Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cryphonectria parasitica is the causal agent of chestnut blight. Infection of this ascomycete with Cryphonectria hypovirus 1 (CHV1) results in reduction of virulence and sporulation of the fungus. The virus affects fungal gene expression and several of the CHV1 downregulated genes encode secreted proteins that contain consensus Kex2 processing signals. Additionally, CHV1 has been shown to colocalize in infected cells primarily with fungal trans-Golgi network vesicles containing the Kex2 protease. We report here the cloning, analysis, and possible role of the C. parasitica Kex2 gene (CpKex2). CpKex2 gene sequence analysis showed high similarity to other ascomycete kexin-like proteins. Southern blot analyses of CpKex2 showed a single copy of this gene in the fungal genome. In order to monitor the expression and evaluate the function of CpKex2, antibodies were raised against expressed protein and Kex2-silenced mutants were generated. Western blots indicate that the Kex2 protein was constitutively expressed. Growth rate of the fungus was not significantly affected in Kex2-silenced strains; however, these strains showed reduced virulence, reduced sexual and asexual sporulation, and reductions in mating and fertility. The reduced virulence was correlated with reduced Kex2 enzymatic activity and reduced relative mRNA transcript levels as measured by real time reverse-transcriptase polymerase chain reaction. These results suggest that secreted proteins processed by Kex2 are important in fungal development and virulence.
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PMID:Silencing of Kex2 significantly diminishes the virulence of Cryphonectria parasitica. 1913 73

The apicomplexan parasite Cryptosporidium is a significant cause of diarrheal disease worldwide. Previously, we reported that a Cryptosporidium parvum subtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity in C. parvum lysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in the C. parvum genome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA from C. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of approximately 64 kDa and approximately 48 kDa, for C. parvum lysates and proteins "shed" during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 by C. parvum lysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity in C. parvum and for processing of gp40/15. Importantly, the recombinant prodomain inhibited C. parvum infection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.
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PMID:Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvum infection in vitro. 1916 60