{"id":{"repo_id":"buffalo","oai_identifier":"oai:ubir.buffalo.edu:10477/83828"},"canonical_url":"https://search.dev.ndltd.org/etd/buffalo/oai:ubir.buffalo.edu:10477/83828","repository":{"repo_id":"buffalo","name":"Buffalo","base_url":"https://ubir.buffalo.edu/oai/request"},"display":{"title":"Studies on the Structure, Function, and Selective Inhibition of the Hsp90 Family of Molecular Chaperones","abstract":"Ph.D.","abstract_html":"Ph.D.","abstract_has_math":false,"creators":["Huck, John; 0000-0002-0755-3850"],"institution":"State University of New York at Buffalo","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Gewirth, Daniel","Structural Biology"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-06-17T19:54:27Z","date_published":"2022-06-17T19:54:27Z","updated_at":"2026-07-27T19:05:28Z","subjects":["biochemistry"],"languages":["eng"],"rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10477/83828","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Gewirth, Daniel","Structural Biology"]},{"key":"dc:creator","label":"Author","values":["Huck, John; 0000-0002-0755-3850"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2022-06-17T19:54:27Z","2020"]},{"key":"dc:publisher","label":"Institution","values":["State University of New York at Buffalo"]},{"key":"dc:type","label":"Dc Type","values":["Text","Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["biochemistry"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/10477/83828"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Ph.D.","Hsp90s are a conserved family of ATP-driven molecular chaperones required for the conformational maturation of a widely diverse set of essential client proteins. This same chaperone function, however, is co-opted in cancer and other diseases to protect a dysregulated proteome, stabilize mutant proteins that would otherwise be degraded, and promote disease progression, making hsp90s attractive therapeutic targets. Humans possess four hsp90 paralogs: Hsp90α and Hsp90β in the cytosol, Grp94 in the endoplasmic reticulum (ER), and Trap1 in the mitochondria. Grp94, which is the main focus of this dissertation, is required for the proper localization of many cell surface receptors, such as toll-like receptors (TLRs) and integrins, along with growth factor receptors such as HER2. The mechanism by which Grp94 engages and matures its client proteins in the ER to ensure their proper localization is almost completely unknown. Overexpression of Grp94 is a hallmark of many cancers and is associated with aggressive behavior and poor prognosis. The oncogenic roles of Grp94 as a cancer chaperone, however, have only recently been established. The overarching goal of this work is to characterize the mechanism of Grp94 action and, in the long-term, to exploit these understandings for therapeutic benefit. Hsp90s form constitutive homodimers via their C-terminal domains and cycle through a series of large conformational shifts between open and closed dimer states. These shifts are driven by the binding of ATP to the N-terminal domain (NTD), which in the ATP-bound state forms the second, closed dimer interface. Cytosolic Hsp90 utilizes a cohort of co-chaperones to regulate this dimer closure, modulate the rate of ATP hydrolysis, and recruit client proteins and other chaperone systems. Interestingly, the ER does not contain homologous co-chaperones. A structure of Grp94 in a closed state has not been described, and the determinants that regulate closure have not been intensively studied. In chapter 2, we present the crystal structure of AMPPNP-bound Grp94 in the closed dimer conformation, capturing Grp94 in its catalytically active state. The structure includes the Pre-N domain, a region that is poorly conserved between hsp90 paralogs that is upstream of the NTD. We show that the Grp94 Pre-N domain is essential for client maturation, and identify this region as an important intrinsic regulatory element of the Grp94 ATPase cycle. In chapter 3, we found that the Pre-N domain is a required for chaperone function due to its protective role in regulating the total levels of N-linked glycosylation. We found that truncations and mutations of the Pre-N domain fundamentally disrupt normal glycosylation, leading to hyper-glycosylation that inactivates the chaperone. Lastly, in chapter 4, we explored the binding of an Hsp90α selective inhibitor to the cytosolic paralogs, revealing the mechanistic basis behind the reported binding preference and identifying an exploitable residue that may aid future efforts to improve selectivity. Together, these studies illuminate key mechanistic questions about Grp94 chaperone function, which will help guide the development of new and improved therapeutics."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Studies on the Structure, Function, and Selective Inhibition of the Hsp90 Family of Molecular Chaperones"]}]}],"canonical_facts":{"dc:contributor":["Gewirth, Daniel","Structural Biology"],"dc:creator":["Huck, John; 0000-0002-0755-3850"],"dc:date":["2022-06-17T19:54:27Z","2020"],"dc:description":["Ph.D.","Hsp90s are a conserved family of ATP-driven molecular chaperones required for the conformational maturation of a widely diverse set of essential client proteins. This same chaperone function, however, is co-opted in cancer and other diseases to protect a dysregulated proteome, stabilize mutant proteins that would otherwise be degraded, and promote disease progression, making hsp90s attractive therapeutic targets. Humans possess four hsp90 paralogs: Hsp90α and Hsp90β in the cytosol, Grp94 in the endoplasmic reticulum (ER), and Trap1 in the mitochondria. Grp94, which is the main focus of this dissertation, is required for the proper localization of many cell surface receptors, such as toll-like receptors (TLRs) and integrins, along with growth factor receptors such as HER2. The mechanism by which Grp94 engages and matures its client proteins in the ER to ensure their proper localization is almost completely unknown. Overexpression of Grp94 is a hallmark of many cancers and is associated with aggressive behavior and poor prognosis. The oncogenic roles of Grp94 as a cancer chaperone, however, have only recently been established. The overarching goal of this work is to characterize the mechanism of Grp94 action and, in the long-term, to exploit these understandings for therapeutic benefit. Hsp90s form constitutive homodimers via their C-terminal domains and cycle through a series of large conformational shifts between open and closed dimer states. These shifts are driven by the binding of ATP to the N-terminal domain (NTD), which in the ATP-bound state forms the second, closed dimer interface. Cytosolic Hsp90 utilizes a cohort of co-chaperones to regulate this dimer closure, modulate the rate of ATP hydrolysis, and recruit client proteins and other chaperone systems. Interestingly, the ER does not contain homologous co-chaperones. A structure of Grp94 in a closed state has not been described, and the determinants that regulate closure have not been intensively studied. In chapter 2, we present the crystal structure of AMPPNP-bound Grp94 in the closed dimer conformation, capturing Grp94 in its catalytically active state. The structure includes the Pre-N domain, a region that is poorly conserved between hsp90 paralogs that is upstream of the NTD. We show that the Grp94 Pre-N domain is essential for client maturation, and identify this region as an important intrinsic regulatory element of the Grp94 ATPase cycle. In chapter 3, we found that the Pre-N domain is a required for chaperone function due to its protective role in regulating the total levels of N-linked glycosylation. We found that truncations and mutations of the Pre-N domain fundamentally disrupt normal glycosylation, leading to hyper-glycosylation that inactivates the chaperone. Lastly, in chapter 4, we explored the binding of an Hsp90α selective inhibitor to the cytosolic paralogs, revealing the mechanistic basis behind the reported binding preference and identifying an exploitable residue that may aid future efforts to improve selectivity. Together, these studies illuminate key mechanistic questions about Grp94 chaperone function, which will help guide the development of new and improved therapeutics."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/10477/83828"],"dc:language":["eng"],"dc:publisher":["State University of New York at Buffalo"],"dc:rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"dc:subject":["biochemistry"],"dc:title":["Studies on the Structure, Function, and Selective Inhibition of the Hsp90 Family of Molecular Chaperones"],"dc:type":["Text","Dissertation"]},"updated_at":"2026-07-27T19:05:28Z"}