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Author(s): Shira L Cramer [1]; Achinto Saha [2]; Jinyun Liu [3]; Surendar Tadi [4]; Stefano Tiziani [4]; Wupeng Yan [5]; Kendra Triplett [1, 5]; Candice Lamb [1, 5]; Susan E [...]

2-chloroacetamidine is an active site-directed, time-dependent inactivator that targets the active-site cysteine residue of DDAH and likely inactivates PAD4 through a similar mechanism. 2-chloroacetamidine may serve as a general pharmacophore for inactivating this superfamily of enzymes and that incorporation of additional functional groups could allow a wide range of application in activity-based protein profiling.

Chromophobe (Ch) renal cell carcinoma (RCC) arises from the intercalated cell in the distal nephron. There are no proven treatments for metastatic ChRCC. A distinguishing characteristic of ChRCC is strikingly high levels of reduced (GSH) and oxidized (GSSG) glutathione. Here, we demonstrate that ChRCC-derived cells exhibit higher sensitivity to ferroptotic inducers compared with clear-cell RCC. ChRCC-derived cells are critically dependent on cystine via the cystine/glutamate antiporter xCT to maintain high levels of glutathione, making them sensitive to inhibitors of cystine uptake and cyst(e)inase. Gamma-glutamyl transferase 1 (GGT1), a key enzyme in glutathione homeostasis, is markedly suppressed in ChRCC relative to normal kidney. Importantly, GGT1 overexpression inhibits the proliferation of ChRCC cells in vitro and in vivo, suppresses cystine uptake, and decreases levels of GSH and GSSG. Collectively, these data identify ferroptosis as a metabolic vulnerability in ChRCC, providing a potential avenue for targeted therapy for these distinctive tumors. [ABSTRACT FROM AUTHOR]

Extensive studies in prostate cancer and other malignancies have revealed that L-methionine (L-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum L-Met, either via partial dietary restriction or with bacterial L-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial L-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode L-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of L-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum L-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis. [ABSTRACT FROM AUTHOR]

Comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I are used to demonstrate the tolerance exhibited by several metal binding ligands towards the amino acid substitutions. The comprehensive saturation mutagenesis analysis of a metallohydrolase active site revealed that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution.

The article explains the structure, bacterial expression, purification, as well as the characterization of the human asparaginase-like protein 1 (hASRGL1). The protein is shown to act as an Ntn hydrolase and hence is shown to be also useful for the removal of accumulation of the toxin peptides in mammalian brain and other tissues.

The mechanism and regulation of dimethylargininase (DDAH) activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. The results support a proposed substrate assisted mechanism for Pseudomonas aeruginosa (Pa) DDAH in which ligand binding modulates the reactivity of the active-site cysteine.

A study claims that N-acylhomoserine lactone (AHL) lactonases from Bacillus species 240B1 is not a metalloprotein. However, the gene for an AHL lactonase from Bacillus thuringiensis is cloned and the protein is expressed, purified and found to bind 2 equiv of zinc.

A study is carried out on the dimethylarginine dimethylaminohydrolase (DDAH) that regulates the concentrations of human endogenous inhibitors of nitric oxide synthase, N(sub omega)-methyl-L-arginine (NMMA), and asymmetric N(sub omega), N(sub omega)-dimethyl-L-arginine (ADMA). The studies demonstrate that covalent adduct was attached to an active site residue and implicates Cys249 as the catalytic nucleophile required for intermediate formation.

Summary Cancer cells reprogram their metabolism, altering both uptake and utilization of extracellular nutrients. We individually depleted amino acid nutrients from isogenic cells expressing commonly activated oncogenes to identify correspondences between nutrient supply and viability. In HME (human mammary epithelial) cells, deprivation of cystine led to increased cell death in cells expressing an activated epidermal growth factor receptor (EGFR) mutant. Cell death occurred via synchronous ferroptosis, with generation of reactive oxygen species (ROS). Hydrogen peroxide promoted cell death, as both catalase and inhibition of NADPH oxidase 4 (NOX4) blocked ferroptosis. Blockade of EGFR or mitogen-activated protein kinase (MAPK) signaling similarly protected cells from ferroptosis, whereas treatment of xenografts derived from EGFR mutant non-small-cell lung cancer (NSCLC) with a cystine-depleting enzyme inhibited tumor growth in mice. Collectively, our results identify a potentially exploitable sensitization of some EGFR/MAPK-driven tumors to ferroptosis following cystine depletion. [ABSTRACT FROM AUTHOR]

The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacterial expression, purification, and biochemical characterization of human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the Cys75Ala point mutation. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for the function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway. [ABSTRACT FROM AUTHOR]

Herein we report the bacterial expression, purification, and enzymatic characterization of the human asparaginase-like protein 1 (hASRGL1). We present evidence that hASRGL1 exhibits β-aspartyl peptidase activity consistent with enzymes designated as plant-type asparaginases, which had thus far been found in only plants and bacteria. Similar to nonmammalian plant-type asparaginases, hASRGL1 is shown to be an Ntn hydrolase for which Thr168 serves as the essential N-terminal nucleophile for intramolecular processing and catalysis, corroborated in part by abolishment of both activities through the Thr168A1a point mutation. In light of the activity profile reported here, ASRGL1s may act synergistically with protein L-isoaspartyl methyl transferase to relieve accumulation of potentially toxic isoaspartyl peptides in mammalian brain and other tissues. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is uniformly lethal due to profound treatment resistance. Altered cellular metabolism is a key mediator of GBM treatment resistance. Uptake of the essential sulfur-containing amino acid methionine is drastically elevated in GBMs compared to normal cells, however, it is not known how this methionine is utilized or whether it relates to GBM treatment resistance. Here, we find that radiation acutely increases the levels of methionine-related metabolites in a variety of treatment-resistant GBM models. Stable isotope tracing studies further revealed that radiation acutely activates methionine to S-adenosyl methionine (SAM) conversion through an active signaling event mediated by the kinases of the DNA damage response. In vivo tumor SAM synthesis increases after radiation, while normal brain SAM production remains unchanged, indicating a tumor- specific metabolic alteration to radiation. Pharmacological and dietary strategies to block methionine to SAM conversion slowed DNA damage response and increased cell death following radiation in vitro. Mechanistically, these effects are due to depletion of DNA repair proteins and are reversed by SAM supplementation. These effects are selective to GBMs lacking the methionine salvage enzyme methylthioadenosine phosphorylase. Pharmacological inhibition of SAM synthesis hindered tumor growth in flank and orthotopic in vivo GBM models when combined with radiation. By contrast, methionine depletion does not reduce tumor SAM levels and fails to radiosensitize intracranial models, indicating depleting SAM, as opposed to simply lowering methionine, is critical for hindering tumor growth in intracranial models of GBM. These results highlight a new signaling link between DNA damage and SAM synthesis and define the metabolic fates of methionine in GBM in vivo . Inhibiting radiation-induced SAM synthesis slows DNA repair and augments radiation efficacy in GBM. Using MAT2A inhibitors to deplete SAM may selectively overcome treatment resistance in GBMs with defective methionine salvage while sparing normal brain.

Background: Hyperactivated protein arginine methyltransferases (PRMTs) are implicated in human cancers. Inhibiting tumor intrinsic PRMT5 was reported to potentiate antitumor immune responses, highlighting the possibility of combining PRMT5 inhibitors (PRMT5i) with cancer immunotherapy. However, global suppression of PRMT5 activity impairs the effector functions of immune cells. Here, we sought to identify strategies to specifically inhibit PRMT5 activity in tumor tissues and develop effective PRMT5i-based immuno-oncology (IO) combinations for cancer treatment, particularly for methylthioadenosine phosphorylase (MTAP)-loss cancer., Methods: Isogeneic tumor lines with and without MTAP loss were generated by CRISPR/Cas9 knockout. The effects of two PRMT5 inhibitors (GSK3326595 and MRTX1719) were evaluated in these isogenic tumor lines and T cells in vitro and in vivo . Transcriptomic and proteomic changes in tumors and T cells were characterized in response to PRMT5i treatment. Furthermore, the efficacy of MRTX1719 in combination with immune checkpoint blockade was assessed in two syngeneic murine models with MTAP-loss tumor., Results: GSK3326595 significantly suppresses PRMT5 activity in tumors and T cells regardless of the MTAP status. However, MRTX1719, a methylthioadenosine-cooperative PRMT5 inhibitor, exhibits tumor-specific PRMT5 inhibition in MTAP-loss tumors with limited immunosuppressive effects. Mechanistically, transcriptomic and proteomic profiling analysis reveals that MRTX1719 successfully reduces the activation of the PI3K pathway, a well-documented immune-resistant pathway. It highlights the potential of MRTX1719 to overcome immune resistance in MTAP-loss tumors. In addition, MRTX1719 sensitizes MTAP-loss tumor cells to the killing of tumor-reactive T cells. Combining MRTX1719 and anti-PD-1 leads to superior antitumor activity in mice bearing MTAP-loss tumors., Conclusion: Collectively, our results provide a strong rationale and mechanistic insights for the clinical development of MRTX1719-based IO combinations in MTAP-loss tumors., Competing Interests: Competing interests: JRA reports non-financial support and reasonable reimbursement for travel from European Society for Medical Oncology and Loxo Oncology; receiving consulting and travel fees from Ellipses Pharma, Molecular Partners, IONCTURA, Sardona, Mekanistic, Amgen, Merus, MonteRosa, Aadi and Bridgebio (including serving on the scientific advisory board); consulting fees from Vall d’Hebron Institute of Oncology/Ministero De Empleo Y Seguridad Social, Chinese University of Hong Kong, Boxer Capital, LLC, Tang Advisors, LLC and Guidepoint, receiving research funding from Blueprint Medicines, Merck Sharp and serving as investigator in clinical trials with Cancer Core Europe, Symphogen, BioAlta, Pfizer, Kelun-Biotech, GlaxoSmithKline, Taiho, Roche Pharmaceuticals, Hummingbird, Yingli, Bicycle Therapeutics, Merus, Aadi Bioscience, ForeBio, Loxo Oncology, Hutchison MediPharma, Ideaya, Amgen, Tango Therapeutics, Mirati Therapeutics, Linnaeus Therapeutics, MonteRosa, Kinnate, Yingli, Debio, BioTheryX, Storm Therapeutics, Beigene, MapKure, Relay, Novartis, FusionPharma, C4 Therapeutics, Scorpion Therapeutics, Incyte, Fog Pharmaceuticals, Tyra, Nuvectis Pharma. MTB is a co-founder of EpiCypher. No potential conflicts of interest were disclosed by other authors., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)