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Chemical Science Feb 2024Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine...
Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues.
PubMed: 38362406
DOI: 10.1039/d3sc06551k -
The Journal of Clinical Investigation Feb 2024Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat...
Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell-produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.
Topics: Infant, Newborn; Humans; Immunoglobulin G; Immunoglobulins, Intravenous; Immunoglobulin Fc Fragments; Inflammation; Receptors, Fc; Glycosylation
PubMed: 38357917
DOI: 10.1172/JCI172980 -
Analytical Chemistry Feb 2024Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid and has been linked to various diseases and health...
Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid and has been linked to various diseases and health conditions. However, current methods for identifying isoAsp sites in proteins often suffer from ambiguity and have not gained widespread adoption. We developed a novel method that exclusively labels isoAsp with deuterium. This method capitalizes on the unique structural characteristics of isoAsp residues, which possess a free α-carboxyl group and can form an oxazolone ring. Once the oxazolone ring forms, it facilitates racemization at the C-position, incorporating a deuteron from a DO solvent. The sites of deuterium-incorporated isoAsp in proteins can be unequivocally determined by comparing the precursor and product ion masses of the peptides from proteins reacted in HO and DO. The effectiveness of this method has been demonstrated through its application to model proteins lysozyme and rituximab. Furthermore, we have confirmed that the isoAsp deuterium-labeling reaction efficiently labels both l- and d-isoAsp without distinction, as well as isoglutamic acid (isoGlu), for which no effective detection methods currently exist.
Topics: Deuterium; Amino Acid Sequence; Oxazolone; Peptides; Mass Spectrometry; Proteins; Isoaspartic Acid
PubMed: 38344941
DOI: 10.1021/acs.analchem.3c05194 -
Plants (Basel, Switzerland) Jan 2024Sweet potato ( (L.) Lam.) is one of the most widely cultivated crops in the world, with outstanding stress tolerance, but drought stress can lead to a significant...
Sweet potato ( (L.) Lam.) is one of the most widely cultivated crops in the world, with outstanding stress tolerance, but drought stress can lead to a significant decrease in its yield. To reveal the response mechanism of sweet potato to drought stress, an integrated physiological, transcriptome and metabolome investigations were conducted in the leaves of two sweet potato varieties, drought-tolerant zhenghong23 (Z23) and a more sensitive variety, jinong432 (J432). The results for the physiological indexes of drought showed that the peroxidase (POD) and superoxide dismutase (SOD) activities of Z23 were 3.68 and 1.21 times higher than those of J432 under severe drought, while Z23 had a higher antioxidant capacity. Transcriptome and metabolome analysis showed the importance of the amino acid metabolism, respiratory metabolism, and antioxidant systems in drought tolerance. In Z23, amino acids such as asparagine participated in energy production during drought by providing substrates for the citrate cycle (TCA cycle) and glycolysis (EMP). A stronger respiratory metabolism ability could better maintain the energy supply level under drought stress. Drought stress also activated the expression of the genes encoding to antioxidant enzymes and the biosynthesis of flavonoids such as rutin, resulting in improved tolerance to drought. This study provides new insights into the molecular mechanisms of drought tolerance in sweet potato.
PubMed: 38337884
DOI: 10.3390/plants13030351 -
Theranostics 2024Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of...
The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.
Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Monobody binding was evaluated by molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4 T, and CD8 T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.
Topics: Animals; Mice; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Tumor Microenvironment; Calreticulin; Molecular Docking Simulation; Reactive Oxygen Species; Neoplasms; Cell Line, Tumor
PubMed: 38323311
DOI: 10.7150/thno.90376 -
Frontiers in Plant Science 2024The use of slow-release fertilizers and seed-fertilizers cause localized high-ammonium (NH ) environments in agricultural fields, adversely affecting wheat growth and...
The use of slow-release fertilizers and seed-fertilizers cause localized high-ammonium (NH ) environments in agricultural fields, adversely affecting wheat growth and development and delaying its yield. Thus, it is important to investigate the physiological responses of wheat and its tolerance to NH stress to improve the adaptation of wheat to high NH environments. In this study, the physiological mechanisms of ammonium tolerance in wheat () were investigated in depth by comparative analysis of two cultivars: NH -tolerant Xumai25 and NH -sensitive Yangmai20. Cultivation under hydroponic conditions with high NH (5 mM NH , AN) and nitrate (5 mM NO , NN), as control, provided insights into the nuanced responses of both cultivars. Compared to Yangmai20, Xumai25 displayed a comparatively lesser sensitivity to NH stress, as evident by a less pronounced reduction in dry plant biomass and a milder adverse impact on root morphology. Despite similarities in NH efflux and the expression levels of and between the two cultivars, Xumai25 exhibited higher NH influx, while maintaining a lower free NH concentration in the roots. Furthermore, Xumai25 showed a more pronounced increase in the levels of free amino acids, including asparagine, glutamine, and aspartate, suggesting a superior NH assimilation capacity under NH stress compared to Yangmai20. Additionally, the enhanced transcriptional regulation of vacuolar glucose transporter and glucose metabolism under NH stress in Xumai25 contributed to an enhanced carbon skeleton supply, particularly of 2-oxoglutarate and pyruvate. Taken together, our results demonstrate that the NH tolerance of Xumai25 is intricately linked to enhanced glucose metabolism and optimized glucose transport, which contributes to the robust NH assimilation capacity.
PubMed: 38318495
DOI: 10.3389/fpls.2024.1339105 -
Frontiers in Plant Science 2023Protein hydrolysates have gained interest as plant biostimulants due to their positive effects on plant performances. They are mainly composed of amino acids, but there...
Protein hydrolysates have gained interest as plant biostimulants due to their positive effects on plant performances. They are mainly composed of amino acids, but there is no evidence of the role of individual of amino acids as biostimulants. In this study we carried out experiments to monitor the development of Arabidopsis seedlings on amino acid containing media in order to analyze the biostimulant properties of the twenty individual proteinogenic amino acids. We demonstrated that proteinogenic amino acids are not good nitrogen sources as compared to nitrate for plant growth. Biostimulant analyses were based on leaf area measurements as a proxy of plant growth. We developed the Amino Acid Use Efficiency index to quantify the biostimulating effect of individual amino acids in the presence of nitrate. This index allowed us to classify amino acids into three groups, characterized by their inhibiting, neutral, and beneficial effects regarding leaf area. Glutamine and asparagine demonstrated the most significant effects in promoting leaf area in the presence of nitrate supply. The stimulating effect was confirmed by using the L and D enantiomeric forms. Both L-glutamine and L-asparagine stimulated leaf area at low concentrations, emphasizing their biostimulating properties. Our plant growth design and AAUE index pave the way for the identification of other bioactive molecules in protein hydrolysates and for the comparison of biostimulant performances.
PubMed: 38317837
DOI: 10.3389/fpls.2023.1281495 -
Amino Acids Feb 2024This study investigated the effect of high-intensity interval exercise on total and individual amino acid concentrations in red blood cells (RBCs) and plasma. Seven...
This study investigated the effect of high-intensity interval exercise on total and individual amino acid concentrations in red blood cells (RBCs) and plasma. Seven males (31 ± 13 yr) provided venous blood samples at rest, immediately and 15 min and 30 min following an 8-min high-intensity exercise bout. The exercise bout was 16 × 15 s cycle efforts at 0.4N/kg of body mass and 90 rpm, interspersed with 15 s passive recovery. Total and individual amino acid concentrations of RBC and plasma and blood cell parameters were analysed. No significant differences for total amino acid concentrations between RBC and plasma were found. Individual amino acid analyses showed significant interaction effects for alanine and α-aminoadipic acid (P < 0.05), with plasma alanine significantly increased from baseline across the recovery period (P < 0.001). Blood fraction (group) effects showed greater concentrations of glycine, serine, asparagine, aspartic acid, glutamic acid, α-aminoadipic acid and ornithine in RBC, while greater concentrations of alanine, α-aminobutyric acid, valine, leucine, isoleucine, threonine, proline, phenylalanine, glutamine, tryptophan and cystine were found in plasma (P < 0.05). Comparable levels of histidine, lysine and tyrosine were observed between blood fractions. Significant differences in the variation of total amino acids in RBC were reported with higher variance at rest compared to following exercise (P = 0.01). Haemoglobin, pack cell volume and white blood cell count significantly increased immediately following exercise (P < 0.05) but returned to baseline after 15 min recovery. These results support the notion of individualised amino acid transportation roles for RBC and plasma during exercise.
Topics: Male; Humans; Amino Acids; Erythrocytes; Plasma; Alanine; Glutamic Acid
PubMed: 38300362
DOI: 10.1007/s00726-023-03378-y -
The Journal of Clinical Investigation Feb 2024Aberrant alternative splicing (AS) events have been identified in a variety of cancers. Although somatic mutations of splicing factors and dysregulation of RNA-binding...
Aberrant alternative splicing (AS) events have been identified in a variety of cancers. Although somatic mutations of splicing factors and dysregulation of RNA-binding proteins (RBPs) have been linked to AS and tumor malignancy, it remains unclear how upstream mechanisms contribute to cancer development via alternative gene splicing. In this issue of the JCI, Wenrui Zhang and colleagues identified the role of asparagine endopeptidase (AEP), an intracellular cysteine endopeptidase, in promoting solid tumor-associated RNA splicing. The authors demonstrated that tumor environmental factors such as oxygen and nutrient deprivation induce the activity of AEP in a HIF1A-dependent manner. The activated AEP, in turn, cleaves an RNA helicase DDX3X to promote its nuclear retention. The authors further showed that this DDX3X nuclear fraction engages with splicing machinery to induce AS events in several cancer cells. These findings suggest that targeting an AEP-dependent aberrant RNA splicing cascade may facilitate therapeutics for solid tumors.
Topics: Humans; Neoplasms; RNA Splicing; Alternative Splicing; DEAD-box RNA Helicases
PubMed: 38299588
DOI: 10.1172/JCI177609 -
Nature Communications Jan 2024Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are...
Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.
Topics: Animals; Humans; Plasmodium falciparum; Asparagine; Aspartate-tRNA Ligase; RNA, Transfer, Amino Acyl; Antimalarials; Mammals
PubMed: 38297033
DOI: 10.1038/s41467-024-45224-z