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IScience Jun 2024Although glutamine addiction in cancer cells is extensively reported, there is controversy on the impact of glutamine metabolism on the immune cells within the tumor...
Although glutamine addiction in cancer cells is extensively reported, there is controversy on the impact of glutamine metabolism on the immune cells within the tumor microenvironment (TME). To address the role of extracellular glutamine, we enzymatically depleted circulating glutamine using PEGylated gamma-glutamyl transferase (PEG-GGT) in syngeneic mouse models of breast and colon cancers. PEG-GGT treatment inhibits growth of cancer cells , but it increases myeloid-derived suppressor cells (MDSCs) and has no significant impact on tumor growth. By deriving a glutamine depletion signature, we analyze diverse human cancers within the TCGA and illustrate that glutamine depletion is not associated with favorable clinical outcomes and correlates with accumulation of MDSC. Broadly, our results help clarify the integrated impact of glutamine depletion within the TME and advance PEG-GGT as an enzymatic tool for the systemic and selective depletion (no asparaginase activity) of circulating glutamine in live animals.
PubMed: 38770139
DOI: 10.1016/j.isci.2024.109817 -
Nature Communications May 2024TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive....
TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive. Asparaginase-like-1 protein (ASRGL1) cleaves isoaspartates, which alter protein folding and susceptibility to proteolysis. ASRGL1 gene harbors a copy of the human endogenous retrovirus HML-2, whose overexpression contributes to ALS pathogenesis. Here we show that ASRGL1 expression was diminished in ALS brain samples by RNA sequencing, immunohistochemistry, and western blotting. TDP-43 and ASRGL1 colocalized in neurons but, in the absence of ASRGL1, TDP-43 aggregated in the cytoplasm. TDP-43 was found to be prone to isoaspartate formation and a substrate for ASRGL1. ASRGL1 silencing triggered accumulation of misfolded, fragmented, phosphorylated and mislocalized TDP-43 in cultured neurons and motor cortex of female mice. Overexpression of ASRGL1 restored neuronal viability. Overexpression of HML-2 led to ASRGL1 silencing. Loss of ASRGL1 leading to TDP-43 aggregation may be a critical mechanism in ALS pathophysiology.
Topics: Amyotrophic Lateral Sclerosis; Animals; Humans; DNA-Binding Proteins; Mice; Female; TDP-43 Proteinopathies; Neurons; Brain; Male; Motor Cortex
PubMed: 38755145
DOI: 10.1038/s41467-024-48488-7 -
International Journal of Molecular... Apr 2024L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with...
L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from , recently revealed as a new fungus of the genus and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from closely matches species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to and enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.
Topics: Asparaginase; Penicillium; Computer Simulation; Amino Acid Sequence; Fungal Proteins; Epitopes, B-Lymphocyte; Epitopes, T-Lymphocyte; Humans; Aspergillus; Escherichia coli; Dickeya chrysanthemi; Models, Molecular
PubMed: 38732010
DOI: 10.3390/ijms25094788 -
Acta Biochimica Polonica 2024This report describes a comprehensive approach to local random mutagenesis of the Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the...
This report describes a comprehensive approach to local random mutagenesis of the Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the randomization series RDM1. Here, random mutagenesis was applied in the center of the EcAIII molecule, i.e., in the region important for substrate binding and its immediate neighborhood (series RDM2, RDM3, RDM7), in the vicinity of the catalytic threonine triplet (series RDM4, RDM5, RDM6), in the linker region (series RDM8), and in the sodium-binding (stabilization) loop (series RDM9). The results revealed that the majority of the new EcAIII variants have abolished or significantly reduced rate of autoprocessing, even if the mutation was not in a highly conserved sequence and structure regions. AlphaFold-predicted structures of the mutants suggest the role of selected residues in the positioning of the linker and stabilization of the scissile bond in precisely correct orientation, enabling the nucleophilic attack during the maturation process. The presented data highlight the details of EcAIII geometry that are important for the autoproteolytic maturation and for the catalytic mechanism in general, and can be treated as a guide for protein engineering experiments with other Ntn-hydrolases.
Topics: Mutagenesis; Amidohydrolases; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Amino Acid Sequence; Mutation
PubMed: 38721302
DOI: 10.3389/abp.2024.12299 -
AMB Express May 2024L-asparaginase is an important therapeutic enzyme that is frequently utilized in the chemotherapy regimens of adults as well as pediatric patients with acute...
L-asparaginase is an important therapeutic enzyme that is frequently utilized in the chemotherapy regimens of adults as well as pediatric patients with acute lymphoblastic leukemia. However, a high rate of hypersensitivity with prolonged use has limited its utilization. Stenotrophomonas maltophilia (S. maltophilia) EMCC2297 isolate was reported as a novel and promising source for L- asparaginase. The present study aimed at the production, purification, and characterization of L- asparaginase from S. maltophilia EMCC2297 isolate. The microbial production of L-asparaginase by the test isolate could be increased by pre-exposure to chloramphenicol at 200 µg/ml concentration. S. maltophilia EMCC2297 L-asparaginase could be purified to homogeneity by ammonium sulphate precipitation and the purified form obtained by gel exclusion chromatography showed total activity of 96.4375 IU/ml and specific activity of 36.251 IU/mg protein. SDS-PAGE analysis revealed that the purified form of the enzyme is separated at an apparent molecular weight of 17 KDa. Michaelis-Menten constant analysis showed a Km value of 4.16 × 10 M with L-asparagine as substrate and Vmax of 10.67 IU/ml. The antitumor activity of the purified enzyme was evaluated on different cell lines and revealed low IC50 of 2.2 IU/ml and 2.83 IU/ml for Hepatocellular cancer cell line (HepG-2), human leukemia cancer cell line (K-562), respectively whereas no cytotoxic effect could be detected on normal human lung fibroblast cells (MRC-5). However, mice treated with native L-asparaginase showed lower IgG titre compared to commercial L-asparaginase. This study highlights the promising characteristics of this enzyme making it a valuable candidate for further research and development to be an adduct in cancer chemotherapy.
PubMed: 38704453
DOI: 10.1186/s13568-024-01700-9 -
World Journal of Stem Cells Apr 2024Leukemia stem cells (LSCs) are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia (AML), as they are protected by the...
BACKGROUND
Leukemia stem cells (LSCs) are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia (AML), as they are protected by the bone marrow microenvironment (BMM) against conventional therapies. Gossypol acetic acid (GAA), which is extracted from the seeds of cotton plants, exerts anti-tumor roles in several types of cancer and has been reported to induce apoptosis of LSCs by inhibiting Bcl2.
AIM
To investigate the exact roles of GAA in regulating LSCs under different microenvironments and the exact mechanism.
METHODS
In this study, LSCs were magnetically sorted from AML cell lines and the CD34CD38 population was obtained. The expression of leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) and forkhead box M1 (FOXM1) was evaluated in LSCs, and the effects of GAA on malignancies and mitochondrial function were measured.
RESULTS
LRPPRC was found to be upregulated, and GAA inhibited cell proliferation by degrading LRPPRC. GAA induced LRPPRC degradation and inhibited the activation of interleukin 6 (IL-6)/janus kinase (JAK) 1/signal transducer and activator of transcription (STAT) 3 signaling, enhancing chemosensitivity in LSCs against conventional chemotherapies, including L-Asparaginase, Dexamethasone, and cytarabine. GAA was also found to downregulate FOXM1 indirectly by regulating LRPPRC. Furthermore, GAA induced reactive oxygen species accumulation, disturbed mitochondrial homeostasis, and caused mitochondrial dysfunction. By inhibiting IL-6/JAK1/STAT3 signaling degrading LRPPRC, GAA resulted in the elimination of LSCs. Meanwhile, GAA induced oxidative stress and subsequent cell damage by causing mitochondrial damage.
CONCLUSION
Taken together, the results indicate that GAA might overcome the BMM protective effect and be considered as a novel and effective combination therapy for AML.
PubMed: 38690512
DOI: 10.4252/wjsc.v16.i4.444 -
Frontiers in Oncology 2024Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have...
Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with -derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR--mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML.
PubMed: 38690164
DOI: 10.3389/fonc.2024.1326754 -
Frontiers in Immunology 2024Asparaginase (ASNase) is a crucial part of acute leukemia treatment, but immune responses to the agent can reduce its effectiveness and increase the risk of relapse....
BACKGROUND
Asparaginase (ASNase) is a crucial part of acute leukemia treatment, but immune responses to the agent can reduce its effectiveness and increase the risk of relapse. Currently, no reliable and validated biomarker predicts ASNase-induced hypersensitivity reactions during therapy. We aimed to identify predictive biomarkers and determine immune cells responsible for anaphylaxis using a murine model of ASNase hypersensitivity.
METHODS
Our preclinical study uses a murine model to investigate predictive biomarkers of ASNase anaphylaxis, including anti-ASNase antibody responses, immune complex (IC) levels, ASNase-specific binding to leukocytes or basophils, and basophil activation.
RESULTS
Our results indicate that mice immunized to ASNase exhibited dynamic IgM, IgG, and IgE antibody responses. The severity of ASNase-induced anaphylaxis was found to be correlated with levels of IgG and IgE, but not IgM. Basophils from immunized mice were able to recognize and activate in response to ASNase ex vivo, and the extent of recognition and activation also correlated with the severity of anaphylaxis observed. Using a multivariable model that included all biomarkers significantly associated with anaphylaxis, independent predictors of ASNase-induced hypersensitivity reactions were found to be ASNase IC levels and ASNase-specific binding to leukocytes or basophils. Consistent with our multivariable analysis, we found that basophil depletion significantly protected mice from ASNase-induced hypersensitivity reactions, supporting that basophils are essential and can be used as a predictive marker of ASNase-induced anaphylaxis.
CONCLUSIONS
Our study demonstrates the need for using tools that can detect both IC- and IgE-mediated hypersensitivity reactions to mitigate the risk of ASNase-induced hypersensitivity reactions during treatment.
Topics: Animals; Asparaginase; Basophils; Mice; Drug Hypersensitivity; Anaphylaxis; Immunoglobulin E; Female; Disease Models, Animal; Biomarkers; Immunoglobulin G; Antineoplastic Agents
PubMed: 38686384
DOI: 10.3389/fimmu.2024.1392099 -
International Journal of Molecular... Apr 2024This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from (TsA)....
This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.
Topics: Asparaginase; Thermococcus; Hydrogen-Ion Concentration; Enzyme Stability; Biocatalysis; Polyethylene Glycols; Temperature; Archaeal Proteins
PubMed: 38673759
DOI: 10.3390/ijms25084174 -
Journal of Feline Medicine and Surgery Apr 2024The aim of this study was to determine response rates, median progression-free intervals (PFIs) and median survival times (MSTs) for cats with intermediate-large cell...
Evaluation of a multiagent chemotherapy protocol combining vincristine, cyclophosphamide, mitoxantrone and prednisolone (CMOP) for treatment of feline intermediate-large cell lymphoma.
OBJECTIVES
The aim of this study was to determine response rates, median progression-free intervals (PFIs) and median survival times (MSTs) for cats with intermediate-large cell lymphoma treated with a vincristine, cyclophosphamide, mitoxantrone and prednisolone (CMOP) protocol. A secondary objective was to determine the tolerability of mitoxantrone used within this multiagent protocol.
METHODS
The medical records of 31 cats treated at a single institution between 2009 and 2022 were reviewed to identify suitable cases. Cats were included in the study if they had a confirmed diagnosis of intermediate-large cell lymphoma, had received a CMOP protocol as first-line treatment and had completed at least one 4-week cycle of this protocol. Modifications allowed in the protocol included the use of l-asparaginase, vinblastine substitution for vincristine, chlorambucil substitution for cyclophosphamide and dexamethasone or methylprednisolone substitution for prednisolone.
RESULTS
The overall response rate was 74% (n = 23), with 45% (n = 14) achieving complete remission (CR), 29% (n = 9) achieving partial remission (PR) and 26% (n = 8) achieving stable disease (SD). The Kaplan-Meier median PFI and MST were 139 days and 206 days, respectively. Responders (CR or PR) had a significantly longer ( <0.001) median PFI and MST compared with non-responders (SD) (176 days vs 62 days, and 251 days vs 61 days, respectively). Cats that achieved CR had a significantly longer median PFI and MST ( <0.001) at 178 days and 1176 days, respectively. The 6-month and 1- and 2-year survival rates in cats with CR were 64%, 57% and 35%, respectively. Treatment with mitoxantrone was generally well tolerated, with no cats experiencing Veterinary Cooperative Oncology Group adverse effects above grade 2.
CONCLUSIONS AND RELEVANCE
The CMOP protocol is an alternative and well-tolerated treatment for cats with intermediate-large cell lymphoma. As demonstrated with previous chemotherapy protocols, cats that respond to treatment, particularly those that achieve CR, are likely to have more durable responses.
Topics: Animals; Cats; Mitoxantrone; Cat Diseases; Vincristine; Cyclophosphamide; Antineoplastic Combined Chemotherapy Protocols; Prednisolone; Male; Female; Retrospective Studies; Treatment Outcome
PubMed: 38647264
DOI: 10.1177/1098612X241234614