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The Journal of Biological Chemistry Oct 2012Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique...
Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.
Topics: Amidohydrolases; Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Bacterial Proteins; Biocatalysis; Catalytic Domain; Diazooxonorleucine; Enzyme Inhibitors; Hydrolysis; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mycobacterium tuberculosis; Structural Homology, Protein
PubMed: 22942282
DOI: 10.1074/jbc.M112.384784 -
Scientific Reports May 2018Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for...
Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for bacteria. Here we demonstrate that the development of B. subtilis biofilms is dependent on the use of glutamine or glutamate as a nitrogen source. We show a differential metabolic requirement within the biofilm; while glutamine is necessary for the dividing cells at the edges, the inner cell mass utilizes lactic acid. Our results indicate that biofilm cells preserve a short-term memory of glutamate metabolism. Finally, we establish that drugs that target glutamine and glutamate utilization restrict biofilm development. Overall, our work reveals a spatial regulation of nitrogen and carbon metabolism within the biofilm, which contributes to the fitness of bacterial complex communities. This acquired metabolic division of labor within biofilm can serve as a target for novel anti-biofilm chemotherapies.
Topics: Aminooxyacetic Acid; Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Biofilms; Carbon; Diazooxonorleucine; Gene Expression Regulation, Bacterial; Glutamic Acid; Glutamine; Mutant Proteins; Nitrogen; Repressor Proteins
PubMed: 29740028
DOI: 10.1038/s41598-018-25401-z -
Scientific Reports Apr 2019Glutamine metabolism and the mechanistic target of rapamycin (mTOR) pathway are activated cooperatively in the differentiation and activation of inflammatory immune...
Glutamine metabolism and the mechanistic target of rapamycin (mTOR) pathway are activated cooperatively in the differentiation and activation of inflammatory immune cells. But the combined inhibition of both pathways was rarely investigated. This study investigated how inhibiting both glutamine metabolism with 6-diazo-5-oxo-L-norleucine (DON) and mTOR with rapamycin affects immune cells and the arthritis in a mouse model. We revealed that rapamycin and DON additively suppressed CD4 T cell proliferation, and both of them inhibited Th17 cell differentiation. While DON inhibited the differentiation of dendritic cells and macrophages and facilitated that of Ly6G granulocytic (G)-MDSCs more strongly than did rapamycin, G-MDSCs treated with rapamycin but not DON suppressed CD4 T cell proliferation in vitro. The combination of rapamycin and DON significantly suppressed the arthritis in SKG mice more strongly than did each monotherapy in vivo. The numbers of CD4 T and Th17 cells in the spleen were lowest in mice treated with the combination therapy. Thus, combined treatment with rapamycin and DON additively ameliorated the arthritis in SKG mice, possibly by suppressing CD4 T cell proliferation and Th17 differentiation. These results suggest the combination of rapamycin and DON may be a potential novel therapy for arthritis.
Topics: Animals; Arthritis; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Dendritic Cells; Diazooxonorleucine; Female; Glutamine; Immunosuppression Therapy; Macrophages; Mice, Inbred BALB C; Myeloid-Derived Suppressor Cells; Sirolimus; TOR Serine-Threonine Kinases; Th17 Cells
PubMed: 31011190
DOI: 10.1038/s41598-019-42932-1 -
Journal of Bacteriology Sep 1987The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense...
The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.
Topics: Alanine; Ammonia; Glutamate Dehydrogenase; Glutamate Synthase; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Gram-Negative Bacteria; Nitrogen; Transaminases
PubMed: 2887545
DOI: 10.1128/jb.169.9.4211-4214.1987 -
Biochimie Sep 1985In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but...
In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.
Topics: Amidohydrolases; Binding, Competitive; Glutaminase; Kinetics; Molecular Weight; Multienzyme Complexes; Saccharomyces cerevisiae; Substrate Specificity
PubMed: 3910115
DOI: 10.1016/s0300-9084(85)80294-7 -
Journal of Virology Oct 2016Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system...
UNLABELLED
Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell-deficient mice and in mice with pharmacological inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of the glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed the onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines, and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease.
IMPORTANCE
Encephalomyelitis due to infection with mosquito-borne alphaviruses is an important cause of death and of long-term neurological disability in those who survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurological disease. DON, a glutamine antagonist, inhibited the proliferation of lymphocytes in response to infection, prevented the development of brain inflammation, and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of the virus from the brain was also prevented. When treatment was stopped, the immune response was generated, brain inflammation occurred, virus was cleared, and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage.
Topics: Alphavirus; Alphavirus Infections; Animals; Cells, Cultured; Central Nervous System; Cytokines; Diazooxonorleucine; Encephalitis; Encephalitis, Viral; Encephalomyelitis; Female; Glutamine; Lymphocytes; Mice; Mice, Inbred C57BL; Paralysis; Sindbis Virus; Virus Replication
PubMed: 27489275
DOI: 10.1128/JVI.01045-16 -
International Journal of Cancer Nov 2010Metastatic cancer is a major cause of morbidity and mortality. Current therapeutic options consist of chemotherapy, radiation or targeted therapies. However, these...
Metastatic cancer is a major cause of morbidity and mortality. Current therapeutic options consist of chemotherapy, radiation or targeted therapies. However, these therapies are often toxic, effective over a small range of cancer types or result in drug resistance. Therefore, a more global, less toxic strategy for the management of metastatic cancer is required. Although most cancers display increased glucose metabolism, glutamine is also a major energy substrate for many cancers. We evaluated the antimetastatic potential of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog, using the new VM mouse model of systemic metastasis. We found that primary tumor growth was ∼20-fold less in DON-treated mice than in untreated control mice. We also found that DON treatment inhibited metastasis to liver, lung and kidney as detected by bioluminescence imaging and histology. Our findings provide proof of concept that metabolic therapies targeting glutamine metabolism can manage systemic metastatic cancer.
Topics: Animals; Antimetabolites, Antineoplastic; Blood Glucose; Body Weight; Brain Neoplasms; Caloric Restriction; Cell Growth Processes; Cell Line, Tumor; Cerebrum; Diazooxonorleucine; Drug Delivery Systems; Female; Glucose; Glutamine; Male; Mice; Neoplasm Metastasis
PubMed: 20473919
DOI: 10.1002/ijc.25431 -
Molecular Cancer Therapeutics Oct 2022Glutamine is a conditionally essential amino acid consumed by rapidly proliferating cancer cells, which deprives the same fuel from immune cells and contributes to tumor...
Glutamine is a conditionally essential amino acid consumed by rapidly proliferating cancer cells, which deprives the same fuel from immune cells and contributes to tumor immune evasion. As such, the broad antagonism of glutamine in tumors and the tumor microenvironment may lead to direct antitumor activity and stimulation of antitumoral immune responses. DRP-104 (sirpiglenastat) was designed as a novel prodrug of the broad-acting glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). DRP-104 is an inactive form that is preferentially converted to DON within tumors. Metabolomic profiling of tumors treated with DRP-104 revealed widespread changes indicative of the disruption of tumor anabolism and canonical cancer metabolism pathways; including altered glutamine metabolism while several immunosuppressive metabolites were decreased. Gene expression profiling revealed broad immunological modulation, confirmed by flow cytometry indicating that DRP-104 treatment resulted in substantial and broad changes in various immune cell infiltrates, such as increased TIL, T, NK, and NK T cells. Functionally, T cells became more proliferative and less exhausted; tumor-associated macrophages were polarized to the M1 phenotype; MDSCs and protumorigenic proteins were decreased in TME. Finally, DRP-104 demonstrated significant antitumor activity as a monotherapy, which was further enhanced in combination with checkpoint blockade therapies, leading to improved survival and long-term durable cures. In summary, DRP-104 broadly remodels the tumor microenvironment by inducing extensive tumor metabolism effects and enhancing the infiltration and function of multiple immune cells distinct from those obtained by checkpoint inhibitor therapy. This unique mechanism of action supports the ongoing clinical development of DRP-104 alone and in combination with checkpoint inhibitors.
Topics: Amino Acids, Essential; Cell Line, Tumor; Diazooxonorleucine; Glutamine; Humans; Immune System; Neoplasms; Prodrugs; Tumor Microenvironment
PubMed: 35930753
DOI: 10.1158/1535-7163.MCT-22-0282 -
Biochemical and Biophysical Research... Jul 2003To elucidate the role of acetyl-L-carnitine in the brain, we used a novel method, 'Bioradiography,' in which the dynamic process could be followed in living slices by...
To elucidate the role of acetyl-L-carnitine in the brain, we used a novel method, 'Bioradiography,' in which the dynamic process could be followed in living slices by use of positron-emitter labeled compounds and imaging plates. We studied the incorporation of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) into rat brain slices incubated in oxygenated Krebs-Ringer solution. Under the glucose-free condition, [18F]FDG uptake rate decreased with time and plateaued within 350 min in the cerebral cortex and cerebellum, and the addition of 1 or 5mM acetyl-L-carnitine did not alter the [18F]FDG uptake rate. When a glutaminase inhibitor, 0.5mM 6-diazo-5-oxo-L-norleucine (DON), was added under the normal glucose condition, [18F]FDG uptake rate decreased. Acetyl-L-carnitine (1mM), which decreased [18F]FDG uptake rate, reversed this DON-induced decrease in [18F]FDG uptake rate in the cerebral cortex. These results suggest that acetyl-L-carnitine can be used for the production of releasable glutamate rather than as an energy source in the brain.
Topics: Acetylcarnitine; Animals; Antimetabolites, Antineoplastic; Brain; Cerebral Cortex; Diazooxonorleucine; Fluorodeoxyglucose F18; Glucose; Glutamic Acid; Male; Models, Biological; Radiography; Rats; Rats, Sprague-Dawley; Time Factors; Tomography, Emission-Computed
PubMed: 12821151
DOI: 10.1016/s0006-291x(03)01103-3 -
Nature Oct 2013Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be...
Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.
Topics: Acetylglucosamine; Animals; Arrhythmias, Cardiac; Benzylamines; Brain; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Diabetes Complications; Diazooxonorleucine; Enzyme Activation; Glucose; Glycosylation; Humans; Hyperglycemia; Mice; Myocardium; Myocytes, Cardiac; Rats; Sarcoplasmic Reticulum; Sulfonamides
PubMed: 24077098
DOI: 10.1038/nature12537