapy options, this can frequently lead to therapy failure and emergence of drug resistance. To improve current treatment strategies, we have combined comprehensive chemical-genomic screening in Saccharomyces cerevisiae and validation in C. albicans with the goal of identifying compounds that can couple with the fungistatic drug fluconazole to make it fungicidal. Among the genes identified in the yeast screen, we found that only AGE3, which codes for an ADP-ribosylation factor GTPase activating effector protein, abrogates fluconazole tolerance in C. albicans. The age3 mutant was more sensitive to other sterols and cell wall inhibitors, including caspofungin. The deletion of AGE3 in drug resistant clinical isolates and in constitutively active calcineurin signaling mutants restored fluconazole sensitivity. We confirmed chemically the AGE3-dependent drug sensitivity by showing a potent fungicidal synergy between fluconazole and brefeldin A (an inhibitor of the guanine nucleotide exchange factor for ADP ribosylation factors) in wild type C. albicans as well as in drug resistant clinical isolates. Addition of calcineurin inhibitors to the fluconazole/brefeldin A combination only initially improved pathogen killing. Brefeldin A synergized with different drugs in non-albicans Candida species as well as Aspergillus fumigatus. Microarray studies showed that core transcriptional responses to two different drug classes are not significantly altered in age3 mutants. The therapeutic potential of inhibiting ARF activities was demonstrated by in vivo studies that showed age3 mutants are avirulent in wild type mice, attenuated in virulence in immunocompromised mice and that fluconazole treatment was significantly more efficacious when ARF signaling was genetically compromised. This work describes a new, widely conserved, broad-spectrum mechanism involved in fungal drug resistance and virulence and offers a potential route for single or improved combination therapies.


Assuntos
Fatores de Ribosilação do ADP/genética , Antifúngicos/farmacologia , Candida albicans/patogenicidade , Farmacorresistência Fúngica/genética , Virulência/genética , Fatores de Ribosilação do ADP/efeitos dos fármacos , Fatores de Ribosilação do ADP/metabolismo , Animais , Brefeldina A/farmacologia , Candida albicans/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Fluconazol/farmacologia , Expressão Gênica/efeitos dos fármacos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas do Sistema de Duplo-Híbrido , Virulência/efeitos dos fármacos
6.
Mol Syst Biol ; 5: 338, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20029371

RESUMO

Chemotherapies, HIV infections, and treatments to block organ transplant rejection are creating a population of immunocompromised individuals at serious risk of systemic fungal infections. Since single-agent therapies are susceptible to failure due to either inherent or acquired resistance, alternative therapeutic approaches such as multi-agent therapies are needed. We have developed a bioinformatics-driven approach that efficiently predicts compound synergy for such combinatorial therapies. The approach uses chemogenomic profiles in order to identify compound profiles that have a statistically significant degree of similarity to a fluconazole profile. The compounds identified were then experimentally verified to be synergistic with fluconazole and with each other, in both Saccharomyces cerevisiae and the fungal pathogen Candida albicans. Our method is therefore capable of accurately predicting compound synergy to aid the development of combinatorial antifungal therapies.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Biologia Computacional , Desenho Assistido por Computador , Desenho de Fármacos , Fluconazol/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Animais , Antifúngicos/química , Antifúngicos/uso terapêutico , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Fluconazol/química , Fluconazol/uso terapêutico , Regulação Fúngica da Expressão Gênica , Humanos , Modelos Moleculares , Estrutura Molecular , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Relação Estrutura-Atividade
7.
Expert Opin Ther Targets ; 23(8): 711-724, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31169041

RESUMO

Introduction: Cystic fibrosis (CF) is the most frequent lethal orphan disease and is caused by mutations in the CFTR gene. The most frequent mutation F508del-CFTR affects multiple organs; infections and subsequent infections and complications in the lung lead to death. Areas covered: This review focuses on new targets and mechanisms that are attracting interest for the development of CF therapies. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) but has some function if it can traffic to the plasma membrane. Cell-based assays have been used to screen chemical libraries for small molecule correctors that restore its trafficking. Pharmacological chaperones are correctors that bind directly to the F508del-CFTR mutant and promote its folding and trafficking. Other correctors fall into a heterogeneous class of proteostasis modulators that act indirectly by altering cellular homeostasis. Expert opinion: Pharmacological chaperones have so far been the most successful correctors of F508del-CFTR trafficking, but their level of correction means that more than one corrector is required. Proteostasis modulators have low levels of correction but hold promise because some can correct several different CFTR mutations. Identification of their cellular targets and the potential for development may lead to new therapies for CF.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Terapia de Alvo Molecular , Animais , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Desenvolvimento de Medicamentos , Retículo Endoplasmático/metabolismo , Humanos , Mutação , Dobramento de Proteína
8.
EMBO Mol Med ; 11(6)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31040128

RESUMO

Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.


Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogênicas/metabolismo , Multimerização Proteica , Proteostase , Animais , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Masculino , Camundongos , Mucoproteínas/genética , Proteínas Oncogênicas/genética
9.
Mol Biol Cell ; 15(12): 5492-502, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15483057

RESUMO

We have determined the transcriptional response of the budding yeast Saccharomyces cerevisiae to cold. Yeast cells were exposed to 10 degrees C for different lengths of time, and DNA microarrays were used to characterize the changes in transcript abundance. Two distinct groups of transcriptionally modulated genes were identified and defined as the early cold response and the late cold response. A detailed comparison of the cold response with various environmental stress responses revealed a substantial overlap between environmental stress response genes and late cold response genes. In addition, the accumulation of the carbohydrate reserves trehalose and glycogen is induced during late cold response. These observations suggest that the environmental stress response (ESR) occurs during the late cold response. The transcriptional activators Msn2p and Msn4p are involved in the induction of genes common to many stress responses, and we show that they mediate the stress response pattern observed during the late cold response. In contrast, classical markers of the ESR were absent during the early cold response, and the transcriptional response of the early cold response genes was Msn2p/Msn4p independent. This implies that the cold-specific early response is mediated by a different and as yet uncharacterized regulatory mechanism.


Assuntos
Adaptação Fisiológica/genética , Temperatura Baixa , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Metabolismo dos Carboidratos , Análise por Conglomerados , Genes Fúngicos/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , Fatores de Tempo , Transcrição Gênica/genética
10.
Biochim Biophys Acta ; 1723(1-3): 265-9, 2005 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-15780982

RESUMO

A new strategy for the rapid creation of DNA deletion libraries using a simple PCR-based method is presented. Unidirectional deletion fragments are created and may be cloned into any vector system without the constraint of using restriction enzymes. Our strategy combines methodologies from DNA sequencing, PCR, and homologous recombination (either in vivo or in vitro) to allow for the creation of a library containing fragments representing all possible deletions of a given cDNA. Using this strategy we have successfully constructed a deletion library of the cDNA encoding for the lumenal domain of yeast Ire1p, and have shown that resulting fragments range from 100 bp to the full length cDNA (1557 bp). This method is simple, inexpensive, and can easily be adapted for automated high-throughput research.


Assuntos
Biblioteca Gênica , Reação em Cadeia da Polimerase/métodos , Deleção de Genes , Recombinação Genética
11.
Methods Mol Biol ; 1356: 31-41, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26519063

RESUMO

Many biological processes are regulated by protein-protein interactions, and the analysis of these interactions has been a productive endeavor contributing to our understanding of cellular organization and function. The yeast two-hybrid technique is a widely used, powerful method of analyzing protein-protein interactions. The currently used formats, however, have inherent limitations, providing an opportunity to develop new alternatives that extend our ability to detect protein-protein interactions of biological relevance. Here we present a two-hybrid system named SRYTH (Ste11p/Ste50p related yeast two-hybrid) based on the Ste11p/Ste50p interaction that uses the activation of the HOG pathway of Saccharomyces cerevisiae as a reporter for interactions. The system is suitable for detecting cytoplasmic protein interactions in their natural subcellular environment, and has been successfully used to investigate protein-protein interactions, including transcription factor associations, in Candida albicans.


Assuntos
Técnicas do Sistema de Duplo-Híbrido , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae
12.
Gene ; 344: 43-51, 2005 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-15656971

RESUMO

We have developed a set of vectors that have enhanced capabilities for efficiently constructing and expressing differentially tagged fusion proteins using Drag&Drop cloning in the yeast Saccharomyces cerevisiae. The pGREG vectors are based on the pRS series with an additional general kanR selection marker. In vivo homologous recombination is used to introduce genes of interest into galactose-inducible expression vectors (pGREGs), permitting the formation of amino-terminal fusions. The vectors all contain common regions for recombination that flank the stuffer fragment. Introduction of common recombination sequences at the end of PCR fragments will permit the cloning of genes without the need for specific restriction sites. In this process, the selectable stuffer HIS3 gene is replaced by successful gene integration, and a screen for loss of the selection marker identifies potential recombinants. Due to the modular structure of the vectors, genes introduced into one vector can be readily transferred by in vivo recombination to all other members of the vector system, thus permitting rapid and easy Drag&Drop construction of a series of tagged proteins. The pGREG series combines features for expression, tagging, integration, localization and library construction with the advantage of obtaining immediate results from sub-sequent experiments. This Drag&Drop system also allows efficient cloning and expression of heterologous genes in large-scale experiments.


Assuntos
Clonagem Molecular/métodos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação Fúngica da Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
13.
Hum Nat ; 15(2): 147-67, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26190411

RESUMO

This study explores the evolutionary-based hypothesis that facial attractiveness (a guiding force in mate selection) is a cue for physical fitness (presumably an important contributor to mate value in ancestral times). Since fluctuating asymmetry, a measure of developmental stability, is known to be a valid cue for fitness in several biological domains, we scrutinized facial asymmetry as a potential mediator between attractiveness and fitness. In our sample of young women, facial beauty indeed indicated physical fitness. The relationships that pertained to asymmetry were in the expected direction. However, a closer analysis revealed that facial asymmetry did not mediate the relationship between fitness and attractiveness. Unexpected problems regarding the measurement of facial asymmetry are discussed.

15.
Mol Biol Cell ; 20(24): 5117-26, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19846660

RESUMO

Activation of the high-osmolarity glycerol (HOG) pathway for osmoregulation in the yeast Saccharomyces cerevisiae involves interaction of the adaptor Ste50p with the cytoplasmic tail of single-transmembrane protein Opy2p. We have determined the solution structure of the Ste50p-RA (Ras association) domain, and it shows an atypical RA fold lacking the beta1 and beta2 strands of the canonical motif. Although the core of the RA domain is fully functional in the pheromone response, an additional region is required for the HOG pathway activation. Two peptide motifs within the intrinsically disordered cytoplasmic tail of Opy2p defined by NMR spectroscopy physically interact with the Step50p-RA domain. These Opy2p-derived peptides bind overlapping regions of the Step50p-RA domain with similarly weak affinities, suggesting a multivalent interaction of these proteins as a crucial point of control of the HOG pathway. As well, overall selection of signaling pathways depends on functionally distinct regions of the Ste50p-RA domain, implicating this element in the control of global regulatory decisions.


Assuntos
Glicerol/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Citoplasma/química , Dados de Sequência Molecular , Concentração Osmolar , Osmose , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Transdução de Sinais , Estresse Fisiológico , Relação Estrutura-Atividade
16.
Science ; 321(5888): 569-72, 2008 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-18653895

RESUMO

Membrane and secretory proteins cotranslationally enter and are folded in the endoplasmic reticulum (ER). Misfolded or unassembled proteins are discarded by a process known as ER-associated degradation (ERAD), which involves their retrotranslocation into the cytosol. ERAD substrates frequently contain disulfide bonds that must be cleaved before their retrotranslocation. Here, we found that an ER-resident protein ERdj5 had a reductase activity, cleaved the disulfide bonds of misfolded proteins, and accelerated ERAD through its physical and functional associations with EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and an ER-resident chaperone BiP. Thus, ERdj5 is a member of a supramolecular ERAD complex that recognizes and unfolds misfolded proteins for their efficient retrotranslocation.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Glutationa/metabolismo , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Cadeias J de Imunoglobulina/química , Cadeias J de Imunoglobulina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Oxirredução , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismo
17.
Genes Dev ; 20(6): 734-46, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16543225

RESUMO

In a variety of yeast cellular pathways, the Ste50p protein regulates the kinase function of the mitogen extracellular signal-regulated kinase kinase (MEKK) Ste11p. Both Ste11p and Ste50p contain sterile alpha motif (SAM) domains; these are interchangeable, and can be replaced by other protein-interacting modules. Furthermore, the function of the Ras association (RA)-like domain of Ste50p can be mimicked by a plasma membrane recruiting signal, and direct plasma membrane targeting of Ste11p bypasses the requirement of Ste50p for Ste11p function. Thus the regulatory role of Ste50p requires both the N-terminal SAM domain to bind Ste11p and the C-terminal RA-like domain to direct kinase localization. We have identified Opy2p, an integral membrane protein that can interact with Ste50p, as a new component in the Sho1p-Ste11p/Ste50p signaling branch of the high-osmolarity glycerol (HOG) pathway. We propose that Opy2p can serve as a membrane anchor for the Ste50p/Ste11p module in the activation of the HOG pathway.


Assuntos
MAP Quinase Quinase Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química
18.
Biochem Biophys Res Commun ; 309(1): 66-70, 2003 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-12943664

RESUMO

Bright luminescent yeast cells with light intensities similar to bacteria containing luciferase (LuxAB) were generated by providing saturating nontoxic levels of the substrates for the bioluminescence reaction (FMNH(2)+O(2) and fatty aldehyde-->light). Z-9-Tetradecenal added to yeast (+luxAB) gave a luminescent signal close to that with decanal with the signal remaining strong for >24h while luminescence of yeast with decanal decayed to less than 0.01% of that with Z-9-tetradecenal after 2min. Moreover, yeast survived in 0.5% (v/v) Z-9-tetradecenal while 0.005% (v/v) decanal was lethal. Luminescence of yeast (+luxAB) was also stimulated 100-fold by transformation with the NADPH-specific FMN reductase (FRP) from Vibrio harveyi. The recognition of the nontoxicity and high luminescence generated by Z-9-tetradecenal and the generation of high levels of FMNH(2) in yeast by transformation with a flavin reductase provide evidence for the strong potential use of bacterial luciferase as the light-emitting sensor of choice in eukaryotic organisms.


Assuntos
Luciferases/química , Medições Luminescentes , Aldeídos/farmacologia , Divisão Celular , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Flavinas/metabolismo , Luz , Luciferases/metabolismo , Oxirredutases/metabolismo , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo
19.
J Biol Chem ; 278(10): 8118-25, 2003 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-12506122

RESUMO

The MAPKKs MEK1 and MEK2 are activated by phosphorylation, but little is known about how these enzymes are inactivated. Here, we show that MEK1 is phosphorylated in vivo at Ser(212), a residue conserved among all MAPKK family members. Mutation of Ser(212) to alanine enhanced the basal activity of MEK1, whereas the phosphomimetic aspartate mutation completely suppressed the activation of both wild-type MEK1 and the constitutively activated MEK1(S218D/S222D) mutant. Phosphorylation of Ser(212) did not interfere with activating phosphorylation of MEK1 at Ser(218)/Ser(222) or with binding to ERK2 substrate. Importantly, mimicking phosphorylation of the equivalent Ser(212) residue of the yeast MAPKKs Pbs2p and Ste7p similarly abrogated their biological function. Our findings suggest that Ser(212) phosphorylation represents an evolutionarily conserved mechanism involved in the negative regulation of MAPKKs.


Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/química , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/química , Serina/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Dados de Sequência Molecular , Mutagênese , Fosforilação , Ratos , Homologia de Sequência de Aminoácidos
20.
Eukaryot Cell ; 2(5): 949-61, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14555477

RESUMO

The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.


Assuntos
Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Caseína Quinases , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Deleção de Genes , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Dados de Sequência Molecular , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Quinases/genética , Precursores de Proteínas/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/farmacologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Serina/metabolismo , Transdução de Sinais , Sorbitol/farmacologia , Frações Subcelulares/química , Treonina/metabolismo
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