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Domain-based Bioinformatics Analysis and Molecular Insights for the Autoregulatory Mechanism of Phafin2

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dc.contributor.authorHasan, Mahmudulen
dc.contributor.committeechairCapelluto, Daniel G.en
dc.contributor.committeememberZhang, Chenmingen
dc.contributor.committeememberKlemba, Michael Wadeen
dc.contributor.committeememberLazar, Maria Iulianaen
dc.contributor.departmentBiological Sciencesen
dc.date.accessioned2024-08-20T08:00:37Zen
dc.date.available2024-08-20T08:00:37Zen
dc.date.issued2024-08-19en
dc.description.abstractPhafin2, an adaptor protein, is involved in various cellular processes, such as apoptosis, autophagy, endosomal cargo transportation, and macropinocytosis. Two domains, namely, PH and FYVE, contribute to Phafin2's cell membrane binding. Phafin2 also contains a poly aspartic acid (polyD) motif in its C-terminal region that can specifically autoinhibit the PH domain binding to membrane phosphatidylinositol 3-phosphate (PtdIns3P). Firstly, the study investigated the domain-based evolutionary pattern of PH, FYVE, and polyD motif of Phafin2 among its orthologs and Phafin2- like proteins. Using different bioinformatics tools and resources, it was concluded that the polyD motif only evolved in Phafin2 and PH- or both PH-FYVE-containing proteins of animals, highlighting the association in cellular functions that might have evolved uniquely in animals. Moreover, PH domain-free FYVE-containing proteins lack polyD motifs. Secondly, intramolecular autoregulatory and membrane binding properties of Phafin2 were studied by employing liposome co-sedimentation assay, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. The residues Gly38, Lys45, Leu45, Lys51, Ala52, and Arg53 of the PH domain form a positively charged binding pocket that can bind the negatively charged polyD motif. The mutated Phafin2 PH domain (K51A/R53C and R53C) was unable to bind to synthetic polyD peptides, establishing the significance of those residues for the interaction between the PH domain and polyD motif. Moreover, the study also concluded that Phafin2-mediated membrane binding is not curvature-dependent.en
dc.description.abstractgeneralPhafin2 is a protein that plays a crucial role in several important cellular functions, including cell death, recycling of cellular components, and transporting materials within cells. The protein's ability to attach to cell membranes is mainly due to two of its specific regions, the PH and FYVE domains. Additionally, Phafin2 has a section called the polyD motif that can block the PH domain from binding to specific cell membrane molecules. This study explored how these regions of Phafin2 have evolved across different species, focusing on the PH, FYVE, and polyD motifs. The findings suggest that the polyD motif is unique to Phafin2 and similar animal proteins, potentially indicating a unique role in animal cell functions. Further experiments examined how Phafin2 regulates itself and binds to cell membranes. The study identified specific amino acids in the PH domain crucial for interacting with the polyD motif. When these amino acids were altered, Phafin2 could no longer bind to synthetic polyD peptides, highlighting their importance. Finally, the research determined that Phafin2's ability to bind to membranes does not depend on the shape or curvature of the membrane.en
dc.description.degreeMaster of Scienceen
dc.format.mediumETDen
dc.identifier.othervt_gsexam:41261en
dc.identifier.urihttps://hdl.handle.net/10919/120963en
dc.language.isoenen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectPhafin2en
dc.subjectMembrane Curvatureen
dc.subjectAutophagyen
dc.subjectAutoregulationen
dc.subjectNMRen
dc.titleDomain-based Bioinformatics Analysis and Molecular Insights for the Autoregulatory Mechanism of Phafin2en
dc.typeThesisen
thesis.degree.disciplineBiological Sciencesen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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