W Todd Lowther - US grants

DESCRIPTION (provided by applicant): The typical 2-Cys peroxiredoxins (Prxs) are key antioxidant enzymes in the detoxification of reactive oxygen species including hydrogen peroxide (H2O2). At lower concentrations, H2O2 has also been recognized as an important mediator of cell signaling. In this context, the high cellular concentration and reactivity of Prxs with H2O2 makes them ideally suited to regulate redox-dependent signaling events. Human 2-Cys Prxs can be inactivated, however, through hyperoxidation to the Cys sulfinic acid (Cys-SO2-), a hallmark of many aging- related diseases and cancer. The sensitivity to hyperoxidation and the repair of these Prxs by the enzyme sulfiredoxin (Srx) differs. The mitochondrial PrxIII is the most resistant to hyperoxidation. Surprisingly, few details are available for the structures of the human Prxs when present in different oxidation states (Cys-SH, Cys-S-S-Cys, and Cys-SO2-), and even less is known about the kinetics of hyperoxidation and Srx-mediated repair for this class of enzymes. We have shown that Srx utilizes a novel nucleotide binding motif and sulfur chemistry to reduce the Prx molecule and were able to identify critical kinetic intermediates in the repair of hyperoxidized PrxII by Srx. PrxIII on the other hand exhibits a unique C-terminal sequence in the region that is expected to make direct contact with Srx, based on our crystal structure of the human Srx7PrxI complex. As such, we hypothesize that PrxIII is not only more resistant to hyperoxidation due to its C-terminus and active site differences, but also that it will have a unique interaction with Srx that may influence the repair process. Preliminary studies on the four human, 2-Cys Prxs (PrxI-IV) have confirmed the results obtained in cell culture and have shown that indeed PrxIII is the most resistant to hyperoxidation. In addition, we have generated preliminary crystals for PrxI-IV in different oxidation states and performed comparative kinetics studies for PrxII and PrxIII hyperoxidation by time-resolved mass spectrometry. These analyses have shown for the first time the formation of an intramolecular Cys sulfenamide intermediate in PrxII. Interestingly, PrxIII did not form this species under the same reaction conditions, identifying one potential scenario that may impart sensitivity to hyperoxidation in PrxI, PrxII, and PrxIV and resistance to hyperoxidation in PrxIII. Given that the transgenic expression of PrxIII and Srx results in protection against oxidative stress-induced apoptosis and tissue damage during myocardial infarction, an understanding of the structural and kinetics bases of Prxs catalysis, hyperoxidation, and repair by Srx will be invaluable for the future design of novel treatment strategies using PrxIII and/or Srx variants in gene therapy. The specific aims of the proposal are to investigate the structural and kinetic determinants of hyperoxidation in human 2-Cys Prxs (Aim I), and to investigate the repair mechanisms of human 2-Cys Prxs by Srx (Aim2). PUBLIC HEALTH RELEVANCE: The inactivation of 2-Cys peroxiredoxins (Prxs) by hydrogen peroxide is a hallmark of many aging-related diseases including cancer, cardiovascular disease, and Alzheimer's disease. Humans possess four Prxs;one of which is unusually resistant to inactivation by hyperoxidation and has been shown to prevent cell death and tissue damage from a heart attack. The purpose of this study is to understand how Prxs become inactivated and how they can be repaired by an enzyme called sulfiredoxin.

[unreadable] DESCRIPTION (provided by applicant): This R21 proposal will investigate the structure-function relationships of human glyoxylate/hydroxypyruvate reductase (GRHPR), a key enzyme in glyoxylate and hydroxypyruvate metabolism. Defects in human GRHPR are present in the rare genetic disease primary hyperoxaluria type 2 (PH2). These mutations ultimately result in the buildup of oxalate and the formation and deposition of urinary tract calcium oxalate kidney stones. An altered GRHPR activity could also contribute to idiopathic stone disease, a common debilitating health problem that impacts daily life and incurs significant health care costs. The historical analysis of GRHPR from other organisms has yielded contradictory evidence for the preference of cofactor, the salt dependence of the reaction, and substrate inhibition. In addition, no structural or biochemical experiments have been reported for human GRHPR. The long-term goals of this research are to characterize the kinetic properties of human GRHPR, to identify the structural features that determine its activity, and to understand how these properties are altered in mutant enzymes causing PH2. The proposed study will (Aim 1) determine the crystal structures of human GRHPR alone and in complex with NADPH and (Aim 2) determine the cofactor and substrate specificity of human GRHPR through the biochemical analysis of wild-type and PH2 mutant enzymes. The structures of human GRHPR will enable the mapping of current and future PH2 variants onto the structure and the prediction of the physiological consequences. The biochemical data will reveal the kinetic parameters associated with the interaction of the enzyme with substrates, cofactors and modulating anions. The cha racterization of human GRHPR will help establish a more precise physiological role for the enzyme and help explain how mutations cause disease. Such studies may ultimately lead to improved treatment strategies for individulas with PH2 and possibly those with idiopathic stone disease. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The reduction of methionine sulfoxide (MetO) is mediated by methionine sulfoxide reductases (Msr). The MsrA and MsrB families can reduce free MetO and MetO within a peptide or protein context. This process is stereospecific with the S- and R-forms of MetO repaired by MsrA and MsrB, respectively. In contrast, fRMsr is a bacterial enzyme specific for the free form of Met-(R)-O. E. coli fRMsr is the first GAF domain family member to show enzymatic activity (Lin et al. 2007 PNAS 104:9597). Other GAF domain proteins substitute the Cys residues and others to specifically bind cyclic nucleotides, chromophores, and many other ligands for signal potentiation. Therefore, Met-(R)-O may represent a signaling molecule in response to oxidative stress and nutrients via the TOR pathway in some organisms. Moreover, fRMsr may play a key role in maintaining the reduced Met pool for protein synthesis in bacteria. The fRMsr enzyme is an attractive target for drug design as humans do not possess this protein. Site-directed mutagenesis and kinetic analyses have shown that three Cys residues are involved in the fRMsr catalytic mechanism, including a Cys sulfenic acid intermediate. While the mechanism appears to be similar to the other Msr enzymes, the structural basis for the recognition of the substrate is novel. Moreover, two of the Cys residues are located on surface loops that upon MetO reduction result in the formation of a disulfide bond (Cys84-Cys118) and complete enclosure of the active site cavity. Current efforts have lead to the crystallographic determination of the Met product complex. In this complex the active site loops show marked structural rearrangement. The focus of this study is to determine the Cys94-Cys118 reaction intermediate, which should show even more dramatic structural rearrangements.

DESCRIPTION (provided by applicant): Primary hyperoxaluria types I and 2 (PH1 and PH2) are characterized by an inability to efficiently metabolize glyoxylate as a consequence of functional defects in alanine-glyoxylate aminotransferase and glyoxylate reductase, respectively. Excessive oxalate synthesis results and may ultimately cause renal failure. To date, there are no therapies that can specifically reduce oxalate synthesis. Hydroxyproline metabolism is the only recognized source of glyoxylate that has been identified to date. We hypothesize that the breakdown of dietary and endogenous hydroxyproline contributes to the bulk of the oxalate synthesized in PH1 and PH2 patients. Confirmation of this hypothesis would suggest that the inhibition of hydroxyproline degradation may be a significant therapeutic opportunity for diminishing endogenous oxalate production in PH patients. As a first step toward testing this hypothesis, we have synthesized homogeneously labeled 13C-hydroxyproline and verified that it can be used to follow the degradation of hydroxyproline to oxalate, glycolate, lactate, and glycine using chromatographic techniques coupled to mass detection. The specific aims of the proposed research are: (1) to quantitate the contribution of hydroxyproline metabolism to endogenous oxalate synthesis in normal human subjects and patients with PH using homogeneously-labeled Hyp, 13C5-hydroxyproline, as a metabolic tracer; (2) to quantitate the contribution of hydroxyproline metabolism to endogenous oxalate synthesis in mouse KO models of PH (Agxt and Grhpr KO) using 13C5-hydroxyproline. Through the quantitation and tracking of the isotopic label within metabolites, we will be able to demonstrate whether or not hydroxyproline degradation contributes to endogenous glycolate, and oxalate synthesis. Moreover, the mouse data will benchmark the contribution of hydroxyproline to urinary glycolate and oxalate in each genetic setting. This latter data will be invaluable for the future testing of therapeutics targeting the unique enzymes of the hydroxyproline degradation pathway. PUBLIC HEALTH RELEVANCE: The synthesis of oxalate, a key component of kidney stones, is influenced by glyoxylate levels. Glyoxylate is produced from hydroxyproline during the normal degradation of collagen within the body and that consumed in the diet. The purpose of this study is to determine whether or not hydroxyproline degradation contributes adversely to the elevated levels of glyoxylate and oxalate production observed in primary hyperoxaluria patients.

DESCRIPTION (provided by applicant): The typical 2-Cys peroxiredoxins (Prxs) are key antioxidant enzymes in the detoxification of reactive oxygen species including hydrogen peroxide (H2O2). At lower concentrations, H2O2 has also been recognized as an important mediator of cell signaling. In this context, the high cellular concentration and reactivity of Prxs with H2O2 makes them ideally suited to regulate redox-dependent signaling events. Human 2-Cys Prxs can be inactivated, however, through hyperoxidation to the Cys sulfinic acid (Cys-SO2-), a hallmark of many aging- related diseases and cancer. The sensitivity to hyperoxidation and the repair of these Prxs by the enzyme sulfiredoxin (Srx) differs. The mitochondrial PrxIII is the most resistant to hyperoxidation. Surprisingly, few details are available for the structures of the human Prxs when present in different oxidation states (Cys-SH, Cys-S-S-Cys, and Cys-SO2-), and even less is known about the kinetics of hyperoxidation and Srx-mediated repair for this class of enzymes. We have shown that Srx utilizes a novel nucleotide binding motif and sulfur chemistry to reduce the Prx molecule and were able to identify critical kinetic intermediates in the repair of hyperoxidized PrxII by Srx. PrxIII on the other hand exhibits a unique C-terminal sequence in the region that is expected to make direct contact with Srx, based on our crystal structure of the human Srx7PrxI complex. As such, we hypothesize that PrxIII is not only more resistant to hyperoxidation due to its C-terminus and active site differences, but also that it will have a unique interaction with Srx that may influence the repair process. Preliminary studies on the four human, 2-Cys Prxs (PrxI-IV) have confirmed the results obtained in cell culture and have shown that indeed PrxIII is the most resistant to hyperoxidation. In addition, we have generated preliminary crystals for PrxI-IV in different oxidation states and performed comparative kinetics studies for PrxII and PrxIII hyperoxidation by time-resolved mass spectrometry. These analyses have shown for the first time the formation of an intramolecular Cys sulfenamide intermediate in PrxII. Interestingly, PrxIII did not form this species under the same reaction conditions, identifying one potential scenario that may impart sensitivity to hyperoxidation in PrxI, PrxII, and PrxIV and resistance to hyperoxidation in PrxIII. Given that the transgenic expression of PrxIII and Srx results in protection against oxidative stress-induced apoptosis and tissue damage during myocardial infarction, an understanding of the structural and kinetics bases of Prxs catalysis, hyperoxidation, and repair by Srx will be invaluable for the future design of novel treatment strategies using PrxIII and/or Srx variants in gene therapy. The specific aims of the proposal are to investigate the structural and kinetic determinants of hyperoxidation in human 2-Cys Prxs (Aim I), and to investigate the repair mechanisms of human 2-Cys Prxs by Srx (Aim2).

Summary The CB1 receptor (CB1R) a therapeutic target for treatment of addictions, neurodegenerative disorders and pain management, but medicinal compounds based upon the CB1R have been limited. The functions of the CB1R to regulate neuronal processes in development, retrograde signaling in neurotransmission, and cellular mechanisms of neuroprotection are critical to brain function. Endocannabinoid ligands 2-arachidonoylglycerol and anandamide are the primary neuromodulators of synaptic activity, but the full understanding of how CB1R signaling can be regulated by associated proteins in specific cell types is just beginning to be appreciated. The Scientific Premise is that CRIP1a modulation of the CB1R can be understood at the structural and functional level such that drug design based on peptide or small molecule interventions can target the CRIP1a-CB1R interaction. Our recently published studies have demonstrated that CRIP1a reduces the density of cell surface CB1R, attenuates the agonist-dependent but not the constitutive internalization processes by competing with ?-arrestins for binding to C-terminal sites, and curtails the trafficking of newly- synthesized CB1R to the cell surface after prolonged WIN55212-2 but not CP55940. Other studies demonstrated that CRIP1a has a critical role in regulating CB1R cellular signaling by altering the preference for coupling from Gi3 & Go, which require the C-terminus for activation, to Gi1 & Gi2, which do not. In unpublished studies, we have determined the high resolution structure from X-ray crystallography, and found that CRIP1a is a member of the family of carriers for myristoylated or isoprenylated proteins. Based upon this major advance in knowledge of the structure and function of CRIP1a, we hypothesize that the function of CRIP1a is to interact with the CB1R?G-protein complex in ways that can be regulated by G-alpha and/or G-gamma subunit specificity, phosphorylation, and interaction with other regulatory proteins that are known to release cargo. We propose to investigate the CB1R associated proteins in the N18TG2 neuroblastoma cell model which endogenously expresses the CB1R and its associated proteins, as well as in in vitro experiments of purified recombinant proteins and peptides derived therefrom. The aims of this project are to investigate: the interaction of CRIP1a with the CB1R; the structural and functional interaction of CRIP1 with G- proteins; and the regulation of CRIP1a function by cargo-releasing proteins and phosphorylation. The results of the proposed investigation should prove to be transformative for the field by providing evidence that CB1R and associated CRIP1a interact to direct cellular signaling pathways. From this understanding, novel peptides and small molecules could be developed as therapeutic agents for neurological diseases in which both CB1R and CRIP1a co-exist in neurons.

CRYSTALLOGRAPHY AND COMPUTATIONAL BIOSCIENCES SHARED RESOURCE Project Summary The Crystallography and Computational Biosciences Shared Resource (CCBSR) is a highly-specialized Resource that provides Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) members with access to expertise, consultation, and state-of-the-art structural and computational biology approaches. The resulting data enhance ongoing, peer-reviewed projects and new funding applications. The CCBSR's scientific importance centers on the need to understand the structural basis for protein-protein, protein-DNA/RNA, and protein-ligand/drug interactions; and how dynamics affect these interactions. The use of the CCBSR has steadily increased, as have the depth and breadth of projects. From 2011?2015, the Co-Directors have worked with 27 laboratories from 11 departments (74% WFBCCC members; all with peer-reviewed funding) to publish 36 manuscripts, solve 9 new structures, perform experiments for 18 existing and newly-funded grants, and collect preliminary data for new funding applications. The CCBSR has three co-Directors (W. Todd Lowther, Ph.D.; Thomas Hollis, Ph.D.; Fred Salsbury, Ph.D.), each with different areas of scientific, experimental, and computational expertise. Therefore, after initial consultation, one or more of the Co-Directors assists the WFBCCC member with the development of their project, matching the project to the expertise of a specific Co- Director. The Specific Aims of the CCBSR are to: 1) Determine the appropriate crystallography and/or computational approach for structural analysis of important biomolecules in cancer research; 2) Conduct initial crystallization trials or simulations to generate preliminary data for grant applications; and 3) Provide facilities and expertise for funded projects to determine the structure of important biomolecules in cancer research. The crystallography component is managed by the WFBCCC but also has institutional funding. The Co-Directors attend the quarterly WFBCCC Shared Resources meeting, which includes discussions on activity, best practices, enhanced capabilities, and administrative management techniques. These meetings provide an overview of operations, finances, and utilization, and facilitate regular evaluation by WFBCCC leadership.