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Understanding the Molecular Basis of MIC‐CAP Syndrome through Structural and Functional Studies of the Deubiquitinase AMSH
Author(s) -
Shrestha Rashmi,
Ronau Judith,
Das Chittaranjan
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.883.7
Subject(s) - deubiquitinating enzyme , ubiquitin , biology , mutant , endocytic cycle , protein fragment complementation assay , microbiology and biotechnology , escrt , endosome , endocytosis , biochemistry , complementation , receptor , gene , intracellular
AMSH, associated molecule with SH3 domain of signal transducing adaptor molecule, is a deubiquitinase recruited by the endosomal sorting complex required for transport (ESCRT) machinery. This machinery executes the endocytic sorting of cell‐surface receptors tagged with Lys63‐linked polyubiquitin chains and delivers them to lysosomes for degradation. Recently, it was found that mutations in AMSH lead to microcephaly‐capillary malformation (MIC‐CAP) syndrome. Since the molecular basis of the MIC‐CAP syndrome is not known, we would like to use fission yeast as a model system, which has an AMSH orthologue, Sst2 to gain insight into the disease. I cloned, purified and solved the crystal structure of Sst2 in free and ubiquitin bound forms. Along with kinetic analysis, these studies led to the conclusion that Sst2 is indeed the AMSH orthologue in fission yeast. I then generated and characterized Sst2 mutants carrying the same mutations found in MIC‐CAP syndrome in AMSH. The disease mutant Thr319Ile showed a defect in its k cat in diubiquitin cleavage assays indicating a loss of interactions during the catalytic transition state. Interestingly, another disease mutation, R433X, far from both the ubiquitin binding site and the active site, showed a severe defect in k cat , the basis of which is being further investigated. In order to functionally characterize these mutants, I am using a deletion S. pombe strain (ubp4Δ1 ubp5Δ ubp9Δ ubp15Δ sst2Δ) lacking endogenous Sst2, which shows a defect in endocytosis. Complementation assay using plasmids carrying Sst2 and its mutants will be used to study their effects on endocytosis .These studies will provide insights into how mutations in the AMSH gene can cause MIC‐CAP Syndrome.