The Identification of Gamma-Glutamyl Hydrolase in Uterine Corpus Endometrial Carcinoma: a Predictive Model and Machine Learning.

BACKGROUND: Poor neoplastic differentiation contributes to the rapid progression of uterine corpus endometrial carcinoma (UCEC). Thus, it is essential to identify candidate genes, clarifying the carcinogenesis and progression of UCEC. METHODS: We screened genes that affect differentiation and prognosis in UCEC. Least absolute selection and shrinkage operator (LASSO) regression, univariate Cox, and multivariate Cox proportional risk regression analyses were performed to screen out γ-glutamyl hydrolase (GGH) as the candidate gene. The clinical value of GGH on prognosis was evaluated. The relationship between GGH and immune infiltration was assessed by CIBERSORT, EPIC, ssGSEA, unsupervised clustering and immunohistochemistry (IHC). Additionally, we investigated the effect of GGH knockdown in vitro. RESULTS: Among the GGH, CDKN2A, and SIX1 genes, the impact of GGH was predominant on immune infiltration in UCEC. A nomogram containing GGH and other clinical features showed good predictive performance via curve analysis (DCA). In the functional analysis, GGH affected differentiation, tumour proliferation, and immune regulation. The immunosuppressive components were enriched in the GGH-high group, with poor immunotherapy efficacy. The study suggests that GGH may influence the progression of UCEC by regulating the glycolytic process. CONCLUSIONS: GGH is closely associated with various immune cell infiltrations. Our study demonstrates the prognostic role of GGH in carcinogenesis in UCEC.

Methotrexate level discrepancy post-glucarpidase: A pediatric case series and review of literature.

Methotrexate is a common component of pediatric oncology treatment and delayed clearance increases risk of significant toxicities. Glucarpidase is indicated for patients with toxic plasma methotrexate concentrations with renal toxicity. Laboratory interference with immunoassay measurement post-glucarpidase administration is well established, with current product labeling indicating this persists for 48 h. However, recent experience in pediatric patients supports this discrepancy persists beyond 48 h. Three cases experienced delayed methotrexate clearance and received glucarpidase with subsequent measurement of methotrexate levels by liquid chromatography tandem mass spectrometry (LC-MS/MS) and/or immunoassay. Within this case series, discrepancies between LC-MS/MS and immunoassay levels persisted significantly longer than 48 h.

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress.

Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase Rad6. How Rad6-mediated ubiquitination of ribosomes affects translation, however, is unclear. We therefore perform Ribo-seq and Disome-seq in Saccharomyces cerevisiae and show that oxidative stress causes ribosome pausing at specific amino acid motifs, which also leads to ribosome collisions. However, these redox-pausing signatures are lost in the absence of Rad6 and do not depend on the ribosome-associated quality control (RQC) pathway. We also show that Rad6 is needed to inhibit overall translation in response to oxidative stress and that its deletion leads to increased expression of antioxidant genes. Finally, we observe that the lack of Rad6 leads to changes during translation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation.

The use of glucarpidase as a rescue therapy for high dose methotrexate toxicity - a review of pharmacological and clinical data.

INTRODUCTION: This review aims to summarize recent data on the pharmacodynamic, pharmacokinetic, and safety of glucarpidase. This is an enzymatic agent that catalyzes the conversion of methotrexate (MTX) into inactive metabolites. Glucarpidase is used to manage high-dose MTX (HDMTX) toxic plasma concentration, especially in patients with impaired renal function. AREAS COVERED: In this review, studies on glucarpidase clinical efficacy as a therapeutic option for patients suffering from MTX kidney toxicity were presented. Pharmacodynamic and pharmacokinetic properties of glucarpidase were included. Moreover, potential interactions and safety issues were discussed. EXPERT OPINION: The use of glucarpidase is an effective therapeutic strategy in both adults and children treated with high doses of MTX for various types of cancer who have developed acute renal failure. Glucarpidase causes MTX to be converted to nontoxic metabolites and accelerates the time for its complete elimination. After administration of glucarpidase, it is possible to resume HDMTX.

Development of a p-Hydroxybenzyl-Alcohol-Linked Glutamate Prodrug for Activation by Pseudomonas Carboxypeptidase G2.

Directed enzyme-prodrug therapies used for targeted drug delivery require prodrugs that are chemically stable and processed efficiently by the activating enzyme. We recently reported the development of AMS-6-Glu (2), a glutamate-masked version of the cytotoxic natural product 5'-O-sulfamoyladenosine (AMS, 1) that can be activated by Pseudomonas carboxypeptidase G2 (CPG2). Herein, we report the development of a second-generation prodrug, AMS-5'-PHOBA-Glu (5), that undergoes cleavage by CPG2 with >160-fold higher efficiency. Use of a p-hydroxybenzyl alcohol (PHOBA) self-immolative linker overcame unexpected chemical instability observed with a conventional p-aminobenzyl alchohol (PABA) linker.

Glucarpidase (carboxypeptidase G2): Biotechnological production, clinical application as a methotrexate antidote, and placement in targeted cancer therapy.

Patients receiving high-dose methotrexate (HDMTX) for malignancies are exposed to diverse complications, including nephrotoxicity, hepatotoxicity, mucositis, myelotoxicity, neurological symptoms, and death. Glucarpidase is a recombinant carboxypeptidase G2 (CPG2) that converts MTX into nontoxic metabolites. In this study, the role of vector type, gene optimization, orientation, and host on the expression of CPG2 is investigated. The effectiveness of various therapeutic regimens containing glucarpidase is classified and perspectives on the dose adjustment based on precision medicine are provided. Conjugation with cell-penetrating peptides, human serum albumin, and polymers such as PEG and dextran for delivery, higher stability, and production of the biobetter variants of CPG2 is highlighted. Conjugation of CPG2 to F(ab՜)2 or scFv antibody fragments against tumor-specific antigens and the corresponding prodrugs for tumor-targeted drug delivery using the antibody-directed enzyme prodrug therapy (ADEPT) is communicated. Trials to reduce the off-target effects and the possibility of repeated ADEPT cycles by adding pro-domains sensitive to tumor-overexpressed proteases, antiCPG2 antibodies, CPG2 mutants with immune-system-unrecognizable epitopes, and protective polymers are reported. Intracellular cpg2 gene expression by gene-directed enzyme prodrug therapy (GDEPT) and the concerns regarding the safety and transfection efficacy of the GDEPT vectors are described. A novel bifunctional platform using engineered CAR-T cell micropharmacies, known as Synthetic Enzyme-Armed KillER (SEAKER) cells, expressing CPG2 to activate prodrugs at the tumor niche is introduced. Taken together, integrated data in this review and recruiting combinatorial strategies in novel drug delivery systems define the future directions of ADEPT, GDEPT, and SEAKER cell therapy and the placement of CPG2 therein.

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6.

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6-HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.

Pharmacokinetics and Pharmacodynamics of Glucarpidase Rescue Treatment After High-dose Methotrexate Therapy Based on Modeling and Simulation.

BACKGROUND: Model-informed approaches are important in drug development, including for dose optimization and the collection of evidence in support of efficacy. MATERIALS AND METHODS: We developed a modified Michaelis-Menten pharmacokinetics/pharmacodynamics model and used it to conduct simulations of glucarpidase at doses between 10 and 80 U/kg rescue treatment after high-dose methotrexate therapy. We carried out a dose-finding modeling and simulation study before a phase II study of glucarpidase. Monte-Carlo simulations were conducted using the deSolve package of R software (version 4.1.2). The proportion of samples in which the plasma methotrexate concentration was less than 0.1 and 1.0 µmol/l at 70 and 120 h after methotrexate treatment was evaluated for each dosage of glucarpidase. RESULTS: The proportion of samples in which the plasma methotrexate concentration was less than 0.1 µmol/l at 70 h after methotrexate treatment was 71.8% and 89.6% at 20 and 50 U/kg of glucarpidase, respectively. The proportion of samples in which the plasma methotrexate concentration was less than 0.1 µmol/l at 120 h after methotrexate treatment was 46.4% and 59.0% at 20 and 50 U/kg of glucarpidase, respectively. CONCLUSION: We determined a recommended glucarpidase dose of 50 U/kg to be ethically acceptable. A rebound in the serum concentration of methotrexate may be observed in many patients after the administration of glucarpidase, and long-term monitoring (over 144 h) of the serum methotrexate concentration may be needed after the administration of glucarpidase. Its validity was confirmed in the phase II study and glucarpidase was approved for manufacturing in Japan.

Evaluation of Intracellular Metabolism of Methotrexate in Hepatocytes and Embryonic Kidney Cells based on Folylpolyglutamate Synthetase and Gamma-Glutamyl Hydrolase Expression.

BACKGROUND: Methotrexate (MTX) is a common folic acid antagonist in clinical medicine, easily inducing a common adverse side effect of liver and kidney injury. It has been found that the expression of Folylpolyglutamate Synthetase (FPGS) and gamma-Glutamyl Hydrolase (GGH) may be closely related to that of related proteins to affect the intracellular metabolism of MTX. OBJECTIVE: The relationship between FPGS/GGH and MTXPGs accumulation in liver and kidney cells was explored by adjusting the expression of FPGS and GGH in cells using UPLC-MS/MS quantitative technology. METHOD: Based on UPLC-MS/MS quantitative techniques, the relationship between MTXPGs accumulation and FPGS/GGH in hepatocytes and embryonic kidney cells was explored by adjusting the expression of FPGS and GGH, and the effect of FPGS/GGH on the intracellular toxicity of MTX was comprehensively analyzed. RESULT: The results showed that the difference in methotrexate polyglutamates (MTXPGs) accumulation in liver and kidney cells was related to the difference in FPGS and GGH expression. The expression of FPGS interacted with that of GGH. These results suggest that the protein abundance ratio of FPGS to GGH (FPGS/GGH) has more potential to be used as a predictor of MTX efficacy than the FPGS or GGH single protein index. This can effectively avoid liver and kidney damage caused by MTX and guides the rational use of drugs in MTX. CONCLUSION: The results prove that there is a positive correlation between the FPGS/GGH and the accumulation of MTXPGS in liver and kidney cells. Summarily, the FPGS/GGH is expected to be a predictor for MTXPGs accumulation and provides an effective method to evaluate the toxicity caused by MTX.

Design of a dual-function agent by fusing a designed anti-VEGF-A binder and CPG-2 enzyme.

Anti-VEGF therapies are common for the treatment of cancer. Carboxypeptidase G (CPG-2) enzyme is a zinc-dependent metalloenzyme that metabolizes non-toxic synthetic 'benzoic mustard prodrugs' to cytotoxic moieties in tumor cells. In this study, we designed a dual-activity agent by combining a designed anti-VEGF- and CPG-2 enzyme to convert methotrexate (MTX). VEGF-A was docked against a set of scaffolds, and suitable inverse rotamers were made. Rosetta design was used for the interface design. The top 1200 binders were chosen by flow cytometry and displayed in yeast. The activity of CPG-2 enzyme was analyzed at different temperature conditions and in the presence of the substrate, MTX. Optimal binders were selected and protein was eluted using immobilized metal affinity chromatography and size-exclusion chromatography. Both, native PAGE and on-yeast flow cytometry confirmed the binding of the binder to VEGF-A. The activity of truncated enzymes was slightly lower than that of full-length enzymes linked to VEGF-A. The method should be generally useful as a dual-activity agent for targeting VEGF-A and combination therapy with the enzyme CPG-2 for metabolizing non-toxic prodrugs to cytotoxic moieties.Communicated by Ramaswamy H. Sarma.

The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import.

Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenic enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and increases the levels of its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in compromised protein import into mitochondria and reduced steady-state levels of mitochondrial proteins. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery to improve mitochondrial protein import during phases of metabolic transition.

Interaction between glycolysis‒cholesterol synthesis axis and tumor microenvironment reveal that gamma-glutamyl hydrolase suppresses glycolysis in colon cancer.

Background: Metabolic reprogramming is a feature of cancer. However, colon cancer subtypes based on the glycolysis‒cholesterol synthesis axis have not been identified, and little is known about connections between metabolic features and the tumor microenvironment. Methods: Data for 430 colon cancer cases were extracted from The Cancer Genome Atlas, including transcriptome data, clinical information, and survival outcomes. Glycolysis and cholesterol synthesis-related gene sets were obtained from the Molecular Signatures Database for a gene set variation analysis. The relationship between the genomic landscape and immune landscape were investigated among four metabolic subtypes. Hub genes were determined. The clinical significance of candidate hub gene was evaluated in 264 clinical samples and potential functions were validated in vitro and in vivo. Results: Colon cancer cases were clustered into four metabolic subtypes: quiescent, glycolytic, cholesterogenic, and mixed. The metabolic subtypes differed with respect to the immune score, stromal score, and estimate score using the ESTIMATE algorithm, cancer-immunity cycle, immunomodulator signatures, and signatures of immunotherapy responses. Patients in the cholesterogenic group had better survival outcomes than those for other subtypes, especially glycolytic. The glycolytic subtype was related to unfavorable clinical characteristics, including high mutation rates in TTN, APC, and TP53, high mutation burden, vascular invasion, right colon cancer, and low-frequency microsatellite instability. GGH, CACNG4, MME, SLC30A2, CKMT2, SYN3, and SLC22A31 were identified as differentially expressed both in glycolytic-cholesterogenic subgroups as well as between colon cancers and healthy samples, and were involved in glycolysis‒cholesterol synthesis. GGH was upregulated in colon cancer; its high expression was correlated with CD4+ T cell infiltration and longer overall survival and it was identified as a favorable independent prognostic factor. The overexpression of GGH in colon cancer-derived cell lines (SW48 and SW480) inhibited PKM, GLUT1, and LDHA expression and decreased the extracellular lactate content and intracellular ATP level. The opposite effects were obtained by GGH silencing. The phenotype associated with GGH was also validated in a xenograft nude mouse model. Conclusions: Our results provide insight into the connection between metabolism and the tumor microenvironment in colon cancer and provides preliminary evidence for the role of GGH, providing a basis for subsequent studies.

Ultrasonically Enhanced ZD2767P-Carboxypeptidase G2 Deactivates Cisplatin-Resistant Human Lung Cancer Cells.

The prodrug-enzyme regimen ZD2767P+CPG2 is limited by low efficacy. Here, ultrasound was used to modulate ZD2767P+CPG2 (i.e., ZD2767P+CPG2+US) against cisplatin-resistant human lung cancer cells. A549 and A549/DDP (resistant subline) cells received ZD2767P+CPG2 or ZD2767P+CPG2+US. Either ZD2767P+CPG2 or ZD2767P+CPG2+US led to cell death and apoptosis, and ZD2767P+CPG2+US produced stronger effects; comet assays revealed that these two means directly caused DNA double-strand break. Z-VAD-fmk and/or ferrostatin-1 increased the cell survival percentage, and Z-VAD-fmk decreased the apoptosis percentage. The level of transferrin was increased in treated cells, but those of ferroportin and glutathione peroxidase 4 (GPX4) were reduced, with higher intracellular levels of reactive oxygen species and of iron. Intracellular pharmacokinetics of ZD2767D (activated drug) indicated that the peak level, area under the drug level vs. time curve, and mean residence time in ZD2767P+CPG2+US were higher than those in ZD2767P+CPG2. Both ZD2767P+CPG2 and ZD2767P+CPG2+US were effective on xenograft tumors in nude mice; inhibitory rates were 39.7% and 63.5% in A549 tumors and 50.0% and 70.1% in A549/DDP tumors, respectively. A higher apoptosis level and a lower GPX4 level were noted in tumors receiving treatments. No severe adverse events were observed. These data demonstrated that ZD2767P+CPG2+US deactivated cancer cells via apoptosis and ferroptosis pathways, being a candidate therapy for cisplatin-resistant lung cancer.

Overexpression of gamma glutamyl hydrolase predicts extranodal extension in squamous cell carcinoma of the oral cavity.

OBJECTIVE: Extranodal extension (ENE) is an important prognostic factor in oral squamous cell carcinoma (OSCC). However, ENE is only confirmed postoperatively by histologic assessment of the lymph nodes after neck dissection. Accurate identification of ENE preoperatively would help in management of OSCC. STUDY DESIGN: We determined the expression of molecular markers gamma glutamyl hydrolase (GGH), cyclin-dependent kinase inhibitor-3 (CDKN3), and chromobox homolog-7 (CBX7) using immunohistochemistry in OSCC clinical samples (n = 35). The intensity of staining was scored using a semiquantitative index (HSCORE). The association between clinicopathologic parameters and expression of molecular markers with ENE status was analyzed using chi-square test. RESULTS: The number of positive nodes and the highest anatomic level of nodal involvement significantly correlated with ENE (P < .05). High GGH expression was significantly associated with ENE (P < .05), with an increased risk for ENE (odds ratio [OR] 9.9, 95% CI 1.08-91.47, P = .04), whereas no significant association was seen for CDKN3 and CBX7 expression with ENE. However, a trend toward significance was observed with a high level of CDKN3 and a low level of CBX7 expression with ENE. CONCLUSIONS: Gamma glutamyl hydrolase offers potential as a predictor for ENE in OSCC, whereas the role of CDKN3 and CBX7 need to be validated in a larger sample.

Ubiquitin Proteasome Gene Signatures in Ependymoma Molecular Subtypes.

The ubiquitin proteasome system (UPS) is critically important for cellular homeostasis and affects virtually all key functions in normal and neoplastic cells. Currently, a comprehensive review of the role of the UPS in ependymoma (EPN) brain tumors is lacking but may provide valuable new information on cellular networks specific to different EPN subtypes and reveal future therapeutic targets. We have reviewed publicly available EPN gene transcription datasets encoding components of the UPS pathway. Reactome analysis of these data revealed genes and pathways that were able to distinguish different EPN subtypes with high significance. We identified differential transcription of several genes encoding ubiquitin E2 conjugases associated with EPN subtypes. The expression of the E2 conjugase genes UBE2C, UBE2S, and UBE2I was elevated in the ST_EPN_RELA subtype. The UBE2C and UBE2S enzymes are associated with the ubiquitin ligase anaphase promoting complex (APC/c), which regulates the degradation of substrates associated with cell cycle progression, whereas UBE2I is a Sumo-conjugating enzyme. Additionally, elevated in ST_EPN_RELA were genes for the E3 ligase and histone deacetylase HDAC4 and the F-box cullin ring ligase adaptor FBX031. Cluster analysis demonstrated several genes encoding E3 ligases and their substrate adaptors as EPN subtype specific genetic markers. The most significant Reactome Pathways associated with differentially expressed genes for E3 ligases and their adaptors included antigen presentation, neddylation, sumoylation, and the APC/c complex. Our analysis provides several UPS associated factors that may be attractive markers and future therapeutic targets for the subtype-specific treatment of EPN patients.

Carboxypeptidase G and pterin deaminase metabolic pathways degrade folic acid in Variovorax sp. F1.

BACKGROUND: Folic acid (FA) is a synthetic vitamin (B9) and the oxidized form of a metabolic cofactor that is essential for life. Although the biosynthetic mechanisms of FA are established, its environmental degradation mechanism has not been fully elucidated. The present study aimed to identify bacteria in soil that degrade FA and the mechanisms involved. RESULTS: We isolated the soil bacterium Variovorax sp. F1 from sampled weed rhizospheres in a grassland and investigated its FA degradation mechanism. Cultured Variovorax sp. F1 rapidly degraded FA to pteroic acid (PA), indicating that FA hydrolysis to PA and glutamate. We cloned the carboxypeptidase G (CPG) gene and found widely distributed paralogs within the Variovorax genus. Recombinant CPG preferred FA and deaminofolic acid as substrates, indicating its involvement in FA degradation by Variovorax. Prolonged culture of Variovorax sp. F1 resulted in decreased rates of deaminofolic acid (DFA) and deaminopteroic acid (DPA) accumulation. This indicated that the deamination reaction also comprised a route of FA degradation. We also identified an F1 gene that was orthologous to the pterin deaminase gene (Arad3529) of Agrobacterium radiobacter. The encoded protein deaminated FA and PA to DFA and DPA, which was consistent with the deamination activity of FA and PA in bacterial cell-free extracts. CONCLUSION: We discovered that the two enzymes required for FA degradation pathways in isolates of Variovorax sp. F1 comprise CPG and pterin deaminase, and that DFA and PA are intermediates in the generation of DPA.

Seeing the unseen: a trifoliate (MYB117) mutant allele fortifies folate and carotenoids in tomato fruits.

In tomato (Solanum lycopersicum), mutations in the gene encoding the R2R3-MYB117 transcription factor elicit trifoliate leaves and initiate the formation of axillary meristems; however, their effects on fruit ripening remain unexplored. The fruits of a new trifoliate (tf) mutant (tf-5) were firmer and had higher °Brix values and higher folate and carotenoid contents. The transcriptome, proteome, and metabolome profiling of tf-5 reflected a broad-spectrum change in cellular homeostasis. The tf-5 allele enhanced the fruit firmness by suppressing cell wall softening-related proteins. tf-5 fruit displayed a substantial increase in amino acids, particularly γ-aminobutyric acid, with a parallel reduction in aminoacyl-tRNA synthases. The increased lipoxygenase protein and transcript levels seemingly elevated jasmonic acid levels. In addition, increased abscisic acid hydrolase transcript levels coupled with reduced precursor supply lowered abscisic acid levels. The upregulation of carotenoids was mediated by modulation of methylerythreitol and plastoquinone pathways and increased the levels of carotenoid isomerization proteins. The upregulation of folate in tf-5 was connoted by the increase in the precursor p-aminobenzoic acid and transcript levels of several folate biosynthesis genes. The reduction in pterin-6-carboxylate levels and γ-glutamyl hydrolase activity indicated that reduced folate degradation in tf-5 increased folate levels. Our study delineates that in addition to leaf development, MYB117 also influences fruit metabolism. The tf-5 allele can be used to increase γ-aminobutyric acid, carotenoid, and folate levels in tomato.