Davies, Christopher Williamson.
Structural and Functional Characterization of the Endosome-associated Deubiquitinating Enzyme AMSH.
Degree: PhD, Chemistry, 2013, Purdue University
The endosomal sorting complexes required for transport (ESCRT) machinery is a ubiquitin-dependent molecular mechanism made of up of four individual complexes: ESCRT-0, -I, -II, III, that is necessary for regulating the degradation of cell surface receptors directed towards the lysosome. Not only are the ESCRTs implicated in endosomal sorting and trafficking of proteins, its members also have roles in other important biological processes such as: cytokinesis, HIV budding, transcriptional regulation, and autophagy. As a function of its involvement in several processes throughout the cell, the ESCRT machinery is implicated in a wide variety of diseases including cancer, neurological disease, bacterial infections, cardiovascular disease, and retroviral infection. Proteins marked for lysosomal degradation (cargo) are first ubiquitinated, and then, shuttled in a sequential mechanism through the complexes. In the last step, ubiquitin is removed from the cargo, which is subsequently encapsulated into intralumenal vesicles (ILV) that will ultimately be transported to and fuse with lysosome, degrading and recycling its contents. Deubiquitination is the removal of ubiquitin, catalyzed by deubiquitinating enzymes (DUBs). The human ESCRT machinery recruits two DUBs: AMSH (associated molecule with a Src homology 3 (SH3) domain of signal transducing adaptor molecule (STAM) or simply, STAM-binding protein (STAMBP)), and UBPY/USP8 (ubiquitin specific protease 8). Both AMSH and USP8 have the same ESCRT-recognition domains facilitating recruitment to ESCRT-0 and ESCRT-III. The Saccharmyces cerevisiae (S. cerevisiae) version of the ESCRT complex employs only one DUB, Doa4 (degradation of alpha 4) that serves to recycle ubiquitin at ESCRT-III, just prior to ILV formation. Therefore, it is not fully understood why the human ESCRT system requires the function of both AMSH and USP8.
The focus of this thesis is to understand the role of AMSH recruitment at ESCRT-0 with hopes of providing further insight into its role within the ESCRT complex. In doing so, I crystallized and determined the structure of catalytic domain of AMSH. Using this structure, I structurally and thermodynamically compared AMSH to the homologous protein, AMSH-LP. Secondly, I characterized AMSH kinetically by introducing individual point mutations within the catalytic domain and carried out a detailed kinetic analysis to understand the catalytic mechanism of AMSH. Finally, using a combination of biophysical and biochemical experiments, I investigated how AMSH is recruited and recognized at ESCRT-0. My studies show that AMSH is structurally identical to AMSH-LP, however, thermodynamically less stable. Also, AMSH has exquisite specificity for Lys63-linked ubiquitin chains because it recognizes a three-residue sequence within its proximal ubiquitin-binding site. Furthermore, two residues within the distal ubiquitin-binding site (Thr313 and Glu316) play significant roles within AMSH's catalytic mechanism, one of which, Thr313, is mutated to Ile in children with…
Advisors/Committee Members: Chittaranjan Das, Chittaranjan Das, Suzanne Bart, Philip Low, Cynthia Stauffacher.
Subjects/Keywords: pure sciences; biological sciences; amsh; biophysics; deubiquitinating enzymes; metalloprotease; ubiquitin; x-ray crystallography; Biochemistry; Biophysics
Davies, Christopher W. Ph.D., Purdue University, December 2013…
to Zotero / EndNote / Reference
APA (6th Edition):
Davies, C. W. (2013). Structural and Functional Characterization of the Endosome-associated Deubiquitinating Enzyme AMSH. (Doctoral Dissertation). Purdue University. Retrieved from https://docs.lib.purdue.edu/open_access_dissertations/200
Chicago Manual of Style (16th Edition):
Davies, Christopher Williamson. “Structural and Functional Characterization of the Endosome-associated Deubiquitinating Enzyme AMSH.” 2013. Doctoral Dissertation, Purdue University. Accessed January 21, 2020.
MLA Handbook (7th Edition):
Davies, Christopher Williamson. “Structural and Functional Characterization of the Endosome-associated Deubiquitinating Enzyme AMSH.” 2013. Web. 21 Jan 2020.
Davies CW. Structural and Functional Characterization of the Endosome-associated Deubiquitinating Enzyme AMSH. [Internet] [Doctoral dissertation]. Purdue University; 2013. [cited 2020 Jan 21].
Available from: https://docs.lib.purdue.edu/open_access_dissertations/200.
Council of Science Editors:
Davies CW. Structural and Functional Characterization of the Endosome-associated Deubiquitinating Enzyme AMSH. [Doctoral Dissertation]. Purdue University; 2013. Available from: https://docs.lib.purdue.edu/open_access_dissertations/200