Metabolic Heterogeneity, Plasticity, and Adaptation to "Glutamine Addiction" in Cancer Cells: The Role of Glutaminase and the GTωA [Glutamine Transaminase-ω-Amidase (Glutaminase II)] Pathway.

Many cancers utilize l-glutamine as a major energy source. Often cited in the literature as "l-glutamine addiction", this well-characterized pathway involves hydrolysis of l-glutamine by a glutaminase to l-glutamate, followed by oxidative deamination, or transamination, to α-ketoglutarate, which enters the tricarboxylic acid cycle. However, mammalian tissues/cancers possess a rarely mentioned, alternative pathway (the glutaminase II pathway): l-glutamine is transaminated to α-ketoglutaramate (KGM), followed by ω-amidase (ωA)-catalyzed hydrolysis of KGM to α-ketoglutarate. The name glutaminase II may be confused with the glutaminase 2 (GLS2) isozyme. Thus, we recently renamed the glutaminase II pathway the "glutamine transaminase-ω-amidase (GTωA)" pathway. Herein, we summarize the metabolic importance of the GTωA pathway, including its role in closing the methionine salvage pathway, and as a source of anaplerotic α-ketoglutarate. An advantage of the GTωA pathway is that there is no net change in redox status, permitting α-ketoglutarate production during hypoxia, diminishing cellular energy demands. We suggest that the ability to coordinate control of both pathways bestows a metabolic advantage to cancer cells. Finally, we discuss possible benefits of GTωA pathway inhibitors, not only as aids to studying the normal biological roles of the pathway but also as possible useful anticancer agents.

The metabolic importance of the glutaminase II pathway in normal and cancerous cells.

In rapidly dividing cells, including many cancer cells, l-glutamine is a major energy source. Utilization of glutamine is usually depicted as: l-glutamine → l-glutamate (catalyzed by glutaminase isozymes; GLS1 and GLS2), followed by l-glutamate → α-ketoglutarate [catalyzed by glutamate-linked aminotransferases or by glutamate dehydrogenase (GDH)]. α-Ketoglutarate is a major anaplerotic component of the tricarboxylic acid (TCA) cycle. However, the glutaminase II pathway also converts l-glutamine to α-ketoglutarate. This pathway consists of a glutamine transaminase coupled to ω-amidase [Net reaction: l-Glutamine + α-keto acid + H2O → α-ketoglutarate + l-amino acid + NH4+]. This review focuses on the biological importance of the glutaminase II pathway, especially in relation to metabolism of cancer cells. Our studies suggest a component enzyme of the glutaminase II pathway, ω-amidase, is utilized by tumor cells to provide anaplerotic carbon. Inhibitors of GLS1 are currently in clinical trials as anti-cancer agents. However, this treatment will not prevent the glutaminase II pathway from providing anaplerotic carbon derived from glutamine. Specific inhibitors of ω-amidase, perhaps in combination with a GLS1 inhibitor, may provide greater therapeutic efficacy.

The metabolic importance of the overlooked asparaginase II pathway.

The asparaginase II pathway consists of an asparagine transaminase [l-asparagine + α-keto acid ⇆ α-ketosuccinamate + l-amino acid] coupled to ω-amidase [α-ketosuccinamate + H2O → oxaloacetate + NH4+]. The net reaction is: l-asparagine + α-keto acid + H2O → oxaloacetate + l-amino acid + NH4+. Thus, in the presence of a suitable α-keto acid substrate, the asparaginase II pathway generates anaplerotic oxaloacetate at the expense of readily dispensable asparagine. Several studies have shown that the asparaginase II pathway is important in photorespiration in plants. However, since its discovery in rat tissues in the 1950s, this pathway has been almost completely ignored as a conduit for asparagine metabolism in mammals. Several mammalian transaminases can catalyze transamination of asparagine, one of which - alanine-glyoxylate aminotransferase type 1 (AGT1) - is important in glyoxylate metabolism. Glyoxylate is a precursor of oxalate which, in the form of its calcium salt, is a major contributor to the formation of kidney stones. Thus, transamination of glyoxylate with asparagine may be physiologically important for the removal of potentially toxic glyoxylate. Asparaginase has been the mainstay treatment for certain childhood leukemias. We suggest that an inhibitor of ω-amidase may potentiate the therapeutic benefits of asparaginase treatment.

HIF1α stabilization in hypoxia is not oxidant-initiated.

Hypoxic adaptation mediated by HIF transcription factors requires mitochondria, which have been implicated in regulating HIF1α stability in hypoxia by distinct models that involve consuming oxygen or alternatively converting oxygen into the second messenger peroxide. Here, we use a ratiometric, peroxide reporter, HyPer to evaluate the role of peroxide in regulating HIF1α stability. We show that antioxidant enzymes are neither homeostatically induced nor are peroxide levels increased in hypoxia. Additionally, forced expression of diverse antioxidant enzymes, all of which diminish peroxide, had disparate effects on HIF1α protein stability. Moreover, decrease in lipid peroxides by glutathione peroxidase-4 or superoxide by mitochondrial SOD, failed to influence HIF1α protein stability. These data show that mitochondrial, cytosolic or lipid ROS were not necessary for HIF1α stability, and favor a model where mitochondria contribute to hypoxic adaptation as oxygen consumers.

High Levels of Glutaminase II Pathway Enzymes in Normal and Cancerous Prostate Suggest a Role in 'Glutamine Addiction'.

Abstract: Many tumors readily convert l-glutamine to α-ketoglutarate. This conversion is almost invariably described as involving deamidation of l-glutamine to l-glutamate followed by a transaminase (or dehydrogenase) reaction. However, mammalian tissues possess another pathway for conversion of l-glutamine to α-ketoglutarate, namely the glutaminase II pathway: l-Glutamine is transaminated to α-ketoglutaramate, which is then deamidated to α-ketoglutarate by ω-amidase. Here we show that glutamine transaminase and ω-amidase specific activities are high in normal rat prostate. Immunohistochemical analyses revealed that glutamine transaminase K (GTK) and ω-amidase are present in normal and cancerous human prostate and that expression of these enzymes increases in parallel with aggressiveness of the cancer cells. Our findings suggest that the glutaminase II pathway is important in providing anaplerotic carbon to the tricarboxylic acid (TCA) cycle, closing the methionine salvage pathway, and in the provision of citrate carbon in normal and cancerous prostate. Finally, our data also suggest that selective inhibitors of GTK and/or ω-amidase may be clinically important for treatment of prostate cancer. In conclusion, the demonstration of a prominent glutaminase II pathway in prostate cancer cells and increased expression of the pathway with increasing aggressiveness of tumor cells provides a new perspective on 'glutamine addiction' in cancers.

N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and can synergize with prostaglandin E2 to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice.

OBJECTIVES: N-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke. METHODS: Hemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC. RESULTS: NAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ). INTERPRETATION: NAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.

Cystamine and cysteamine as inhibitors of transglutaminase activity in vivo.

Cystamine is commonly used as a transglutaminase inhibitor. This disulphide undergoes reduction in vivo to the aminothiol compound, cysteamine. Thus, the mechanism by which cystamine inhibits transglutaminase activity in vivo could be due to either cystamine or cysteamine, which depends on the local redox environment. Cystamine inactivates transglutaminases by promoting the oxidation of two vicinal cysteine residues on the enzyme to an allosteric disulphide, whereas cysteamine acts as a competitive inhibitor for transamidation reactions catalyzed by this enzyme. The latter mechanism is likely to result in the formation of a unique biomarker, N-(γ-glutamyl)cysteamine that could serve to indicate how cyst(e)amine acts to inhibit transglutaminases inside cells and the body.

Sulfur amino acid restriction-induced changes in redox-sensitive proteins are associated with slow protein synthesis rates.

The mechanisms underlying life span extension by sulfur amino acid restriction (SAAR) are unclear. Cysteine and methionine are essential for the biosynthesis of proteins and glutathione (GSH), a major redox buffer in the endoplasmic reticulum (ER). We hypothesized that SAAR alters protein synthesis by modulating the redox milieu. Male F344-rats were fed control (CD: 0.86% methionine without cysteine) and SAAR diets (0.17% methionine without cysteine) for 12 weeks. Growth rates, food intake, cysteine and GSH levels, proteins associated with redox status and translation, and fractional protein synthesis rates (FSRs) were determined in liver. Despite a 40% higher food intake, growth rates for SAAR rats were 27% of those fed CD. Hepatic free cysteine in SAAR rats was 55% compared with CD rats. SAAR altered tissue distribution of GSH, as hepatic and erythrocytic levels were 56% and 196% of those in CD rats. Lower GSH levels did not induce ER stress (i.e., unchanged expression of Xbp1s , Chop, and Grp78), but activated PERK and its substrates eIF2-α and NRF2. SAAR-induced changes in translation-initiation machinery (higher p-eIF2-α and 4E-BP1, and lower eIF4G-1) resulted in slower protein synthesis rates (53% of CD). Proteins involved in the antioxidant response (NRF2, KEAP1, GCLM, and NQO1) and protein folding (PDI and ERO1-α) were increased in SAAR. Lower FSR and efficient protein folding might be improving proteostasis in SAAR.

Application of open-access databases to determine functional connectivity between resveratrol-binding protein QR2 and colorectal carcinoma.

Colorectal cancer (CRC) is a major cause of cancer-associated deaths worldwide. Recently, oral administration of resveratrol (trans-3,5,4'-trihydroxystilbene) has been reported to significantly reduce tumor proliferation in colorectal cancer patients, however, with little specific information on functional connections. The pathogenesis and development of colorectal cancer is a multistep process that can be categorized using three phenotypic pathways, respectively, chromosome instability (CIN), microsatellite instability (MSI), and CpG island methylator (CIMP). Targets of resveratrol, including a high-affinity binding protein, quinone reductase 2 (QR2), have been identified with little information on disease association. We hypothesize that the relationship between resveratrol and different CRC etiologies might be gleaned using publicly available databases. A web-based microarray gene expression data-mining platform, Oncomine, was selected and used to determine whether QR2 may serve as a mechanistic and functional biotarget within the various CRC etiologies. We found that QR2 messenger RNA (mRNA) is overexpressed in CRC characterized by CIN, particularly in cells showing a positive KRAS (Kirsten rat sarcoma viral oncogene homolog) mutation, as well as by the MSI but not the CIMP phenotype. Mining of Oncomine revealed an excellent correlation between QR2 mRNA expression and certain CRC etiologies. Two resveratrol-associated genes, adenomatous polyposis coli (APC) and TP53, found in CRC were further mined, using cBio portal and Colorectal Cancer Atlas which predicted a mechanistic link to exist between resveratrol→QR2/TP53→CIN. Multiple web-based data mining can provide valuable insights which may lead to hypotheses serving to guide clinical trials and design of therapies for enhanced disease prognosis and patient survival. This approach resembles a BioGPS, a capability for mining web-based databases that can elucidate the potential links between compounds to provide correlations of these interactions with specific diseases.

Chymase-dependent production of angiotensin II: an old enzyme in old hearts.

Age-dependent alteration of the renin-angiotensin system (RAS) and generation of angiotensin II (Ang II) are well documented. By contrast, RAS-independent generation of Ang II in aging and its responses to exercise have not been explored. To this end, we examined the effects of chymase, a secretory serine protease, on the angiotensin-converting enzyme (ACE)-independent conversion of Ang I to Ang II. We hypothesized that age-dependent alteration of cardiac Ang II formation is chymase dependent in nature and is prevented by exercise training. Experiments were conducted on hearts isolated from young (3 mo), aged sedentary (24 mo), and aged rats chronically exercised on a treadmill. In the presence of low Ang I levels and downregulation of ACE expression/activity, cardiac Ang II levels were significantly higher in aged than young rats, suggesting an ACE-independent response. Aged hearts also displayed significantly increased chymase expression and activity, as well as upregulation of tryptase, a biological marker of mast cells, confirming a mast cell-sourced increase in chymase. Coincidently, cardiac superoxide produced from NADPH oxidase (Nox) was significantly enhanced in aged rats and was normalized by exercise. Conversely, a significant reduction in cardiac expression of ACE2 followed by lower Ang 1-7 levels and downregulation of the Mas receptor (binding protein of Ang 1-7) in aged rats were completely reversed by exercise. In conclusion, local formation of Ang II is increased in aged hearts, and chymase is primarily responsible for this increase. Chronic exercise is able to normalize the age-dependent alterations via compromising chymase/Ang II/angiotensin type 1 receptor/Nox actions while promoting ACE2/Ang 1-7/MasR signaling. NEW & NOTEWORTHY: Aging increases angiotensin-converting enzyme (ACE)-independent production of cardiac angiotensin II (Ang II), a response that is driven by chymase in an exercise-reversible manner. These findings highlight chymase, in addition to ACE, as an important therapeutic target in the treatment and prevention of Ang II-induced deterioration of cardiac function in the elderly.

ω-Amidase: an underappreciated, but important enzyme in L-glutamine and L-asparagine metabolism; relevance to sulfur and nitrogen metabolism, tumor biology and hyperammonemic diseases.

In mammals, two major routes exist for the metabolic conversion of L-glutamine to α-ketoglutarate. The most widely studied pathway involves the hydrolysis of L-glutamine to L-glutamate catalyzed by glutaminases, followed by the conversion of L-glutamate to α-ketoglutarate by the action of an L-glutamate-linked aminotransferase or via the glutamate dehydrogenase reaction. However, another major pathway exists in mammals for the conversion of L-glutamine to α-ketoglutarate (the glutaminase II pathway) in which L-glutamine is first transaminated to α-ketoglutaramate (KGM) followed by hydrolysis of KGM to α-ketoglutarate and ammonia catalyzed by an amidase known as ω-amidase. In mammals, the glutaminase II pathway is present in both cytosolic and mitochondrial compartments and is most prominent in liver and kidney. Similarly, two routes exist for the conversion of L-asparagine to oxaloacetate. In the most extensively studied pathway, L-asparagine is hydrolyzed to L-aspartate by the action of asparaginase, followed by transamination of L-aspartate to oxaloacetate. However, another pathway also exists for the conversion of L-asparagine to oxaloacetate (the asparaginase II pathway). In this pathway, L-asparagine is first transaminated to α-ketosuccinamate (KSM), followed by hydrolysis of KSM to oxaloacetate by the action of ω-amidase. One advantage of both the glutaminase II and the asparaginase II pathways is that they are irreversible, and thus are important in anaplerosis by shuttling 5-C (α-ketoglutarate) and 4-C (oxaloacetate) units into the TCA cycle. In this review, we briefly mention the importance of the glutaminase II and asparaginase II pathways in microorganisms and plants. However, the major emphasis of the review is related to the importance of these pathways (especially the common enzyme component of both pathways--ω-amidase) in nitrogen and sulfur metabolism in mammals and as a source of anaplerotic carbon moieties in rapidly dividing cells. The review also discusses a potential dichotomous function of ω-amidase as having a role in tumor progression. Finally, the possible role of KGM as a biomarker for hyperammonemic diseases is discussed.

Fluorination at the 4 position alters the substrate behavior of L-glutamine and L-glutamate: Implications for positron emission tomography of neoplasias.

Two 4-fluoro-L-glutamine diastereoisomers [(2S,4R)-4-FGln, (2S,4S)-4-FGln] were previously developed for positron emission tomography. Label uptake into two tumor cell types was greater with [18F](2S,4R)-4-FGln than with [18F](2S,4S)-4-FGln. In the present work we investigated the enzymology of two diastereoisomers of 4-FGln, two diastereoisomers of 4-fluoroglutamate (4-FGlu) (potential metabolites of the 4-FGln diastereoisomers) and another fluoro-derivative of L-glutamine [(2S,4S)-4-(3-fluoropropyl)glutamine (FP-Gln)]. The two 4-FGlu diastereoisomers were found to be moderate-to-good substrates relative to L-glutamate of glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase. Additionally, alanine aminotransferase was shown to catalyze an unusual γ-elimination reaction with both 4-FGlu diastereoisomers. Both 4-FGlu diastereoisomers were shown to be poor substrates, but strong inhibitors of glutamine synthetase. Both 4-FGln diastereoisomers were shown to be poor substrates compared to L-glutamine of glutamine transaminase L and α-aminoadipate aminotransferase. However, (2S,4R)-4-FGln was found to be a poor substrate of glutamine transaminase K, whereas (2S,4S)-4-FGln was shown to be an excellent substrate. By contrast, FP-Gln was found to be a poor substrate of all enzymes examined. Evidently, substitution of H in position 4 by F in L-glutamine/L-glutamate has moderate-to-profound effects on enzyme-catalyzed reactions. The present results: 1) show that 4-FGln and 4-FGlu diastereoisomers may be useful for studying active site topology of glutamate- and glutamine-utilizing enzymes; 2) provide a framework for understanding possible metabolic transformations in tumors of 18F-labeled (2S,4R)-4-FGln, (2S,4S)-4-FGln, (2S,4R)-4-FGlu or (2S,4S)-4-FGlu; and 3) show that [18F]FP-Gln is likely to be much less metabolically active in vivo than are the [18F]4-FGln diastereoisomers.

Activation of NQO1 in NQO1*2 polymorphic human leukemic HL-60 cells by diet-derived sulforaphane.

BACKGROUND: The NAD(P)H: quinone oxidoreductase (NQO1) confers protection against semiquinones and also elicits oxidative stress. The C609T polymorphism of the NQO1 gene, designated NQO1*2, significantly reduces its enzymatic activity due to rapid degradation of protein. Since down regulation of NQO1 mRNA expression correlates with increased susceptibility for developing different types of cancers, we investigated the link between leukemia and the NQO1*2 genotype by mining a web-based microarray dataset, ONCOMINE. Phytochemicals prevent DNA damage through activation of phase II detoxification enzymes including NQO1. Whether NQO1 expression/activity in leukemia cells that carry the labile NQO1*2 genotype can be induced by broccoli-derived phytochemical sulforaphane (SFN) is currently unknown. METHODS AND RESULTS: The ONCOMINE query showed that: (1) acute lymphoblastic leukemia and chronic myelogenous leukemia are associated with reduced NQO1 levels, and (2) under-expressed NQO1 was found in human HL-60 leukemia cell line containing the heterozygous NQO1*2 polymorphism. We examined induction of NQO1 activity/expression by SFN in HL-60 cells. A dose-dependent increase in NQO1 level/activity is accompanied by upregulation of the transcription factor, Nrf2, following 1-10 µM SFN treatment. Treatment with 25 µM SFN drastically reduced NQO1 levels, inhibited cell proliferation, caused sub-G1 cell arrest, and induced apoptosis, and a decrease in the levels of the transcription factor, nuclear factor-κB (NFκB). CONCLUSIONS: Up to 10 µM of SFN increases NQO1 expression and suppresses HL-60 cell proliferation whereas ≥ 25 µM of SFN induces apoptosis in HL-60 cells. Further, SFN treatment restores NQO1 activity/levels in HL-60 cells expressing the NQO1*2 genotype.

Role of homocysteinylation of ACE in endothelial dysfunction of arteries.

The direct impact of de novo synthesis of homocysteine (Hcy) and its reactive metabolites, Hcy-S-S-Hcy and Hcy thiolactone (HCTL), on vascular function has not been fully elucidated. We hypothesized that Hcy synthesized within endothelial cells affects activity of angiotensin-converting enzyme (ACE) by direct homocysteinylation of its amino- and/or sulfhydryl moieties. This covalent modification enhances ACE reactivity toward angiotensin II (ANG II)-NADPH oxidase-superoxide-dependent endothelial dysfunction. Mesenteric and coronary arteries isolated from normal rats were incubated for 3 days with or without exogenous methionine (Met, 0.1-0.3 mM), a precursor to Hcy. Incubation of arteries in Met-free media resulted in time-dependent decreases in vascular Hcy formation. By contrast, vessels incubated with Met produced Hcy in a dose-dependent manner. There was a notably greater de novo synthesis of Hcy from endothelial than from smooth muscle cells. Enhanced levels of Hcy production significantly impaired shear stress-induced dilation and release of nitric oxide, events that are associated with elevated production of vascular superoxide. Each of these processes was attenuated by ANG II type I receptor blocker or ACE and NADPH oxidase inhibitors. In addition, in vitro exposure of purified ACE to Hcy-S-S-Hcy/HCTL resulted in formation of homocysteinylated ACE and an enhanced ACE activity. The enhanced ACE activity was confirmed in isolated coronary and mesenteric arteries that had been exposed directly to Hcy-S-S-Hcy/HCTL or after Met incubation. In conclusion, vasculature-derived Hcy initiates endothelial dysfunction that, in part, may be mediated by ANG II-dependent activation of NADPH oxidase in association with homocysteinylation of ACE.

Resveratrol encapsulated in novel fusogenic liposomes activates Nrf2 and attenuates oxidative stress in cerebromicrovascular endothelial cells from aged rats.

Resveratrol (3,4',5-trihydroxystilbene) is a plant-derived polyphenolic trans-stilbenoid, which exerts multifaceted antiaging effects. Here, we propose a novel delivery system for resveratrol, which significantly increases its cellular uptake into aged cells. Combination of resveratrol with a positively charged lipid component to "conventional" liposomes converts these lipid vesicles to a robust fusogenic system. To study their cellular uptake and cellular effects, we treated primary cerebromicrovascular endothelial cells isolated from aged F344xBN rats with resveratrol encapsulated in fusogenic liposomes (FL-RSV). To demonstrate effective cellular uptake of FL-RSV, accumulation of the lipophilic tracer dye, DiR, and resveratrol in cerebromicrovascular endothelial cells was confirmed using flow cytometry and confocal microscopy and high-performance liquid chromatography electrochemical detection. Treatment of aged cerebromicrovascular endothelial cells with FL-RSV activated Nrf2 (assessed with a reporter gene assay), significantly decreased cellular production of reactive oxygen species (assessed by a flow cytometry-based H2DCFDA fluorescence method), and inhibited apoptosis. Taken together, encapsulation of resveratrol into novel fusogenic liposomes significantly enhances the delivery of resveratrol into aged cells, which subsequently results in rapid activation of cellular Nrf2-driven antioxidant defense mechanisms. Our studies provide proof-of-concept for the development of a novel, translationally relevant interventional strategy for prevention and/or control of oxidative stress-related pathophysiological conditions in aging.

Hydroxamic acid-based histone deacetylase (HDAC) inhibitors can mediate neuroprotection independent of HDAC inhibition.

Histone deacetylase (HDAC) inhibition improves function and extends survival in rodent models of a host of neurological conditions, including stroke, and neurodegenerative diseases. Our understanding, however, of the contribution of individual HDAC isoforms to neuronal death is limited. In this study, we used selective chemical probes to assess the individual roles of the Class I HDAC isoforms in protecting Mus musculus primary cortical neurons from oxidative death. We demonstrated that the selective HDAC8 inhibitor PCI-34051 is a potent neuroprotective agent; and by taking advantage of both pharmacological and genetic tools, we established that HDAC8 is not critically involved in PCI-34051's mechanism of action. We used BRD3811, an inactive ortholog of PCI-34051, and showed that, despite its inability to inhibit HDAC8, it exhibits robust neuroprotective properties. Furthermore, molecular deletion of HDAC8 proved insufficient to protect neurons from oxidative death, whereas both PCI-34051 and BRD3811 were able to protect neurons derived from HDAC8 knock-out mice. Finally, we designed and synthesized two new, orthogonal negative control compounds, BRD9715 and BRD8461, which lack the hydroxamic acid motif and showed that they stably penetrate cell membranes but are not neuroprotective. These results indicate that the protective effects of these hydroxamic acid-containing small molecules are likely unrelated to direct epigenetic regulation via HDAC inhibition, but rather due to their ability to bind metals. Our results suggest that hydroxamic acid-based HDAC inhibitors may mediate neuroprotection via HDAC-independent mechanisms and affirm the need for careful structure-activity relationship studies when using pharmacological approaches.

Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate ß-lyase activity and can transaminate L-selenomethionine.

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate ß-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no ß-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate ß-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate ß-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to ß-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the ß-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze ß-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites.