REGULATION OF PHASE II DETOXIFICATION PATHWAYS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017555
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Biochemistry

Non Technical Summary
The proposed research is focused on cellular detoxification pathways. Glutathione, an abundant low molecular weight thiol, is largely responsible for the tightly controlled maintenance of intracellular reduction-oxidation status essential for normal cellular function. Despite its ubiquitous distribution and critical functions in mammalian systems, numerous aspects of glutathione homeostasis remain unresolved. We will use a variety of biophysical techniques, including protein crystallography, enzymology, and protein chemistry, to examine the mechanisms of the enzymes responsible for maintaining reduced glutathione reservoirs. Our work will provide a structural framework to examine regulation of glutathione metabolism at a molecular level, with the ultimate goal of controlling flux through the glutathione pathway.Similarly, we will examine how small molecule metabolites involved in cellular detoxification pathways, are directed to a given metabolic fate. The product of the enzyme UDP-glucose dehydrogenase, UDP-glucuronate, has similar detoxification functions as glutathione. We will determine the regulatory mechanisms that dictate partitioning of UDP-glucuronate to one of its three fates: hyaluronan production, proteoglycan biosynthesis, and detoxification by UDP-glucuronosyltransferases.Using recently developed eukaryotic model systems, we will validate our results in relevant cellular contexts.?

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3999	1000	40%
311	3999	1000	30%
306	7010	1000	30%

Knowledge Area
302 - Nutrient Utilization in Animals; 311 - Animal Diseases; 306 - Environmental Stress in Animals;

Subject Of Investigation
3999 - Animal research, general; 7010 - Biological Cell Systems;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

glutathione

oxidative stress

detoxification

sulfur metabolism

udp-glucose dehydrogenase

udp-glucuronate

reactive oxygen species

redox biology

Goals / Objectives
Goals / ObjectivesMy research program is centered on the production and partitioning of two essential metabolites: glutathione and UDP-glucuronate. Both are central to Phase II detoxification, but each is also involved in other vital metabolic pathways.Currently, our work is focused on defining UDP-glucuronate partitioning mechanisms, with the ultimate goal of identifying regulatory mechanisms that dictate metabolic fates.The approaches rely on photoactivatable crosslinking of transiently interacting proteins through unnatural amino acid incorporation, and quantification of downstream metabolites by mass spectrometry, competitive binding analysis, and click chemistry.Our second goal is to define the regulation of the enzyme-mediated synthesis and salvage of glutathione, a major biological redox buffer, both in normal conditions and in disease progression.This grant will expand on results from prior funded projects, and are focused on structure/function studies of glutamate cysteine ligase, 5-oxoprolinase, and the recently identified ChaC1, a novel glutathione degrading enzyme. The outcomes of the biophysical characterizations of these enzymes and their regulation will then be evaluated in the context of mammalian cell cultures.Objective 1.Investigate the structural and mechanistic features of post-translational regulation of glutamate cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis.Objective 2.Determine how 5-oxoproline production and degradation influence glutathione levels.Objective 3.Elucidate partitioning mechanisms of UDP-glucuronate by novel biochemical and structural methods.

Project Methods
Objective 1.Investigate the structural and mechanistic features of post-translational regulation of glutamate cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis.Investigate the regulation of GCL activity at the structural level.In mammals, GCL is typically observed as a heterodimer, comprised of a ~70 kDa catalytic subunit, GCLC, and a ~30 kDa regulatory subunit, GCLM.Although the active site of the enzyme is contained entirely in the GCLC subunit, GCLM is required for optimal activity.We will characterize the catalytic properties of GCLC alone and in the presence of GCLM, and use mutagenesis to identify residues critical to the regulation of GCL activity.These studies will elucidate aspects of redox regulated GSH production dependent on GCL, potentially identifying a novel oxidation-sensitive protein regulatory paradigm.Crystal structure determination of GCLC and GCLM.We will identify suitable crystallization conditions for GCLCand GCLMusing standard sparse matrix screening approaches.Crystallization conditions will be optimized and diffraction data collected either in house or at an appropriate synchrotron radiation source.Phase information fwill be obtained by molecular replacement or by multi-wavelength anomalous dispersion (MAD) with a selenomethionine-substituted enzyme.A comprehensive investigation of GCLC and GCLM structure/function relationships will allow us to define the catalytic mechanism of the enzyme.Structural information will undoubtedly reveal additional amino acids that are important in substrate recognition and/or catalysis and these residues will be examined using site directed mutagenesis and kinetic characterizations.Determine the structure of the GCLC/GCLM complex.Efforts to understand GCL catalytic function and regulation would be greatly facilitated by structural information.We have initiated crystallization screens for the GCL complex and have identified several potential crystal growth conditions.We are currently refining conditions. Once optimized, diffraction data will be collected either in house or at an appropriate synchrotron radiation source. Successful structure determination of the complex would provide tremendous insight into catalysis by and regulation of GCL.Objective 2.Determine how 5-oxoproline production and degradation influence glutathione levels.Structurally and kinetically characterize OPLAH.Eukaryotic 5-oxoprolinases are homodimeric enzymes with two hydantoinase domains (HyuA and HyuB) per subunit.Hydantoinases can act on a number of 5- and 6-membered ring substrates, perhaps indicating that OPLAH may hydrolyze other substrates besides 5-oxoproline.Studies on the enzymatic properties of OPLAH have been relatively limited, and a large range of kinetic constants has been observed.The enzyme appears to require both monovalent and divalent cations to maintain catalytic activity and is stabilized by the presence of 5-oxoproline.The impacts of the reported nitrosylation of the enzyme activity and stability have yet to be elucidated, but it is possible that some of the 24 cysteine residues found in human OPLAH may be sites of post-translational regulation.Additional studies are warranted to define the breadth of reactions catalyzed by OPLAH.We have purified recombinant OPLAH to homogeneity and will use standard sparse matrix screens to identify initial conditions and optimize using fine grid custom screens.Once structural information is available, we begin to dissect the catalytic mechanism of the enzyme using site directed mutagenesis, enzyme kinetics, and other biophysical approaches.Functionally characterize the 5-oxoproline biosynthetic enzyme, ChaC1.ChaC1 was initially identified as part of the Unfolded Protein Response (UPR) cascade, downstream of ATF4.Overexpression of ChaC1 led to apoptosis whereas knockdown by siRNA attenuated apoptosis in HEK293 cells, consistent with its involvement in the UPR.Its ability to cleave theg-glutamyl peptide bond of glutathione likely facilitates apoptosis, as lower GSH levels are a hallmark of this process.To inform our functional characterizations, we will perform crystallization trials as described above to obtain diffraction quality crystals of ChaC1. The structure will be solved in apo form and in complexes with putative substrates. Structural information will be used to identify key active site residues, which will be validated by site-directed mutagenesis and characterization of reaction kinetics in mutant enzyme forms.Manipulate protein expression by targeted gene knockdown or overexpression and determine effects of altered 5-oxoproline levels on proliferation, apoptosis, and glutathione production.Although ChaC1 has been overexpressed and characterized as a pro-apoptotic factor in HEK293 cells, its role in glutathione production had not yet been reported, so there is limited understanding of its role in cell survival. We will use our expertise in GSH quantification andg-glutamyl cycle flux analysis to examine levels of GSH, 5-oxoproline, and other cycle intermediates in control and ChaC1 overexpressing cells. These data will be compared to proliferation and apoptosis assays of the cells, which will be performed to confirm the prior reports of ChaC1 pro-apoptotic activity and to extend the mechanistic analysis to impacts on GSH metabolism. Should the pro-apoptotic effects of overexpression be too toxic to obtain reliable data on glutathione metabolites, we will use a doxycycline inducible expression construct and do comparative overexpression experiments in a dox-inducible cell line.Objective 3.Elucidate partitioning mechanisms of UDP-glucuronate by novel biochemical and structural methods.Determine the functional outcome of specific UGDH interactions with components of the androgen elimination pathway.We predict that hexameric versus dimeric variants of UGDH will interact with different proteins in a cellular context, and that these interactions will be important for regulation of enzyme activity and/or cellular processes that depend on ready availability of UDP-glucuronate. We will directly measure interactions of UGDH with hyaluronan synthase, the Golgi UDP-xylose transporter, and the ER UDP- glucuronate transporter, as the three proteins that share the demand for UDP-glucuronate flux. We will quantify UDP-glucuronate directly by LC/MS. Localization of UGDH to the plasma membrane and incorporation ofUDP-glucuronate into glycosaminoglycan production will be assessed by a competitive binding assay for hyaluronan quantification. Partitioning to the Golgi for proteoglycan secretion will be reported as notch glycosylation and chondroitin sulfate proteoglycan. Functional distribution of UGDH to the ER for androgen elimination will be measured by mass spectrometric quantification of DHT-glucuronide.Characterize and validate the UGDH interactome using an unbiased approach.We will employ two approaches to identify both stable and transient UGDH interactions more comprehensively: (1) HPLC fractionation followed by mass spectrometric analysis of whole cell lysates from cells lentivirally transduced with constructs for wild-type UGDH or the T325D mutant, which is an obligate dimer; (2) immunoprecipitation of cell lysates in which UGDH has been expressed with an unnatural amino acid substitution that inserts a photo-activatable crosslinker, followed by mass spectrometric analysis of cross- linked proteins. Putative interacting proteins will be validated in vitro and in a cellular context by co-transfecting UGDH with its putative interacting protein(s) in cells. We will quantify UDP-glucuronate production directly, and again assay effects on UDP- glucuronate use for steroid elimination by DHT-glucuronide levels, for incorporation into hyaluronan, and for proteoglycan synthesis by immunoblotting of chondroitin sulfate.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:The goals of the project add to the general scientific knowledge about redox homeostasis and cellular detoxification. It hasthe potential to impact diverse fields from fertility, aging, and immune function. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The proposed studies are being conducted with the laboratory of Dr. Melanie Simpson (NC State) and provides ample training opportunities for undergraduate, graduate, and post-doctoral researchers. I havea graduate studentand a technician, who is concurrently pursuing her Masters degree,in my laboratory andDr. Simpson has three graduate students working on aspects of these projects. A number of undergraduate students also make contributions. Students are trainined broadly in analytical, structural, and cell based approaches. Students also participate in weekly research presentations and are encouraged to participate in other professional development activities, such as the A2I (Accelerate to Industry) program. How have the results been disseminated to communities of interest?One manuscript has been published and in progress work has been presented in departmental and University forums in seminars and poster conferences. Another review article will be in press in the coming weeks. What do you plan to do during the next reporting period to accomplish the goals?We will continue to build upon the novel approaches we have developed. In particular, we will focus on identifying and confirming potential UGDH protein binding partners. We anticipate completing our investigation GCL heterodimer formation, with the hope of generating a stable GCLC/GCLM complex suitable for structural characterizations by X-ray crystallography. We will also focus on developing rigorous mass spectrometry based approaches to quantify the downstream incorporation of UDP-glucose into metabolic products. The genetic tools we have developed should allow us to more rapidly optimize our analytical approaches.

Impacts
What was accomplished under these goals? We have also developed a more robust method to study protein-protein interactions between GCLM and GCLC (Objective 1). We are using isothermal titration calorimetry (ITC) to directly measure binding affinities. We have characterized >10 GCLC/GCLM complexes with targeted mutations in each protein. We are mapping the likely GCLC/GCLM interface using this approach in conjunction with structural models. We are just beginning kinetic characterizations of GCL complexes with altered stabilities. In the published article, we used genetic manipulations to investigate how modifications in glucuronidation potential impact downstream phenotypic traits, suggesting that partitioning of essential essential precursors can influence cellular function (Objective 3). This past year, we continued protein-protein interaction studies using site specific crosslinking strategies coupled with mass spectrometry to identify novel UGDH binding partners. Validation of targets is ongoing. We have also started a ab initio approach to address this question. We have tethered UGDH to BirA, which can tag nearby proteins with biotin and can then be isolated using streptavidin, to identify transient interaction partners. These studies are in the early validation stages.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Zimmer BM, Howell ME, Ma L, Enders JR, Lehman D, Corey E, Barycki JJ, and Simpson MA. (2021) Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer. Oncotarget 12: 1886-1902

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The goals of the project add to the general scientific knowledge about redox homeostasis and cellular detoxification. It has the potential to impact diverse fields from fertility, aging, and immune function. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The proposed studies are being conducted with the laboratory of Dr. Melanie Simpson (NC State) and provides ample trainingopportunities for undergraduate, graduate, and post-doctoral researchers. I have recently recruited two graduate students tomy laboratory and Dr. Simpson has two graduate students working on aspects of these projects. A number of undergraduatestudents also make contributions. Students are trainined broadly in analytical, structural, and cell based approaches.Students also participate in weekly research presentations and are encouraged to participate in other professionaldevelopment activities, such as the A2I (Accelerate to Industry) program. How have the results been disseminated to communities of interest?One manuscript has been published and in progress work has been presented in departmental and University forums inseminars and poster conferences. What do you plan to do during the next reporting period to accomplish the goals?In the comining year, we will continue to build upon the novel approaches we have developed. In particular, we will focus on identifying and confirmingpotential UGDH protein binding partners and further investigatingGCL heterodimer formation as a regulatory mechanism. We will also begin to develop mass spectrometry based approaches to quantify the downstream incorporation of UDP-glucose into downstream metabolic proucts.

Impacts
What was accomplished under these goals? One collaborative review article has been published that describes the partitioning of UDP-glucose into its metabolic fates. Other major advances in the lab include: Last year we developed robust protein production protocols for several eukaryotic forms of glutamate cysteine ligase (both catalytic and regulatory subunits). This year we have developed two methodologies to examine the formation of the heterocomplex based on sedimentation velocity and surface plasmon resonance. The importance of several amino acid residues to complex formation is being examined. Crystallization trials are ongoing. Last year, we constructedseveral UGDH plasmids that incorporate photoactivatable amino acids at select positions for cross-linking experiments. This year, we have initiated protein-protein interaction studies using site specific crosslinking stategies coupled with mass spectromtry to identify novl protein binding partners. These studies have been expanded to include more sites of crosslinking. Validation of targets is ongoing. A similar targeted approach is currently being deployed to specifically examine the interactions of GCLM and GCLC.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase. Zimmer BM, Barycki JJ, Simpson MA. J Histochem Cytochem. 2021 Jan;69(1):13-23. doi: 10.1369/0022155420947500. Epub 2020 Aug 4. PMID: 32749901

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The goals of the project add to the general scientific knowledge about redox homeostasis and cellular detoxification. It has the potential to impact diverse fields from fertility, aging, and immune function. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The proposed studies are being conducted with the laboratory of Dr. Melanie Simpson (NC State) and provides ample training opportunities for undergraduate, graduate, and post-doctoral researchers. I have recently recruited two graduate students to my laboratory and Dr. Simpson has two graduate students working on aspects of these projects. A number of undergraduate students also make contributions. Students are trainined broadly in analytical, structural, and cell based approaches. Students also participate in weekly research presentations and are encouraged to participate in other professional development activities, such as the A2I (Accelerate to Industry) program. How have the results been disseminated to communities of interest?One manuscript has been published and in progress work has been presented in departmental and University forums in seminars and poster conferences. What do you plan to do during the next reporting period to accomplish the goals?Major efforts will focus on: Optimization of protein crystallization conditions for UGDH and GCL projects. Identification of interaction sites and protein partners via mass spectrometry. We will rely on the expertise of the METRIC core facilty on campus with respect to MS analysis. Identification of the regulatory binding sites of UGDH using kinetic, crystallographic and modeling approaches.

Impacts
What was accomplished under these goals? As noted, one collaborative paper has been published that examined the impact of clinical mutations on UGDH structure and function. Other major advances in the lab include: The development of robust protein production protocols for several eukaryotic forms of glutamate cysteine ligase (both catalytic and regulatory subunits). The construction of several UGDH plasmids that incorporate photoactivatable amino acids as select positions for cross-linking experiments. The development of mass spectrometry methods to detectinteracting proteins that associate with UGDH and GCL.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy. Hengel H1,2, Bosso-Lef�vre C3,4, Grady G5, Szenker-Ravi E3, Li H6, Pierce S7, Lebigot �8, Tan TT9, Eio MY9, Narayanan G9, Utami KH10, Yau M11, Handal N12, Deigendesch W12, Keimer R13, Marzouqa HM12, Gunay-Aygun M14, Muriello MJ14, Verhelst H15, Weckhuysen S16,17,18, Mahida S19, Naidu S19, Thomas TG20, Lim JY21,22,23, Tan ES21,22,23, Haye D24, Willemsen MAAP25, Oegema R26, Mitchell WG27, Pierson TM28, Andrews MV29, Willing MC29, Rodan LH30, Barakat TS31, van Slegtenhorst M31, Gavrilova RH32, Martinelli D33, Gilboa T34, Tamim AM35, Hashem MO36, AlSayed MD37, Abdulrahim MM37, Al-Owain M37, Awaji A38, Mahmoud AAH39, Faqeih EA40, Asmari AA40, Algain SM41, Jad LA39, Aldhalaan HM42, Helbig I43, Koolen DA44, Riess A45, Kraegeloh-Mann I46, Bauer P45, Gulsuner S7, Stamberger H16,17,18, Ng AYJ47, Tang S48, Tohari S47, Keren B49, Schultz-Rogers LE32, Klee EW32, Barresi S33, Tartaglia M33, Mor-Shaked H50, Maddirevula S36, Begtrup A51, Telegrafi A51, Pfundt R44, Sch�le R1,2, Ciruna B11, Bonnard C3, Pouladi MA10,52,53, Stewart JC47, Claridge-Chang A47,54, Lefeber DJ55,56, Alkuraya FS36, Mathuru AS6,47, Venkatesh B4,47, Barycki JJ5, Simpson MA5, Jamuar SS21,22,23,57, Sch�ls L58,59, Reversade B60,61,62,63,64. Nat Commun. 2020 Jan 30;11(1):595. doi: 10.1038/s41467-020-14360-7.