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International Journal of Molecular... Apr 2024The involvement of the second pair of chlorophylls, termed A and A, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at...
The involvement of the second pair of chlorophylls, termed A and A, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A and A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700-P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700-P700) FTIR DS cannot be due to only the P and P pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A and A pigments are modified upon P700 formation. Previously, we showed that the A pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.
Topics: Photosystem I Protein Complex; Spectroscopy, Fourier Transform Infrared; Synechocystis; Mutation; Chlorophyll; Electron Transport; Chlorophyll A
PubMed: 38732056
DOI: 10.3390/ijms25094839 -
Frontiers in Immunology 2024Hereditary angioedema (HAE) is a rare, life-threatening autosomal dominant genetic disorder caused by a deficient and/or dysfunctional C1 esterase inhibitor (C1-INH)...
INTRODUCTION
Hereditary angioedema (HAE) is a rare, life-threatening autosomal dominant genetic disorder caused by a deficient and/or dysfunctional C1 esterase inhibitor (C1-INH) (type 1 and type 2) leading to recurrent episodes of edema. This study aims to explore HAE patients' metabolomic profiles and identify novel potential diagnostic biomarkers for HAE. The study also examined distinguishing HAE from idiopathic angioedema (AE).
METHODS
Blood plasma samples from 10 HAE (types 1/2) patients, 15 patients with idiopathic AE, and 20 healthy controls were collected in Latvia and analyzed using LC-MS based targeted metabolomics workflow. T-test and fold change calculation were used to identify metabolites with significant differences between diseases and control groups. ROC analysis was performed to evaluate metabolite based classification model.
RESULTS
A total of 33 metabolites were detected and quantified. The results showed that isovalerylcarnitine, cystine, and hydroxyproline were the most significantly altered metabolites between the disease and control groups. Aspartic acid was identified as a significant metabolite that could differentiate between HAE and idiopathic AE. The mathematical combination of metabolites (hydroxyproline * cystine)/(creatinine * isovalerylcarnitine) was identified as the diagnosis signature for HAE. Furthermore, glycine/asparagine ratio could differentiate between HAE and idiopathic AE.
CONCLUSION
Our study identified isovalerylcarnitine, cystine, and hydroxyproline as potential biomarkers for HAE diagnosis. Identifying new biomarkers may offer enhanced prospects for accurate, timely, and economical diagnosis of HAE, as well as tailored treatment selection for optimal patient care.
Topics: Humans; Female; Male; Angioedemas, Hereditary; Adult; Biomarkers; Metabolomics; Middle Aged; Metabolome; Young Adult; Case-Control Studies; Complement C1 Inhibitor Protein; Adolescent
PubMed: 38726011
DOI: 10.3389/fimmu.2024.1324671 -
Frontiers in Molecular Biosciences 2024Traditional Chinese medicine (TCM) has a long history and particular advantages in the diagnosis and treatment of diabetic foot gangrene (DFG). Patients with DFG are...
Traditional Chinese medicine (TCM) has a long history and particular advantages in the diagnosis and treatment of diabetic foot gangrene (DFG). Patients with DFG are mainly divided into two subtypes, tendon lesion with edema (GT) and ischemic lesion without edema (GI), which are suitable for different medical strategies. Metabolomics has special significance in unravelling the complexities of multifactorial and multisystemic disorders. This study acquired the serum metabolomic profiles of two traditional Chinese medicine subtypes of DFG to explore potential molecular evidence for subtype characterization, which may contribute to the personalized treatment of DFG. A total of 70 participants were recruited, including 20 with DM and 50 with DFG (20 with GI and 30 with GT). Conventional gas chromatography-mass spectrometry (GC-MS) followed by orthogonal partial least-squares discriminant analysis (OPLS-DA) were used as untargeted metabolomics approaches to explore the serum metabolomic profiles. Kyoto encyclopedia of genes and genomes (KEGG) and MetaboAnalyst were used to identify the related metabolic pathways. Compared with DM patients, the levels of 14 metabolites were altered in the DFG group, which were also belonged to the differential metabolites of GI (13) and GT (7) subtypes, respectively. Among these, urea, α-D-mannose, cadaverine, glutamine, L-asparagine, D-gluconic acid, and indole could be regarded as specific potential metabolic markers for GI, as well as L-leucine for GT. In the GI subtype, D-gluconic acid and L-asparagine are positively correlated with activated partial thromboplastin time (APTT) and fibrinogen (FIB). In the GT subtype, L-leucine is positively correlated with the inflammatory marker C-reactive protein (CRP). Arginine and proline metabolism, glycine, serine and threonine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis are the most important metabolic pathways associated with GI. The main metabolic pathways related to GT include pyrimidine metabolism, glutathione metabolism, biosynthesis of valine, leucine, and isoleucine, as well as valine, serine, and isoleucine with metabolites. The results of this study indicate that patients with different DFG subtypes have distinct metabolic profiles, which reflect the pathological characteristics of each subtype respectively. These findings will help us explore therapeutic targets for DFG and develop precise treatment strategies.
PubMed: 38725871
DOI: 10.3389/fmolb.2024.1384307 -
Journal of the American Chemical Society May 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 Aminoacyl-tRNA Ligase (PEARL). The indole of 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 from two gene clusters, which show that the previously proposed pathway is incorrect and that Nature's route toward pyrroloiminoquinones is much more complicated. We demonstrate that, surprisingly, amino groups in pyrroloiminoquinones are derived from (at least) three different sources, glycine, asparagine, and leucine, all introduced in a tRNA-dependent manner. We also show that an FAD-dependent putative glycine oxidase (Amm14) is required for the process that incorporates the nitrogens from glycine and leucine and that a quinone reductase is required for the incorporation of 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.
Topics: Pyrroloiminoquinones; Multigene Family; Biosynthetic Pathways
PubMed: 38719200
DOI: 10.1021/jacs.4c03677 -
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 -
PloS One 2024Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact...
Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase.
Topics: Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; Bacterial Proteins; Proteomics; Algorithms; Proteins
PubMed: 38701058
DOI: 10.1371/journal.pone.0299287 -
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 -
Human Genomics Apr 2024A major obstacle faced by families with rare diseases is obtaining a genetic diagnosis. The average "diagnostic odyssey" lasts over five years and causal variants are...
BACKGROUND
A major obstacle faced by families with rare diseases is obtaining a genetic diagnosis. The average "diagnostic odyssey" lasts over five years and causal variants are identified in under 50%, even when capturing variants genome-wide. To aid in the interpretation and prioritization of the vast number of variants detected, computational methods are proliferating. Knowing which tools are most effective remains unclear. To evaluate the performance of computational methods, and to encourage innovation in method development, we designed a Critical Assessment of Genome Interpretation (CAGI) community challenge to place variant prioritization models head-to-head in a real-life clinical diagnostic setting.
METHODS
We utilized genome sequencing (GS) data from families sequenced in the Rare Genomes Project (RGP), a direct-to-participant research study on the utility of GS for rare disease diagnosis and gene discovery. Challenge predictors were provided with a dataset of variant calls and phenotype terms from 175 RGP individuals (65 families), including 35 solved training set families with causal variants specified, and 30 unlabeled test set families (14 solved, 16 unsolved). We tasked teams to identify causal variants in as many families as possible. Predictors submitted variant predictions with estimated probability of causal relationship (EPCR) values. Model performance was determined by two metrics, a weighted score based on the rank position of causal variants, and the maximum F-measure, based on precision and recall of causal variants across all EPCR values.
RESULTS
Sixteen teams submitted predictions from 52 models, some with manual review incorporated. Top performers recalled causal variants in up to 13 of 14 solved families within the top 5 ranked variants. Newly discovered diagnostic variants were returned to two previously unsolved families following confirmatory RNA sequencing, and two novel disease gene candidates were entered into Matchmaker Exchange. In one example, RNA sequencing demonstrated aberrant splicing due to a deep intronic indel in ASNS, identified in trans with a frameshift variant in an unsolved proband with phenotypes consistent with asparagine synthetase deficiency.
CONCLUSIONS
Model methodology and performance was highly variable. Models weighing call quality, allele frequency, predicted deleteriousness, segregation, and phenotype were effective in identifying causal variants, and models open to phenotype expansion and non-coding variants were able to capture more difficult diagnoses and discover new diagnoses. Overall, computational models can significantly aid variant prioritization. For use in diagnostics, detailed review and conservative assessment of prioritized variants against established criteria is needed.
Topics: Humans; Rare Diseases; Genome, Human; Genetic Variation; Computational Biology; Phenotype
PubMed: 38685113
DOI: 10.1186/s40246-024-00604-w -
EMBO Molecular Medicine Jun 2024Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations,...
Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD.
Topics: Polycystic Kidney Diseases; Animals; Aspartate-Ammonia Ligase; Mice; Disease Models, Animal; Oligonucleotides, Antisense; Disease Progression; Humans; Kidney
PubMed: 38684863
DOI: 10.1038/s44321-024-00071-9 -
Genes Mar 2024Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems....
Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems. Interactions between the calcium sensor calcineurin B-like protein (CBL) and its target, CBL-interacting protein kinase (CIPK), play important roles in the plant's response to various environmental stresses. As a food crop with important economic and research value, turnip ( var. ) has been well adapted to the environment of the Tibetan Plateau and become a traditional crop in the region. The gene in turnip has not been characterized. In this study, two duplicated genes, and ., were screened from the turnip genome. Based on the phylogenetic analysis, and . were found located in different sub-branches on the phylogenetic tree. Real-time fluorescence quantitative PCR analyses revealed their differential expression levels between the leaves and roots and in response to various stress treatments. The differences in their interactions with BrrCBLs were also revealed by yeast two-hybrid analyses. The results indicate that and . have undergone Asparagine-alanine-phenylalanine (NAF) site divergence during turnip evolution, which has resulted in functional differences between them. Furthermore, responded to high-pH (pH 8.5) stress, while retained its ancestral function (low K), thus providing further evidence of their functional divergence. These functional divergence genes facilitate turnip's good adaptation to the extreme environment of the Tibetan Plateau. In summary, the results of this study reveal the characteristics of the duplicated genes and provide a basis for further functional studies of in turnip.
Topics: Brassica rapa; Plant Proteins; Phylogeny; Gene Duplication; Gene Expression Regulation, Plant; Genes, Duplicate; Stress, Physiological
PubMed: 38674340
DOI: 10.3390/genes15040405