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THE ALLOSTERIC REGULATION OF GLUTAMINASE ENZYMES: DRIVING GLUTAMINE CATABOLISM IN CANCER CELLS

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Abstract

Metabolic reprogramming has been established as a major hallmark for cancer progression. Many cancer cells become dependent on glutamine catabolism, which thus has gained a great deal of attention for being a potentially important therapeutic target. The mitochondrial glutaminase enzymes are upregulated in many cancer cells to catalyze the hydrolysis of glutamine to glutamate, initiating glutamine catabolism. Therefore, mechanistic understanding of the regulation of these enzymes has important implications for the development of strategies to inhibit malignant growth. Here in my thesis, I will first present a novel mechanism for the activation of glutaminase C (GAC), an isoform implicated in the progression of many aggressive cancers. GAC acquires maximal catalytic activity upon binding to anionic activators such as inorganicphosphate. Using a combination of biophysical approaches, I showed that in the absence of phosphate, glutamine binding to the GAC tetramer exhibits positive cooperativity. This cooperativity is coupled to the transition between the open and closed conformation of the ‘lid’, a tyrosine residue located at the edge of the catalytic site, which controls substrate access to this site. Phosphate allows glutamine to independently bind with high affinity to all subunits, which then undergo simultaneous catalysis with all the lids in a uniform conformation. This mechanism shows how the regulated transitioning between different conformational states of GAC ensures the maximal catalytic activity of the enzyme is acquired in cancer cells only when an allosteric activator is available. Secondly, I will discuss the underlying basis for the differences in potencies exhibited by members of the BPTES/CB-839 class of inhibitors, which selectively target GAC. The mechanism of inhibition remained poorly understood as the differences in potency could not be explained with standard cryo-cooled X-ray crystal structures of GAC bound to these compounds. However, using an emerging technique, serial room temperature crystallography, I showed that clear differences were observed between the binding conformations of inhibitors with significantly different potencies. The results were further corroborated using recently established fluorescence assays that directly read-out inhibitor binding to GAC. Collectively, these findings have highlighted novel strategies for designing more potent GAC inhibitors as potential cancer therapeutics.

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152 pages

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2022-08

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Keywords

anti-cancer drugs; cancer; crystallography; glutaminase; structure‐function; Warburg effects

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Committee Chair

Cerione, Richard A.

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Baird, Barbara A.
Crane, Brian

Degree Discipline

Chemistry and Chemical Biology

Degree Name

Ph. D., Chemistry and Chemical Biology

Degree Level

Doctor of Philosophy

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Government Document

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Attribution 4.0 International

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dissertation or thesis

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