RESUMEN
Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.
Asunto(s)
Hongos/enzimología , Proteínas Tirosina Fosfatasas/clasificación , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cisteína/metabolismo , Disulfuros/metabolismo , Fosfatasas de Especificidad Dual , Histidina/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/clasificación , Fosfoproteínas Fosfatasas/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/clasificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
Lanosterol 14alpha-demethylase (14DM) is the target of the azole antifungals, and alteration of the 14DM sequence leading to a decreased affinity of the enzyme for azoles is one of several potential mechanisms for resistance to these drugs in Candida albicans. In order to identify such alterations the authors investigated a collection of 19 C. albicans clinical isolates demonstrating either frank resistance (MICs > or = 32 microg ml(-1)) or dose-dependent resistance (MICs 8-16 microg ml(-1)) to fluconazole. In cell-free extracts from four isolates, including the Darlington strain ATCC 64124, sensitivity of sterol biosynthesis to inhibition by fluconazole was greatly reduced, suggesting that alterations in the activity or affinity of the 14DM could contribute to resistance. Cloning and sequencing of the 14DM gene from these isolates revealed 12 different alterations (two to four per isolate) leading to changes in the deduced amino acid sequence. Five of these mutations have not previously been reported. To demonstrate that these alterations could affect fungal susceptibility to azoles, the 14DM genes from one sensitive and three resistant C. albicans strains were tagged at the carboxyl terminus with a c-myc epitope and expressed in Saccharomyces cerevisiae under control of the endogenous promoter. Transformants receiving 14DM genes from resistant strains had fluconazole MICs up to 32-fold higher than those of transformants receiving 14DM from a sensitive strain, although Western blot analysis indicated that the level of expressed 14DM was similar in all transformants. Amino acid substitutions in the 14DM gene from the Darlington strain also conferred a strong cross-resistance to ketoconazole. In conclusion, multiple genetic alterations in C. albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates.
Asunto(s)
Sustitución de Aminoácidos , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Sistema Enzimático del Citocromo P-450/genética , Oxidorreductasas/genética , Secuencia de Aminoácidos , Antifúngicos/metabolismo , Candida albicans/enzimología , Candida albicans/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Farmacorresistencia Microbiana , Resistencia a Múltiples Medicamentos , Fluconazol/metabolismo , Fluconazol/farmacología , Cetoconazol/metabolismo , Cetoconazol/farmacología , Datos de Secuencia Molecular , Mutación/genética , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Esterol 14-DesmetilasaRESUMEN
The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we have studied the pheromone response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. We demonstrate this approach by investigating the role of Rgs1, a member of the Regulator of G protein Signalling (RGS) family of proteins. The rgs1 gene was identified through the Sz. pombe genome sequencing project (accession number Q09777) and recognized as having similarity to RGS proteins [Tesmer et al. (1997) Cell 89: 251-261], but this is the first report concerning the activity of the protein. Strains lacking rgs1 (Deltargs1) are hypersensitive to pheromone stimulation and unable to conjugate with a mating partner. Inhibition of mating occurs at a relative late stage in the process as Deltargs1 strains exhibit pheromone-dependent transcription and form shmoos. Expression of SST2 (an RGS protein that regulates pheromone signalling in the budding yeast Saccharomyces cerevisiae) overcomes the hypersensitivity of the Deltargs1 strains but fails to rescue their mating defect.