Argininosuccinate Synthetase Research Articles

Optimization of L-Citrulline Operon in Corynebacterium glutamicum for L-Citrulline Production

L-citrulline plays important roles in many physiological processes, and its application range is expanding rapidly. Corynebacterium glutamicum strains have the potential to be efficient L-citrulline producers. In this study, we performed optimization of L-citrulline biosynthesis operon in C. glutamicum ATCC13032 for L-citrulline production. Chromosomal integration of the integral argBEc gene from Escherichia coli (encoding natively insensitive N-acetylglutamate kinase), the deletion of the argR gene (encoding repressor ArgR), and the deletion of the argG gene (encoding argininosuccinate synthase) were achieved simultaneously by one-step genome modification and by obtaining the L-citrulline-producing strain. Then, plasmid-based overexpression of the optimized L-citrulline operon was carried out and the L-citrulline production was further improved. In fed-batch fermentation, the L-citrulline production and yield from glucose of the final strain reached 26.7 g/L and 0.18 g/g, respectively. These results indicate that optimization of L-citrulline operon in C. glutamicum is effective to construct the L-citrulline over-producing strain.

Hepatic urea, creatinine and uric acid metabolism in dairy cows with divergent milk urea concentrations

Milk urea concentration is an indicator for dietary nitrogen (N)-supply and urinary N-excretion. Dairy cows with high (HMU) compared to low milk urea (LMU) concentration have greater plasma urea, creatinine and uric acid concentrations, but if the liver metabolism accounts for these differences is unknown. Eighteen HMU and 18 LMU cows were fed a diet with a low (LP) or normal (NP) crude protein concentration. A N balance study was performed and a 13C-urea bolus was administered to measure urea pool size. Liver samples were analyzed by 2D-gel-based proteomics and RT-qPCR. Although HMU cows had a greater urea pool, plasma urea, uric acid, and hippuric acid concentrations, these differences were not associated with altered expressions of genes related to urea cycling or N-metabolism. Instead, HMU cows had higher oxidative stress levels. Conclusively, other factors than hepatic urea metabolism account for milk urea concentrations. Despite higher plasma urea concentrations and argininosuccinate synthase 1 protein expression on the LP diet, urea cycle mRNA expressions were not affected, indicating that its activity is not controlled at transcriptional level. Feeding the LP diet resulted in increased expressions of enzymes catabolizing fatty acids, but the reason remains to be investigated in future studies.

Enhancement of anti-tumor activity in melanoma using arginine deiminase fused with 30Kc19α protein.

Arginine deiminase (ADI) is a microbial-derived enzyme which catalyzes the conversion of L-arginine into L-citrulline. ADI originating from Mycoplasma has been reported to present anti-tumor activity against arginine-auxotrophic tumors, including melanoma. Melanoma cells are sensitive to arginine depletion due to reduced expression of argininosuccinate synthase 1 (ASS1), a key enzyme for arginine biosynthesis. However, clinical applications of recombinant ADI for melanoma treatment present some limitations. Since recombinant ADI is not human-derived, it shows instability, proteolytic degradation, and antigenicity in human serum. In addition, there is a problem of drug resistance issue due to the intracellular expression of once-silenced ASS1. Moreover, recombinant ADI proteins are mainly expressed as inclusion body forms in Escherichia coli and require a time-consuming refolding process to turn them back into active form. Herein, we propose fusion of recombinant ADI from Mycoplasma hominis and 30Kc19α, a cell-penetrating protein which also increases stability and soluble expression of cargo proteins, to overcome these problems. We inserted matrix metalloproteinase-2 cleavable linker between ADI and 30Kc19α to increase enzyme activity in melanoma cells. Compared to ADI, ADI-LK-30Kc19α showed enhanced solubility, stability, and cell penetration. The fusion protein demonstrated selective cytotoxicity and reduced drug resistance in melanoma cells, thus would be a promising strategy for the improved efficacy in melanoma treatment. KEY POINTS: • Fusion of ADI with 30Kc19α enhances soluble expression and productivity of recombinant ADI in E. coli • 30Kc19α protects ADI from the proteolytic degradation by shielding effect, helping ADI to remain active • Intracellular delivery of ADI by 30Kc19α overcomes ADI resistance in melanoma cells by degrading intracellularly expressed arginine.

One Step Forward with Dry Surface Biofilm (DSB) of Staphylococcus aureus: TMT-Based Quantitative Proteomic Analysis Reveals Proteomic Shifts between DSB and Hydrated Biofilm.

The Gram-positive bacterium Staphylococcus aureus is responsible for serious acute and chronic infections worldwide and is well-known for its biofilm formation ability. Recent findings of biofilms on dry hospital surfaces emphasise the failures in current cleaning practices and disinfection and the difficulty in removing these dry surface biofilms (DSBs). Many aspects of the formation of complex DSB biology on environmental surfaces in healthcare settings remains limited. In the present study, we aimed to determine how the protein component varied between DSBs and traditional hydrated biofilm. To do this, biofilms were grown in tryptic soy broth (TSB) on removable polycarbonate coupons in the CDC biofilm reactor over 12 days. Hydrated biofilm (50% TSB for 48 h, the media was then changed every 48 h with 20% TSB, at 37 °C with 130 rpm). DSB biofilm was produced in 5% TSB for 48 h at 35 °C followed by extended periods of dehydration (48, 66, 42 and 66 h at room temperature) interspersed with 6 h of 5% TSB at 35 °C. Then, we constructed a comprehensive reference map of 12-day DSB and 12-day hydrated biofilm associated proteins of S. aureus using a high-throughput tandem mass tag (TMT)-based mass spectrometry. Further pathway analysis of significantly differentially expressed identified proteins revealed that proteins significantly upregulated in 12-day DSB include PTS glucose transporter subunit IIBC (PtaA), UDP-N-acetylmuramate-L-alanine ligase (MurC) and UDP-N-acetylenolpyruvoylglucosamine (MurB) compared to 12-day hydrated biofilm. These three proteins are all linked with peptidoglycan biosynthesis pathway and are responsible for cell-wall formation and thicker EPS matrix deposition. Increased cell-wall formation may contribute to the persistence of DSB on dry surfaces. In contrast, proteins associated with energy metabolisms such as phosphoribosyl transferase (PyrR), glucosamine--fructose-6-phosphate aminotransferase (GlmS), galactose-6-phosphate isomerase (LacA), and argininosuccinate synthase (ArgG) were significantly upregulated whereas ribosomal and ABC transporters were significantly downregulated in the 12-day hydrated biofilm compared to DSB. However, validation by qPCR analysis showed that the levels of gene expression identified were only partially in line with our TMT-MS quantitation analysis. For the first time, a TMT-based proteomics study with DSB has shed novel insights and provided a basis for the identification and study of significant pathways vital for biofilm biology in this reference microorganism.

Citrullinemia type I in Chinese children: Identification of two novel argininosuccinate synthetase gene mutations.

BackgroundIn this study, we evaluated the clinical characteristics, prognosis, and gene mutations of five children with citrullinemia type I (CTLN1) diagnosed in our department and identified two novel ASS1 gene mutations.MethodsWe examined the clinical characteristics, prognosis, and gene mutations of the five children through data collection, tandem mass spectrometry, and whole-exon sequencing. MutationTaster, regSNP-intron, and SWISS-MODEL were used for bioinformatic analysis to evaluate the two novel gene mutations. We analyzed differences in blood ammonia and citrulline levels based on clinical phenotypes. Finally, we reviewed the medical literature describing Chinese children with CTLN1.ResultsASS1 C773 + 6T > G and c.848 delA as well as c.952_953 del insTT and c.133G > A have not been previously reported in the Human Gene Mutation Database. Using MutationTaster and regSNP-intron, we predicted that these mutations affected protein function. The 3D structure obtained using SWISS-MODEL supported this prediction. Through comparative analysis showed that the ammonia level of the neonatal type was markedly higher than that of other types, whereas citrulline levels did not differ between groups.ConclusionWe identified two novel mutations that cause disease. The blood ammonia level of neonatal form citrullinemia was markedly higher than that of other types. The genotype-phenotype association in Chinese patients remains unclear and should be further evaluated in genetic studies of larger sample sizes.

Abstract IA022: Arginine, serine, xCT and ME, the therapeutic metabolism of different sarcomas

Abstract The absolute diversity of the biology of the sarcomas makes therapeutic development challenging. As most of tumor metabolism is composed of transporters and enzymes, finding metabolic dependencies should allow for small molecule targeting. Due to the rapid metabolic evolution that tumors undergo in response to the targeting of any one metabolic pathway, a deep understanding of sarcoma metabolism is needed. First, the most common metabolic adaptation that sarcomas undergo is loss of expression of argininosuccinate synthetase 1 (ASS1), which is silenced in ~90% of cases by methylation. This results in sarcomas being arginine auxotrophic and sensitive to arginine starvation therapies, such as with arginine deiminase (ADI-PEG20). Not only does arginine starvation alter the Warburgian biology of sarcomas making them dependent of glutamine, but it can be used to upregulate the expression of cell surface transports such as hENT, which allows to gemcitabine internalization. In addition, mouse modeling has demonstrated that vesicular trafficking is key to overcoming initial arginine starvation in vivo, a process that can be blocked with chloroquine. This arginine dependency is likely related to the mesenchymal origin of sarcomas, which would explain its high recurrence rate of ASS1 silencing across histologies. Next, due to the upregulation of 3-phosphoglycerate dehydrogenase (PHGDH), osteosarcoma preferentially utilizes glucose to make the serine that enters the folate cycle, as opposed to completing lower glycolysis. In addition, as osteosarcoma does not seem to depend on extracellular serine import, inhibition of PHGDH leads to pro-survival compensation by the mTORC pathway. This can be targeted on both the AMP Kinase and the AKT dependent parts of the MTORC pathway that converge at FOXO3. When both are inhibited approach demonstrates unique triple synergy and therapeutic strategy for osteosarcoma due to its unique serine biology. Finally, synovial sarcoma lack the expression of malic enzyme 1 (ME1), also likely due to its cell of origin. This leads to reduced glucose oxidation, enhanced glycolysis, and compensatory increased flux through the pentose phosphate pathway for cytoplasmic NADPH production. Additionally, absence of ME1 in SS results in significant reductions in the GSH/GSSG ratio as well as a reduced glutathione synthesis. Sensitivity to GSH pathway inhibition is also reduced while sensitivity to inhibition of the thioredoxin system is significantly increased. ME1 absence results in increases in the labile iron pool that sensitizes synovial sarcoma to ferroptosis. Therefore, ME1 null SS is exquisitely sensitive to induction of ferroptosis with xCT inhibition. This can be accomplished by erastin analogs in vitro and ACXT-3102 (a tumor targeted erastin) in vivo. As we learn more about each sarcoma, we must understand the underlying metabolism of each subtype and their cell of origin. Given the targetable nature of metabolic enzymes and transporters, an ever deeper understanding of sarcoma metabolism is warranted. Citation Format: Brian A. Van Tine. Arginine, serine, xCT and ME, the therapeutic metabolism of different sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA022.

Evidence for high breakpoint variability in 46, XX, SRY-positive testicular disorder and frequent ARSE deletion that may be associated with short stature.

The translocation of SRY onto one of the two X chromosomes results in a 46,XX testicular disorder of sex development; this is supposedly because of non-allelic homologous recombination between the protein kinase X gene (PRKX) and the inverted protein kinase Y pseudogene (PRKY). Although 46,XX SRY-positive men are infertile, the literature data indicate that some of these individuals are of short stature (relative to the general population). We sought to determine whether short stature was linked to additional, more complex chromosomal rearrangements. Twelve laboratories gathered detailed clinical, anthropomorphic, cytogenetic and genetic data (including chromosome microarray data) on patients with 46,XX SRY-positive male syndrome. SRY was present (suggesting a der(X)t(X;Y)) in 34 of the 38 cases (89.5%). When considering only the 20 patients with chromosome microarray data, we identified several chromosomal rearrangements and breakpoints, especially on the X chromosome. In the five cases for whom the X chromosome breakpoint was located in the pseudoautosomal region, there was partial duplication of the derivate X chromosome. In contrast, in the 15 cases for whom the breakpoint was located downstream of the pseudoautosomal region, part of the derivate X chromosome had been deleted (included the arylsulfatase E [ARSE] gene in 11 patients). For patients with versus without ARSE deletion, the mean height was, respectively, 167.7±4.5 and 173.1±4.0cm; this difference was not statistically significant (p =0.1005). Although 46,XX SRY-positive male syndromes were mainly because of imbalanced crossover between the X and Y chromosome during meiosis, the breakpoints differed markedly from one patient to another (especially on the X chromosome); this suggests the presence of a replication-based mechanism for recombination between non-homologous sequences. In some patients, the translocation of SRY to the X chromosome was associated with ARSE gene deletion, which might have led to short stature. With a view to explaining this disorder of sex development, whole exome sequencing could be suggested for SRY-negative patients.

Arginine Deprivation in SCLC: Mechanisms and Perspectives for Therapy.

Arginine deprivation has gained increasing traction as a novel and safe antimetabolite strategy for the treatment of several hard-to-treat cancers characterised by a critical dependency on arginine. Small cell lung cancer (SCLC) displays marked arginine auxotrophy due to inactivation of the rate-limiting enzyme argininosuccinate synthetase 1 (ASS1), and as a consequence may be targeted with pegylated arginine deiminase or ADI-PEG20 (pegargiminase) and human recombinant pegylated arginases (rhArgPEG, BCT-100 and pegzilarginase). Although preclinical studies reveal that ASS1-deficient SCLC cell lines are highly sensitive to arginine-degrading enzymes, there is a clear disconnect with the clinic with minimal activity seen to date that may be due in part to patient selection. Recent studies have explored resistance mechanisms to arginine depletion focusing on tumor adaptation, such as ASS1 re-expression and autophagy, stromal cell inputs including macrophage infiltration, and tumor heterogeneity. Here, we explore how arginine deprivation may be combined strategically with novel agents to improve SCLC management by modulating resistance and increasing the efficacy of existing agents. Moreover, recent work has identified an intriguing role for targeting arginine in combination with PD-1/PD-L1 immune checkpoint inhibitors and clinical trials are in progress. Thus, future studies of arginine-depleting agents with chemoimmunotherapy, the current standard of care for SCLC, may lead to enhanced disease control and much needed improvements in long-term survival for patients.

Clinical Characteristics of Pediatric Patients with Urea Cycle Disorders

Objective: Urea cycle disorders (UCDs) are inherited deficiencies of the enzymes or transport molecules involved in the cellular excretion of excess ammonia produced during protein metabolism. The aim of this study was to evaluate the clinical characteristics and long-term outcome of pediatric patients with UCDs. Material and Methods: Our research was conducted between September 2020-March 2021 in Dr. Sami Ulus Maternity and Child Health Training and Research Hospital. Clinical characteristics in 16 patients with UCDs [carbamoyl phosphate synthetase I deficiency (n=1), N-acetylglutamate synthase deficiency(n=1), argininosuccinate lyase deficiency (n=4), argininosuccinate synthetase deficiency (n=4), arginase deficiency (n=2), ornithine transcarbamylase deficiency (n=2), hyperammonemia hyperornithinemia homocitrullinuria syndrome (n=2)] were defined. The term “neonatal-onset” UCD was used if symptoms occurred within 28 days of life, and “late-onset” if symptoms started after the neonatal period. Results: Eight patients presented with acute metabolic crisis during newborn period. Core clinical phenotype in neonatal-onset UCDs included sepsis-like findings, whereas epilepsy and mental retardation was predominant in lateonset UCDs. For patients with neonatal-onset UCDs, hyperammonemia was more severe at the initial period. Conclusion: Despite evolving treatment opportunities, still high mortality rates were found in neonatal-onset UCD. UCDs should be suspected in pediatric patients with hyperammonemia and metabolic investigations should be performed immediately to enlighten diagnosis. Neonatal-onset UCD usually present with symptoms of acute hyperammonemia, while moresubtle neurological manifestations are frequent initial findings in the late onset UCD.

Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18−/− syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells

BackgroundIFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown.MethodsIn this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry.ResultsThe targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control.ConclusionsThese results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Carnosine Synthase 1 Contributes to Interferon Gamma-Induced Arginine Depletion via Mitogen-activated Protein Kinase 11 Signaling in Bovine Mammary Epithelial Cells.

Arginine is one of the host semiessential amino acids with diverse biological activities, and arginine depletion is associated with the incidence of many diseases. Arginine depletion induced by diet-derived interferon gamma (IFN-γ) leads to malignant transformation and impaired milk quality in healthy lactating bovine mammary epithelial cells (BMECs). However, the molecular mechanism of IFN-γ-induced arginine depletion is unclear. In this study, the BMEC cell line, mammary alveolar cells-large T antigen cells (MAC-T), was stimulated with IFN-γ (10 ng/mL) for 24 h, and cellular arginine and ornithine quantified by liquid chromatography-tandem mass spectrometry. Carnosine synthase 1 (CARNS1) was identified from RNA-seq data, CARNS1 knockdown was achieved using an shRNA interfering plasmid. The expression levels of CARNS1, argininosuccinate synthetase 1 (ASS1), mitogen-activated protein kinase 11 (p38 MAPK), and phosphorylated (p)-p38, and their cognate genes, were analyzed by Western blotting and real-time quantitative polymerase chain reaction. The results showed that IFN-γ inhibited the biosynthesis of arginine, but enhanced its catalysis via disruption of key enzymes involved in arginine metabolism. IFN-γ also inhibited the expression of CARNS1, ASS1, and cationic amino acid transporter 1, while activating the expression and phosphorylation of p38. However, knockdown of CARNS1 reduced arginine level and ASS1 expression and block of either the IFN-γ receptor IFN-γ receptor 2 or p38 relieved both the expression of Carnosine synthase 1 (CARNS1) and ASS1. In summary, these results indicate that IFN-γ induced arginine depletion through inhibition of CARNS1 signaling via activation of p38 in BMECs. These findings provide a novel insight for IFN-γ-related disease control strategies in dairy cows.

Mono-PEGylated thermostable Bacillus caldovelox arginase mutant (BCA-M-PEG20) induces apoptosis, autophagy, cell cycle arrest and growth inhibition in gastric cancer cells

Gastric cancer is one of the most common malignant solid tumors in the world, especially in Asia with high mortality due to a lack of effective treatment. The potential usage of the newly constructed arginine-depleting enzyme—mono-PEGylated Bacillus caldovelox arginase mutant (BCA-M-PEG20), an effective drug against multiple cancer cell lines such as cervical and lung cancers, for the treatment of gastric cancer was demonstrated. Our results indicated that BCA-M-PEG20 significantly inhibited argininosuccinate synthetase (ASS)-positive gastric cancer cells, MKN-45 and BGC-823, while another arginine-depleting enzyme, arginine deiminase (ADI, currently under Phase III clinical trial), failed to suppress the growth of gastric cancer cells. In vitro studies demonstrated that BCA-M-PEG20 inhibited MKN-45 cells by inducing autophagy and cell cycle arrest at the S phase under 0.58 U/mL (IC50 values). Significant caspase-dependent apoptosis was induced in MKN-45 after the treatment with 2.32 U/mL of BCA-M-PEG20. In vivo studies showed that administrations of BCA-M-PEG20 at 250 U/mouse twice per week significantly suppressed about 50% of tumor growth in the MKN-45 gastric cancer xenograft model. Taken together, BCA-M-PEG20 demonstrated a superior potential to be an anti-gastric cancer drug.

Role of 3'-Deoxy-3'-[18F] Fluorothymidine Positron Emission Tomography-Computed Tomography as a Predictive Biomarker in Argininosuccinate Synthetase 1-Deficient Thoracic Cancers Treated With Pegargiminase

IntroductionPegargiminase (ADI-PEG 20I) degrades arginine in patients with argininosuccinate synthetase 1-deficient malignant pleural mesothelioma (MPM) and NSCLC. Imaging with proliferation biomarker 3'-deoxy-3'-[18F] fluorothymidine (18F-FLT) positron emission tomography (PET)-computed tomography (CT) was performed in a phase 1 study of pegargiminase with pemetrexed and cisplatin (ADIPemCis). The aim was to determine whether FLT PET-CT predicts treatment response earlier than CT. MethodsA total of 18 patients with thoracic malignancies (10 MPM; eight NSCLC) underwent imaging. FLT PET-CT was performed at baseline (PET1), 24 hours post-pegargiminase monotherapy (PET2), post one cycle of ADIPemCis (PET3), and at end of treatment (EOT, PET4). CT was performed at baseline (CT1) and EOT (CT4). CT4 (modified) Response Evaluation Criteria in Solid Tumors (RECIST) response was compared with treatment response on PET (changes in maximum standardized uptake value [SUVmax] on European Organisation for Research and Treatment of Cancer–based criteria). Categorical responses (progression, partial response, and stable disease) for PET2, PET3, and PET4 were compared against CT using Cohen’s kappa. ResultsADIPemCis treatment response resulted in 22% mean decrease in size between CT1 and CT4 and 37% mean decrease in SUVmax between PET1 and PET4. PET2 agreed with CT4 response in 62% (8 of 13) of patients (p = 0.043), although decrease in proliferation (SUVmax) did not precede decrease in size (RECIST). Partial responses on FLT PET-CT were detected in 20% (3 of 15) of participants at PET2 and 69% (9 of 13) at PET4 with good agreement between modalities in MPM at EOT. ConclusionsEarly FLT imaging (PET2) agrees with EOT CT results in nearly two-thirds of patients. Both early and late FLT PET-CT provide evidence of response to ADIPemCis therapy in MPM and NSCLC. We provide first-in-human FLT PET-CT data in MPM, indicating it is comparable with modified RECIST.

The argininosuccinate synthetase can differentially regulate nitric oxide synthase in yellow catfish Pelteobagrus fulvidraco

Fish are at high risk of exposure to ammonia in aquaculture systems. When ammonia stress occurs, fish are more prone to disease outbreaks, but the mechanism is not very clear. The argininosuccinate synthetase (ASS) plays an important role in the regulation of urea synthesis and nitric oxide synthesis. We speculated that there must be some relationship between ASS expression and disease outbreak. In this study, ASS was cloned from the yellow catfish. The full-length cDNAs of ASS was 1558 bp, with open reading frames of 1236 bp. The mRNA expression of ASS gene was the highest in liver, kidney and brain. This study consists of two parts: 1) For ammonia challenge in vivo, yellow catfish (15.00 ± 1.50 g) were divided into control group, low ammonia group (1/10 96 h LC50), and high ammonia group (1/2 96 h LC50). The experiment continued for 192 h. The results showed that ammonia stress elevated serum ammonia content, and inhibited urea synthesis enzymes activities but up-regulated the expression levels of related genes except ARG, and induced arginine accumulation and nitric oxide synthase (nNOS and iNOS) different expression, and decreased resistance to Aeromonas hydrophage; 2) For ammonia challenge in vitro, the primary culture of liver cell was divided into four groups: control group, BPP group (Bj-BPP-10c was added as ASS activator), Amm group (96 h LC50), and Amm + BPP group. The experiment continued for 96 h. The results showed that the Bj-BPP-10c can inhibit nNOS activity and improve cell survival rate, and enhance iNOS activity and immune response (lysozyme, complement, respiratory burst, and phagocytic index) by activate ASS when ammonia stress occurred. Our results indicated that targeted regulation of ASS can improve iNOS activity, and enhance the immune response of yellow catfish under ammonia stress.

Argininosuccinate synthase 1, arginine deprivation therapy and cancer management.

Metabolic reprogramming is an emerging hallmark of tumor cells. In order to survive in the nutrient-deprived environment, tumor cells rewire their metabolic phenotype to provide sufficient energy and build biomass to sustain their transformed state and promote malignant behaviors. Amino acids are the main compositions of protein, which provide key intermediate substrates for the activation of signaling pathways. Considering that cells can synthesize arginine via argininosuccinate synthase 1 (ASS1), arginine is regarded as a non-essential amino acid, making arginine depletion as a promising therapeutic strategy for ASS1-silencing tumors. In this review, we summarize the current knowledge of expression pattern of ASS1 and related signaling pathways in cancer and its potential role as a novel therapeutic target in cancer. Besides, we outline how ASS1 affects metabolic regulation and tumor progression and further discuss the role of ASS1 in arginine deprivation therapy. Finally, we review approaches to target ASS1 for cancer therapies.

Targeted Arginine Metabolism Therapy: A Dilemma in Glioma Treatment.

Efforts in the treatment of glioma which is the most common primary malignant tumor of the central nervous system, have not shown satisfactory results despite a comprehensive treatment model that combines various treatment methods, including immunotherapy. Cellular metabolism is a determinant of the viability and function of cancer cells as well as immune cells, and the interplay of immune regulation and metabolic reprogramming in tumors has become an active area of research in recent years. From the perspective of metabolism and immunity in the glioma microenvironment, we elaborated on arginine metabolic reprogramming in glioma cells, which leads to a decrease in arginine levels in the tumor microenvironment. Reduced arginine availability significantly inhibits the proliferation, activation, and function of T cells, thereby promoting the establishment of an immunosuppressive microenvironment. Therefore, replenishment of arginine levels to enhance the anti-tumor activity of T cells is a promising strategy for the treatment of glioma. However, due to the lack of expression of argininosuccinate synthase, gliomas are unable to synthesize arginine; thus, they are highly dependent on the availability of arginine in the extracellular environment. This metabolic weakness of glioma has been utilized by researchers to develop arginine deprivation therapy, which ‘starves’ tumor cells by consuming large amounts of arginine in circulation. Although it has shown good results, this treatment modality that targets arginine metabolism in glioma is controversial. Exploiting a suitable strategy that can not only enhance the antitumor immune response, but also “starve” tumor cells by regulating arginine metabolism to cure glioma will be promising.

PGAM1 regulation of ASS1 contributes to the progression of breast cancer through the cAMP/AMPK/CEBPB pathway.

Phosphoglycerate mutase 1 (PGAM1) is a crucial glycolytic enzyme, and its expression status has been confirmed to be associated with tumor progression and metastasis. However, the precise role and other biological functions of PGAM1 remain unclear. Here, we report that PGAM1 expression is upregulated and related to poor prognosis in patients with breast cancer (BC). Functional experiments showed that knockdown of PGAM1 could suppress the proliferation, invasion, migration, and epithelial–mesenchymal transition of BC cells. Through RNA sequencing, we found that argininosuccinate synthase 1 (ASS1) expression was markedly upregulated in BC cells following PGAM1 knockdown, and it is required to suppress the malignant biological behavior of BC cells. Importantly, we demonstrated that PGAM1 negatively regulates ASS1 expression through the cAMP/AMPK/CEBPB axis. In vivo experiments further validated that PGAM1 promoted tumor growth in BC by altering ASS1 expression. Finally, immunohistochemical analysis showed that downregulated ASS1 levels were associated with PGAM1 expression and poor prognosis in patients with BC. Our study provides new insight into the regulatory mechanism of PGAM1‐mediated BC progression that might shed new light on potential targets and combination therapeutic strategies for BC treatment.

Characterisation of Expression the Arginine Pathway Enzymes in Childhood Brain Tumours to Determine Susceptibility to Therapeutic Arginine Depletion.

Despite significant improvements in treatment and survival in paediatric cancers, outcomes for children with brain tumours remain poor. Novel therapeutic approaches are needed to improve survival and quality of survival. Extracellular arginine dependency (auxotrophy) has been recognised in several tumours as a potential therapeutic target. This dependency is due to the inability of cancer cells to recycle or synthesise intracellular arginine through the urea cycle pathway compared to normal cells. Whilst adult glioblastoma exhibits this dependency, the expression of the arginine pathway enzymes has not been delineated in paediatric brain tumours. We used immunohistochemical (IHC) methods to stain for arginine pathway enzymes in paediatric high-grade glioma (pHGG), low-grade glioma (pLGG), ependymoma (EPN), and medulloblastoma (MB) tumour tissue microarrays (TMAs). The antibodies detected protein expression of the metaboliser arginase (Arg1 and Arg2); recycling enzymes ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS1), and argininosuccinate lyase (ASL); and the transporter SLC7A1. Deficiency of OTC, ASS1, and ASL was seen in 87.5%, 94%, and 79% of pHGG samples, respectively, consistent with an auxotrophic signature. Similar result was obtained in pLGG with 96%, 93%, and 91% of tumours being deficient in ASL, ASS1, and OTC, respectively. 79%, 88%, and 85% of MB cases were ASL, ASS1, and OTC deficient whilst ASL and OTC were deficient in 57% and 91% of EPN samples. All tumour types highly expressed SLC7A1 and Arginase, with Arg2 being the main isoform, demonstrating that they could transport and utilise arginine. Our results show that pHGG, pLGG, EPN, and MB demonstrate arginine auxotrophy based on protein expression and are likely to be susceptible to arginine depletion. Pegylated arginase (BCT-100) is currently in phase I/II trials in relapsed pHGG. Our results suggest that therapeutic arginine depletion may also be useful in other tumour types and IHC analysis of patient tumour samples could help identify patients likely to benefit from this treatment.

Asymptomatic ASS1 carriers with high blood citrulline levels.

IntroductionCitrullinemia Type 1 (CTLN1) is an autosomal recessive disorder caused by variants in the ASS1 gene. This study intends to clarify the etiology of false positives in newborn screening for citrullinemia.MethodNewborns who had elevated dried‐blood spot citrulline levels were enrolled, and medical records were reviewed retrospectively. Common ASS1 variants were screened using high‐resolution melting analysis.ResultBetween 2011 and 2021, 130 newborns received confirmatory testing for citrullinemia, 4 were found to be patients for CTLN1; 11 were patients with citrin deficiency; and 49 newborns were confirmed to be carrying one pathogenic ASS1 variant. The incidence of CTLN1 was 1 in 188,380 (95% confidence interval: 1 in 73,258 to 1 in 484,416). All ASS1 variants studied in this cohort were located in exons 11 to 15, which encode the tetrameric interface regions of the ASS1 protein. Among 10 ASS1 carriers with elevated citrulline levels and complete sequence data, four (40%) revealed additional non‐benign ASS1 variants; in contrast, only 2 of the 26 controls (7.7%), with normal citrulline levels, had additional ASS1 variants.ConclusionHeterozygote ASS1 variants may lead to a mild elevation of blood citrulline levels: about 2–6 times the population mean. Molecular testing and family studies remain critical for precise diagnosis, genetic counseling, and management.