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Rice (New York, N.Y.) Apr 2024Preharvest sprouting (PHS) is an undesirable trait that decreases yield and quality in rice production. Understanding the genes and regulatory mechanisms underlying PHS...
Preharvest sprouting (PHS) is an undesirable trait that decreases yield and quality in rice production. Understanding the genes and regulatory mechanisms underlying PHS is of great significance for breeding PHS-resistant rice. In this study, we identified a mutant, preharvest sprouting 39 (phs39), that exhibited an obvious PHS phenotype in the field. MutMap analysis and transgenic experiments demonstrated that OsAAH, which encodes allantoate amidohydrolase, is the causal gene of phs39 and is essential for PHS resistance. OsAAH was highly expressed in roots and leaves at the heading stage and gradually increased and then weakly declined in the seed developmental stage. OsAAH protein was localized to the endoplasmic reticulum, with a function of hydrolyzing allantoate in vitro. Disruption of OsAAH increased the levels of ureides (allantoate and allantoin) and activated the tricarboxylic acid (TCA) cycle, and thus increased energy levels in developing seeds. Additionally, the disruption of OsAAH significantly increased asparagine, arginine, and lysine levels, decreased tryptophan levels, and decreased levels of indole-3-acetic acid (IAA) and abscisic acid (ABA). Our findings revealed that the OsAAH of ureide catabolism is involved in the regulation of rice PHS via energy and hormone metabolisms, which will help to facilitate the breeding of rice PHS-resistant varieties.
PubMed: 38622442
DOI: 10.1186/s12284-024-00706-y -
BMC Chemistry Apr 2024Cyclin-dependent kinase 8 (CDK8) has emerged as a promising target for inhibiting cancer cell function, intensifying efforts towards the development of CDK8 inhibitors...
Cyclin-dependent kinase 8 (CDK8) has emerged as a promising target for inhibiting cancer cell function, intensifying efforts towards the development of CDK8 inhibitors as potential cancer therapeutics. Mutations in CDK8, a protein kinase, are also implicated as a primary factor associated with tumor formation. In this study, we identified potential inhibitors through virtual screening for CDK8 and single amino acid mutations in CDK8, namely D173A (Aspartate 173 mutate to Alanine), D189N (Aspartate 189 mutate to Asparagine), T196A (Threonine 196 mutate to Alanine) and T196D (Threonine 196 mutate to Aspartate). Four databases (CHEMBEL, ZINC, MCULE, and MolPort) containing 65,209,131 molecules have been searched to identify new inhibitors for CDK8 and its single mutations. In the first step, structure-based pharmacophore modeling in the Pharmit server was used to select the compounds to know the inhibitors. Then molecules with better predicted drug-like molecule properties were selected. The final filter used to select more effective inhibitors among the previously selected molecules was molecular docking. Finally, 13 hits for CDK8, 11 hits for D173A, 11 hits for D189N, 15 hits for T196A, and 12 hits for T196D were considered potential inhibitors. A majority of the virtual screening hits exhibited satisfactorily predict pharmacokinetic characteristics and toxicity properties.
PubMed: 38615023
DOI: 10.1186/s13065-024-01175-6 -
Avicenna Journal of Medical... 2024Asparagine is an amino acid that can be converted into aspartic acid and ammonia by the enzyme L-asparaginase. Some forms of cancer, such Acute Lymphoblastic Leukaemia...
BACKGROUND
Asparagine is an amino acid that can be converted into aspartic acid and ammonia by the enzyme L-asparaginase. Some forms of cancer, such Acute Lymphoblastic Leukaemia (ALL) and Non-Hodgkin Lymphoma (NHL), respond well to this enzyme when employed as a chemotherapeutic drug. The purpose of this research was to find bacteria that can manufacture the enzymes L-asparaginasein marine slattern sediment which can be employed in commercial and industrial scale production.
METHODS
All of the strains were identified as . by biochemical and molecular testing. The strain belongs to the genus, according to nutritional, biochemical, PCR and 16srRNA sequencing data.
RESULTS
According to the findings of this research, have the potential to create a substance that is helpful in a variety of medical applications. The results of this study hint to the possibility that bacteria have the ability to produce antimicrobial compounds, which have the potential to be successful in a wide variety of environments.
CONCLUSION
Numerous opportunities may arise for researchers interested in utilizing the medical potential of enzyme-producing bacteria if they are successfully isolated and screened from aquatic and terrestrial habitats.
PubMed: 38605737
DOI: 10.18502/ajmb.v16i1.14170 -
Trends in Molecular Medicine Jun 2024Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive form of pancreatic cancer, known for its challenging diagnosis and limited treatment options. The focus on... (Review)
Review
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive form of pancreatic cancer, known for its challenging diagnosis and limited treatment options. The focus on metabolic reprogramming as a key factor in tumor initiation, progression, and therapy resistance has gained prominence. In this review we focus on the impact of metabolic changes on the interplay among stromal, immune, and tumor cells, as glutamine and branched-chain amino acids (BCAAs) emerge as pivotal players in modulating immune cell functions and tumor growth. We also discuss ongoing clinical trials that explore metabolic modulation for PDAC, targeting mitochondrial metabolism, asparagine and glutamine addiction, and autophagy inhibition. Overcoming challenges in understanding nutrient effects on immune-stromal-tumor interactions holds promise for innovative therapeutic strategies.
Topics: Humans; Pancreatic Neoplasms; Animals; Carcinoma, Pancreatic Ductal; Glutamine; Tumor Microenvironment; Mitochondria; Amino Acids, Branched-Chain; Autophagy; Energy Metabolism
PubMed: 38604929
DOI: 10.1016/j.molmed.2024.03.008 -
Frontiers in Bioengineering and... 2024Investigating the metabolic behaviour of different cellular phenotypes, i.e., good/bad grower and/or producer, in production culture is important to identify the key... (Review)
Review
Investigating the metabolic behaviour of different cellular phenotypes, i.e., good/bad grower and/or producer, in production culture is important to identify the key metabolite(s)/pathway(s) that regulate cell growth and/or recombinant protein production to improve the overall yield. Currently, LC-MS, GC-MS and NMR are the most used and advanced technologies for investigating the metabolome. Although contributed significantly in the domain, each technique has its own biasness towards specific metabolites or class of metabolites due to various reasons including variability in the concept of working, sample preparation, metabolite-extraction methods, metabolite identification tools, and databases. As a result, the application of appropriate analytical technique(s) is very critical. This review provides a state-of-the-art technological insights and overview of metabolic mechanisms involved in regulation of cell growth and/or recombinant protein production for improving yield from CHO cultures. In this review, the advancements in CHO metabolomics over the last 10 years are traced based on a bibliometric analysis of previous publications and discussed. With the technical advancement in the domain of LC-MS, GC-MS and NMR, metabolites of glycolytic and nucleotide biosynthesis pathway (glucose, fructose, pyruvate and phenylalanine, threonine, tryptophan, arginine, valine, asparagine, and serine, etc.) were observed to be upregulated in exponential-phase thereby potentially associated with cell growth regulation, whereas metabolites/intermediates of TCA, oxidative phosphorylation (aspartate, glutamate, succinate, malate, fumarate and citrate), intracellular NAD+/NADH ratio, and glutathione metabolic pathways were observed to be upregulated in stationary-phase and hence potentially associated with increased cell-specific productivity in CHO bioprocess. Moreover, each of technique has its own bias towards metabolite identification, indicating their complementarity, along with a number of critical gaps in the CHO metabolomics pipeline and hence first time discussed here to identify their potential remedies. This knowledge may help in future study designs to improve the metabolomic coverage facilitating identification of the metabolites/pathways which might get missed otherwise and explore the full potential of metabolomics for improving the CHO bioprocess performances.
PubMed: 38600943
DOI: 10.3389/fbioe.2024.1347138 -
BioRxiv : the Preprint Server For... Mar 2024The bioenergetic demand of photoreceptors rivals that of cancer cells, and numerous metabolic similarities exist between these cells. Glutamine (Gln) anaplerosis via the...
The bioenergetic demand of photoreceptors rivals that of cancer cells, and numerous metabolic similarities exist between these cells. Glutamine (Gln) anaplerosis via the tricarboxylic acid (TCA) cycle provides biosynthetic intermediates and is a hallmark of cancer metabolism. In this process, Gln is first converted to glutamate via glutaminase (GLS), which is a crucial pathway in many cancer cells. To date, no study has been undertaken to examine the role of Gln metabolism in photoreceptors. Here, mice lacking GLS in rod photoreceptors were generated. Animals lacking GLS experienced rapid photoreceptor degeneration with concomitant functional loss. Gln has multiple roles in metabolism including redox balance, biosynthesis of nucleotides and amino acids, and supplementing the TCA cycle. Few alterations were noted in redox balance. Unlabeled targeted metabolomics demonstrated few changes in glycolytic and TCA cycle intermediates, which corresponded with a lack of significant changes in mitochondrial function. GLS deficiency in rod photoreceptors did decrease the fractional labelling of TCA cycle intermediates when provided uniformly labeled C-Gln . However, supplementation with alpha-ketoglutarate provided only marginal rescue of photoreceptor degeneration. Nonessential amino acids, glutamate and aspartate, were decreased in the retina of mice lacking GLS in rod photoreceptors. In accordance with this amino acid deprivation, the integrated stress response (ISR) was found to be activated with decreased global protein synthesis. Importantly, supplementation with asparagine delayed photoreceptor degeneration to a greater degree than alpha-ketoglutarate. These data show that GLS-mediated Gln catabolism is essential for rod photoreceptor amino acid biosynthesis, function, and survival.
PubMed: 38586045
DOI: 10.1101/2024.03.26.582525 -
Scientific Reports Apr 2024Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In...
Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.
Topics: Humans; Female; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; RNA, Messenger; Breast Neoplasms; Gene Editing
PubMed: 38582932
DOI: 10.1038/s41598-024-58765-6 -
Biochemical and Biophysical Research... May 2024Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was...
Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 μg of crude extract, which is the equivalent of 5 × 10 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.
Topics: Humans; Congenital Disorders of Glycosylation; Cytosol; Glycosylation; Glycoproteins; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
PubMed: 38581946
DOI: 10.1016/j.bbrc.2024.149826 -
BioRxiv : the Preprint Server For... Mar 2024Pyrroloiminoquinone containing natural products have long been known for their biological activities. They are derived from tryptophan, but their biosynthetic pathways...
Pyrroloiminoquinone containing natural products have long been known for their biological activities. They are derived from tryptophan, but their biosynthetic pathways have remained elusive. Studies on the biosynthetic gene cluster (BGC) that produces the ammosamides revealed that the first step is attachment of Trp to the C-terminus of a scaffold peptide in an ATP and tRNA dependent manner catalyzed by a PEptide Amino-acyl tRNA ligase (PEARL). The indole of the Trp is then oxidized to a hydroxyquinone. We previously proposed a chemically plausible and streamlined pathway for converting this intermediate to the ammosamides using additional enzymes encoded in the BGC. In this study, we report the activity of four additional enzymes that show that the proposed pathway is incorrect and that Nature's route towards pyrroloiminoquinones is much more complicated. We demonstrate that, surprisingly, the amino groups in pyrroloiminoquinones are derived from three different sources, glycine, asparagine, and leucine, all introduced in a tRNA dependent manner. We also show that an FAD-dependent putative glycine oxidase is required for the process that incorporates the nitrogens from glycine and leucine, and that a quinone reductase is required for the incorporation of the asparagine. Additionally, we provide the first insights into the evolutionary origin of the PEARLs as well as related enzymes such as the glutamyl-tRNA dependent dehydratases involved in the biosynthesis of lanthipeptides and thiopeptides. These enzymes appear to all have descended from the ATP-GRASP protein family.
PubMed: 38559119
DOI: 10.1101/2024.03.12.584671 -
MAbs 2024Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are common degradation pathways that affect the stability of therapeutic antibodies. These...
Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are common degradation pathways that affect the stability of therapeutic antibodies. These modifications can pose a significant challenge in the development of biopharmaceuticals. As such, the early engineering and selection of chemically stable monoclonal antibodies (mAbs) can substantially mitigate the risk of subsequent failure. In this study, we introduce a novel in silico approach for predicting deamidation and isomerization sites in therapeutic antibodies by analyzing the structural environment surrounding asparagine and aspartate residues. The resulting quantitative structure-activity relationship (QSAR) model was trained using previously published forced degradation data from 57 clinical-stage mAbs. The predictive accuracy of the model was evaluated for four different states of the protein structure: (1) static homology models, (2) enhancing low-frequency vibrational modes during short molecular dynamics (MD) runs, (3) a combination of (2) with a protonation state reassignment, and (4) conventional full-atomistic MD simulations. The most effective QSAR model considered the accessible surface area (ASA) of the residue, the pKa value of the backbone amide, and the root mean square deviations of both the alpha carbon and the side chain. The accuracy was further enhanced by incorporating the QSAR model into a decision tree, which also includes empirical information about the sequential successor and the position in the protein. The resulting model has been implemented as a plugin named "Forecasting Reactivity of Isomerization and Deamidation in Antibodies" in MOE software, completed with a user-friendly graphical interface to facilitate its use.
Topics: Isomerism; Asparagine; Antibodies, Monoclonal; Amides; Software
PubMed: 38546837
DOI: 10.1080/19420862.2024.2333436