RESUMO
The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.
Assuntos
Sistemas CRISPR-Cas , Debaryomyces/enzimologia , Debaryomyces/genética , Edição de Genes/métodos , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genética , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Extremófilos/enzimologia , Extremófilos/genética , Regulação da Expressão Gênica , Genoma Fúngico , Organismos Geneticamente Modificados , Osmorregulação/genética , Estresse Oxidativo/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Salino/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The transcription factor ScRpn4 coordinates the expression of Saccharomyces cerevisiae proteasomal genes. ScRpn4 orthologues are found in a number of other Saccharomycetes yeasts. Their functions, however, have not yet been characterised experimentally in vivo . We expressed the Debaryomyces hansenii DEHA2D12848 gene encoding an ScRpn4 orthologue (DhRpn4), in an S. cerevisiae strain lacking RPN4 . We showed that DhRpn4 activates transcription of proteasomal genes using ScRpn4 binding site and provides resistance to various stresses. The 43-238 aa segment of DhRpn4 contains an unique portable transactivation domain. Similar to the ScRpn4 N-terminus, this domain lacks a compact structure Moreover, upon overexpression in D. hansenii , DhRpn4 upregulates protesomal genes. Thus, we show that DhRpn4 is the activator for proteasomal genes.
Assuntos
Regulação Fúngica da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomycetales/enzimologia , Fatores de Transcrição/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Previous studies have demonstrated the importance of the ubiquitin-proteasome pathway in the immune response to bacterial pathogens. To investigate the role of this system in the context of leprosy, Mycobacterium leprae-stimulated peripheral blood mononuclear cells (PBMC) were treated with the proteasome inhibitor MG132 to assess the levels of apoptosis and cytokine secretion. The results showed that the inhibition of proteasome activity significantly reduced M. leprae-mediated cell death. In addition, MG132 treatment led to a significant decrease in M. leprae-induced TNF-alpha and IL-10 secretion. Together, these results suggest that modulations of the ubiquitin-proteasome pathway may participate in the human response to M. leprae.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Citocinas/biossíntese , Leupeptinas/farmacologia , Mycobacterium leprae/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Apoptose , Morte Celular/efeitos dos fármacos , Humanos , Interleucina-10/metabolismo , Hanseníase/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Mycobacterium leprae/fisiologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195-199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenomenon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3.