RESUMEN
Ubiquitination is an important posttranslational modification of proteins in eukaryotic cells, wherein ubiquitin molecules are conjugated to target proteins. Ubiquitination is catalyzed by the cascade of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). The number of E2s encoded in eukaryotes partly explains their contribution to the inherent specificity of the ubiquitin system. The ubiquitin conjugating enzyme UBC1 of Saccharomyces cerevisiae participates the degradation of short-lived and abnormal proteins. UBC1 consists of two well-defined domains separated by a long flexible linker. E2-25K, the human homolog of UBC1 is crucial to neurons and its failure leads to neurodegenerative disorders. The linker of UBC1 is of 22 amino acids, while that of E2-25K has 6 amino acids. To understand the importance of the linker, the chimeric protein, cUBC1 was constructed by substituting the linker of E2-25K in UBC1. cUBC1 shows minor changes in its secondary structure. cUBC1 expression in ubc1 deletion mutants showed no effect over growth, thermotolerance and resistance to antibiotic stress. However, survival under heat stress was enhanced with cUBC1. Western blot analysis of the enzymatic activity showed cUBC1 performed equally well as UBC1. Hence, cUBC1 demonstrates that the shorter linker increased the stability of UBC1.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aminoácidos/metabolismo , Humanos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Ubiquitin-activating enzyme (E1) is the first enzyme of the ubiquitination pathway and is required to activate ubiquitin. E1 of Saccharomyces cerevisiae is a large multidomain monomeric protein. There are no studies available on the domains of yeast E1 as independent entities. Four domains of E1 namely, first catalytic cysteine half-domain (FCCH), four-helix bundle (4HB), second catalytic cysteine half-domain (SCCH) and ubiquitin fold domain (UFD) were characterised to understand their structural and functional independence vis-a-vis full length E1. Spectroscopic characterisation using circular dichroism and fluorescence suggested that these domains can act as independent folding units and attain native-like secondary structure. The structural features obtained with the peptides SCCH and FCCH of S. cerevisiae bear a high degree of structural similarity to the corresponding fragments of mouse from literature. Nearly 50% of the residues of the 4HB domain of the S. cerevisiae sample showed helical conformation. They displayed a high degree of conservation when compared with 4HB of mouse with respect to their identity and arrangement. The fragment UFD of yeast formed an α/ß domain as in the whole protein and exhibited 45% homology with that of mouse, showing a similar arrangement of α and ß elements in its secondary structure. Overexpression of the domains in vivo indicated that the SCCH domain and to some extent UFD apparently interfere with cellular functions such as survival under various stresses.
Asunto(s)
Saccharomyces cerevisiae/enzimología , Enzimas Activadoras de Ubiquitina/química , Ubiquitina/química , Animales , Proliferación Celular , Dicroismo Circular , Escherichia coli/metabolismo , Ratones , Pruebas de Sensibilidad Microbiana , Dominios Proteicos , Estructura Secundaria de Proteína , Espectrometría de FluorescenciaRESUMEN
Ubiquitination is tightly regulated to control degradation, localization and function of various proteins. Ubiquitination is catalysed by three enzymes, namely E1, E2 and E3. The specificity shown by E2s for E3s holds key to regulation of ubiquitination. Here we focussed on the E2 enzymes, UBC4 and UBC5 of Saccharomyces cerevisiae, which are almost identical differing only by 11 residues. They show functional complementation in protein degradation, especially during stress response. Existence of two almost identical proteins suggests specialized requirement of one of them under selective conditions. To understand the reasons for the residue differences between them, mutations were introduced in the UBC4 gene to generate single residue variants by swapping with codons from UBC5. Though the variants are found to be functionally active in Δubc4Δubc5 strain of yeast, they cause reduced growth under normal conditions, altered survival under heat and antibiotic stresses, when compared with UBC4. The variants indicated decrease in protein stability theoretically. Hence, the residues of UBC5 individually do not confer any structural advantage to UBC4. Interactive proteins of UBC4 are nearly three times more than those of UBC5. UBC5, therefore, is a functionally minimized version, evolved as another means of regulation to meet cell stage specific needs.