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Carbohydrate Research Jun 2024High-mannose-type glycan structure of N-glycoproteins plays important roles in the proper folding of proteins in sorting glycoprotein secretion and degradation of...
High-mannose-type glycan structure of N-glycoproteins plays important roles in the proper folding of proteins in sorting glycoprotein secretion and degradation of misfolded proteins in the endoplasmic reticulum (ER). The GlcManGlcNAc (G1M9)-type N-glycan is one of the most important signaling molecules in the ER. However, current chemical synthesis strategies are laborious, warranting more practical approaches for G1M9-glycopeptide development. Wang et al. reported the procedure to give G1M9-Asn-Fmoc through chemical modifications and purifications from 40 chicken eggs, but only 3.3 mg of G1M9-glycopeptide was obtained. Therefore, better methods are needed to obtain more than 10 mg of G1M9-glycopeptide. In this study, we report the preparation of G1M9-glycopeptide (13.2 mg) linking Asn-Gly-Thr triad as consensus sequence from 40 chicken eggs. In this procedure, λ-carrageenan treatment followed by papain treatment was used to separate the Fc region of IgY antibody that harbors high-mannose glycans. Moreover, cotton hydrophilic interaction liquid chromatography was adapted for easy purification. The resulting G1M9-Asn(Fmoc)-Gly-Thr was identified by nuclear magnetic resonance and mass spectroscopy. G1M9-Asn(Fmoc)-Gly, G1M9-Asn(Fmoc), and G1M9-OH were also detected by mass spectroscopy. Here, our developed G1M9-tripeptide might be useful for the elucidation of glycoprotein functions as well as the specific roles of the consensus sequence.
Topics: Animals; Chickens; Egg Yolk; Oligosaccharides; Asparagine; Mannose; Threonine; Consensus Sequence; Glycine; Glycopeptides
PubMed: 38703662
DOI: 10.1016/j.carres.2024.109138 -
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 -
Journal of AAPOS : the Official... Jun 2024NGLY1 deficiency is a rare autosomal recessive disorder with core features of global developmental delay, liver enzyme abnormalities, movement disorder, polyneuropathy,...
BACKGROUND
NGLY1 deficiency is a rare autosomal recessive disorder with core features of global developmental delay, liver enzyme abnormalities, movement disorder, polyneuropathy, and hypo- or alacrima. We characterized the full spectrum and evolution of the ocular phenotype in a prospective natural history of NGLY1 deficiency.
METHODS
We collected ophthalmological data on 29 individuals with NGLY1 deficiency in a natural history study. Medical records were reviewed to confirm caregiver-reported symptoms. Of the 29, 15 participants appeared for at least one ophthalmological examination.
RESULTS
Caregivers reported at least one ocular sign or symptom in 90% of participants (26/29), most commonly decreased tears, refractive error, and chronic infection. Daily eye medication, including artificial tears, ophthalmic ointment, and topical antibiotics were used by 62%. Ophthalmological examination confirmed refractive errors in 93% (14/15) and corneal abnormalities in 73% (11/15).
CONCLUSIONS
Given nearly universal hypolacrima and additional prominent ocular findings in NGLY1 deficiency, a targeted ocular history and ophthalmologic examination may facilitate prompt diagnosis and early initiation of preventive eye care, preserving vision and overall ocular health.
Topics: Adolescent; Adult; Child; Child, Preschool; Female; Humans; Infant; Male; Young Adult; Eye Diseases; Longitudinal Studies; Phenotype; Prospective Studies; Refractive Errors; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
PubMed: 38697387
DOI: 10.1016/j.jaapos.2024.103925 -
Pancreas May 2024Amino acids play an essential role in protein synthesis, metabolism and survival of pancreatic acini. Adequate nutritional support is important for acute pancreatitis...
OBJECTIVES
Amino acids play an essential role in protein synthesis, metabolism and survival of pancreatic acini. Adequate nutritional support is important for acute pancreatitis treatment. However, high concentrations of arginine and lysine may induce acute pancreatitis. The study aimed to identify the most suitable L-amino acids as safe energy sources for pancreatic acinar cells.
METHODS
Pancreatic acini were isolated from male Wistar rats. Effects of amino acids (0.1-20 mM) on uncoupled respiration of isolated acini were studied with a Clark electrode. Cell death was evaluated with fluorescent microscopy and DNA gel electrophoresis.
RESULTS
Among the tested amino acids, glutamate, glutamine, alanine, lysine and aspartate were able to stimulate the uncoupled respiration rate of isolated pancreatic acini, while arginine, histidine and asparagine were not. Lysine, arginine and glutamine (20 mM) caused complete loss of plasma membrane integrity of acinar cells after 24 h of incubation. Glutamine also caused early (2-4 h) cell swelling and blebbing. Aspartate, asparagine and glutamate only moderately decreased the number of viable cells, while alanine and histidine were not toxic. DNA fragmentation assay and microscopic analysis of nuclei showed no evidence of apoptosis in cells treated with amino acids.
CONCLUSIONS
Alanine and glutamate are safe and effective energy sources for mitochondria of pancreatic acinar cells.
PubMed: 38696385
DOI: 10.1097/MPA.0000000000002350 -
Journal of Agricultural and Food... May 2024Maillard reaction (MR) plays a pivotal role in the food flavor industry, including a cascade of reactions starting with the reaction between amino compounds and reducing...
Maillard reaction (MR) plays a pivotal role in the food flavor industry, including a cascade of reactions starting with the reaction between amino compounds and reducing sugars, and thus provides various colors and flavors. A new group of volatile compounds called pyrazinones found in MR are now getting more attention. In this study, eight volatile pyrazinones were found in the asparagine MR systems, in which 3,5-dimethyl- and 3,6-dimethyl-2(1)-pyrazinones were reported for the first time. The major formation pathways were the reactions between asparagine and α-dicarbonyls, with decarboxylation as a critical step. Besides, novel alternative pathways involving alanine amidation and successive reactions with α-dicarbonyls were explored and successfully formed eight pyrazinones. The major differences between alanine-amidated pathways and decarboxylation pathways are the amidation step and absence of the decarboxylation step. For the alanine-amidated pathways, the higher the temperature, the better the amidation effect. The optimal amidation temperature was 200 °C in this study. The reaction between the alanine amide and α-dicarbonyls after amidation can happen at low temperatures, such as 35 and 50 °C, proposing the possibility of pyrazinone formation in real food systems. Further investigations should be conducted to investigate volatile pyrazinones in various food systems as well as the biological effects and kinetic formation differences of the volatile pyrazinones.
Topics: Maillard Reaction; Pyrazines; Alanine; Asparagine; Volatile Organic Compounds; Flavoring Agents
PubMed: 38695891
DOI: 10.1021/acs.jafc.4c02079 -
Biochemistry May 2024Lipoxygenases (LOXs) from pathogenic fungi are potential therapeutic targets for defense against plant and select human diseases. In contrast to the canonical LOXs in...
Lipoxygenases (LOXs) from pathogenic fungi are potential therapeutic targets for defense against plant and select human diseases. In contrast to the canonical LOXs in plants and animals, fungal LOXs are unique in having appended -linked glycans. Such important post-translational modifications (PTMs) endow proteins with altered structure, stability, and/or function. In this study, we present the structural and functional outcomes of removing or altering these surface carbohydrates on the LOX from the devastating rice blast fungus, , LOX. Alteration of the PTMs did notinfluence the active site enzyme-substrate ground state structures as visualized by electron-nuclear double resonance (ENDOR) spectroscopy. However, removal of the eight -linked glycans by asparagine-to-glutamine mutagenesis nonetheless led to a change in substrate selectivity and an elevated activation energy for the reaction with substrate linoleic acid, as determined by kinetic measurements. Comparative hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of wild-type and Asn-to-Gln LOX variants revealed a regionally defined impact on the dynamics of the arched helix that covers the active site. Guided by these HDX results, a single glycan sequon knockout was generated at position 72, and its comparative substrate selectivity from kinetics nearly matched that of the Asn-to-Gln variant. The cumulative data from model glyco-enzyme LOX showcase how the presence, alteration, or removal of even a single -linked glycan can influence the structural integrity and dynamics of the protein that are linked to an enzyme's catalytic proficiency, while indicating that extensive glycosylation protects the enzyme during pathogenesis by protecting it from protease degradation.
Topics: Glycosylation; Lipoxygenase; Substrate Specificity; Protein Conformation; Catalytic Domain; Protein Processing, Post-Translational; Fungal Proteins; Models, Molecular; Polysaccharides; Kinetics; Enzyme Activation
PubMed: 38690768
DOI: 10.1021/acs.biochem.4c00109 -
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: Animals; Humans; Mice; Aspartate-Ammonia Ligase; Disease Models, Animal; Disease Progression; Kidney; Oligonucleotides, Antisense; Polycystic Kidney Diseases
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