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Molecular mechanisms governing Hexokinase 2 nuclear shuttling and resistance to 2-deoxyglucose

Lesko, Mitchell A (2024) Molecular mechanisms governing Hexokinase 2 nuclear shuttling and resistance to 2-deoxyglucose. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Hexokinases represent the gateway enzyme of glucose metabolism by regulating cellular glucose uptake and glycolytic rate. Interestingly, some mammalian hexokinase isoforms shuttle into the nucleus in response to nutrient starvation, necessitating further investigation into the regulation and function of nuclear hexokinases. Here, using Saccharomyces cerevisiae and quantitative live-cell imaging, we gain insight into this phenomenon. In agreement with current mammalian models, we demonstrate nuclear accumulation of hexokinase 2 (Hxk2) in response to glucose starvation. Through this approach, we identify a NUAK-family kinase, Tda1, as crucial for inducing Hxk2 nuclear accumulation. Additionally, we identify a lysine residue located in the Hxk2 N-terminal tail, that maintains nuclear exclusion in glucose-replete conditions. This study advances our understanding of the mechanisms regulating hexokinase nuclear accumulation, with potential implications for disease, as increased hexokinase expression is linked to cancer progression, making hexokinases promising drug targets. 2-deoxyglucose (2DG) is a hexokinase inhibitor that elicits a starvation response in cancer cells. Despite its therapeutic potential, cancer cells gain resistance to 2DG by suppressing hexokinase activity. Yeast studies show that spontaneous loss-of-function mutations in the HXK2 gene confer 2DG resistance. To understand how Hxk2 mutations confer 2DG resistance we conducted lab evolution of wild-type yeast, identifying a novel HXK2 mutation (Hxk2G238V) in resistant cells. Biochemical and phenotypic analyses reveal this mutant encodes a loss-of-function allele, though the affected residue does not interact with substrates. Molecular dynamics simulations predict that Hxk2G238V impedes glucose binding by altering the stability of the glucose binding pocket and large-scale domain closures required for catalysis. In past screens, we identified the Hxk2G55V allele that also conferred 2DG resistance. Hxk2G55V encodes an unstable enzyme that accumulates in the nucleus regardless of glucose availability. Though far from the enzymatic pocket, this mutation impacted the dynamics of a key “hinge-point,” influencing Hxk2 domain closures and hindering stable glucose binding. Collectively, these findings present the first atomistic models describing the impact of Hxk2 mutations on enzyme dynamics and advance our understanding of substrate-induced conformational changes in hexokinases.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Lesko, Mitchell Amal344@pitt.edumal3440000-0002-7788-0005
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee CoChairO'Donnell, Allyson Fallyod@pitt.eduallyod
Committee CoChairDurrant, Jacob Ddurrantj@pitt.edudurrantj
Committee MemberBerman, Andrea Jajb190@pitt.eduajb190
Committee MemberBrodsky, Jeffrey Ljbrodsky@pitt.edujbrodsky
Committee MemberBoyle, Jon Pboylej@pitt.eduboylej
Committee MemberSchmidt, Martin Cmcs2@pitt.edumcs2
Date: 13 May 2024
Date Type: Publication
Defense Date: 21 February 2024
Approval Date: 13 May 2024
Submission Date: 14 March 2024
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 399
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Hexokinase 2-deoxyglucose Confocal microscopy Quantitative fluorescence microscopy Molecular Dynamics Glycolysis 2DG Drug resistance Nuclear shuttling Microscopy
Date Deposited: 13 May 2024 13:52
Last Modified: 13 May 2024 13:52


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