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Nature Communications Jul 2024Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot...
Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5-3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.
Topics: Humans; Sodium-Coupled Vitamin C Transporters; Ascorbic Acid; Cryoelectron Microscopy; Biological Transport; Sodium; Models, Molecular; Protein Multimerization; Protein Binding; HEK293 Cells; Protein Conformation
PubMed: 38956111
DOI: 10.1038/s41467-024-49899-2 -
Nature Communications Jul 2024Iron plays a fundamental role in multiple brain disorders. However, the genetic underpinnings of brain iron and its implications for these disorders are still lacking....
Iron plays a fundamental role in multiple brain disorders. However, the genetic underpinnings of brain iron and its implications for these disorders are still lacking. Here, we conduct an exome-wide association analysis of brain iron, measured by quantitative susceptibility mapping technique, across 26 brain regions among 26,789 UK Biobank participants. We find 36 genes linked to brain iron, with 29 not being previously reported, and 16 of them can be replicated in an independent dataset with 3,039 subjects. Many of these genes are involved in iron transport and homeostasis, such as FTH1 and MLX. Several genes, while not previously connected to brain iron, are associated with iron-related brain disorders like Parkinson's (STAB1, KCNA10), Alzheimer's (SHANK1), and depression (GFAP). Mendelian randomization analysis reveals six causal relationships from regional brain iron to brain disorders, such as from the hippocampus to depression and from the substantia nigra to Parkinson's. These insights advance our understanding of the genetic architecture of brain iron and offer potential therapeutic targets for brain disorders.
Topics: Humans; Iron; Brain; Exome Sequencing; Male; Female; Mendelian Randomization Analysis; Genome-Wide Association Study; Parkinson Disease; Middle Aged; Genetic Predisposition to Disease; Aged; Nerve Tissue Proteins; Adult; Alzheimer Disease
PubMed: 38956042
DOI: 10.1038/s41467-024-49702-2 -
Journal of Food Science Jul 2024The wide biological activity of the Moringa oleifera represents a potential opportunity for developing selective cancer treatment drugs. The bioactive phytochemicals in...
The wide biological activity of the Moringa oleifera represents a potential opportunity for developing selective cancer treatment drugs. The bioactive phytochemicals in Moringa seed extract (MSE) indicated large numbers of phytochemicals (21 compounds) with dominant abundance for cycloisolongifolene, 8,9-dehydro-9-vinyl, and chamazulene accounting for 12.7% and 12.19% of the total detected compounds. The MSE showed a potent anticancer effect toward Caco-2, MDA, and HepG-2 cells with half-maximal inhibitory concentration (IC) values of 9.15 ± 1.18, 4.85 ± 0.11, and 7.36 ± 0.22 µg/mL, respectively, with higher safety (≥31-folds) toward normal human cells (IC of 150.7 ± 11.11 µg/mL). It appears that MSE stimulates selective-dose-dependent cell shrinkage, and nuclear condensation in the tumor cells, which finally induces the apoptosis pathway to increase its anticancer action. Additionally, MSE showed a potent capability to stimulate cell cycle arrest in both main checkpoint phases (G0/G1 and G2/M) of cell population growth. The apoptotic death stimulation was confirmed through upregulation of tumor protein p53 (p53) and cyclin-dependent kinase inhibitor p21 (p21) expression by more than three- to sixfold and downregulation of B-cell lymphoma 2 expression (threefold) in MSE-treated cells compared to 5-fluorouracil (5-FU)-treated tumor cells. Furthermore, the MSE revealed strong anti-inflammatory activity with significant antioxidant activity by lowering nitric oxide levels and enhancing the superoxide dismutase activity. On the other hand, the MSE revealed broad-spectrum antibacterial activity in a dose-dependent manner against Staphylococcus aureus minimum inhibitory concentration (MIC of 1.25 mg/mL), followed by Salmonella typhimurium (MIC of 1.23 mg/mL), whereas Escherichia coli was the least sensitive to MSE activity (MIC of 22.5 mg/mL) with significant antibiofilm activity against sensitive pathogens.
PubMed: 38955793
DOI: 10.1111/1750-3841.17223 -
Journal of Medical Genetics Jul 2024Transport protein particle (TRAPP) is a multiprotein complex that functions in localising proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in...
BACKGROUND
Transport protein particle (TRAPP) is a multiprotein complex that functions in localising proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in diseases affecting muscle, brain, eye and to some extent liver. We present three patients who are compound heterozygotes for a missense variant and a structural variant in the gene. structural variants have not yet been described in association with a disease. In order to reveal the estimated genesis of identified structural variants, we performed sequencing of individual breakpoint junctions and analysed the extent of homology and the presence of repetitive elements in and around the breakpoints.
METHODS
Biochemical methods including isoelectric focusing on serum transferrin and apolipoprotein C-III, as well as mitochondrial respiratory chain complex activity measurements, were used. Muscle biopsy samples underwent histochemical analysis. Next-generation sequencing was employed for identifying sequence variants associated with neuromuscular disorders, and Sanger sequencing was used to confirm findings.
RESULTS
We suppose that non-homologous end joining is a possible mechanism of deletion origin in two patients and non-allelic homologous recombination in one patient. Analyses of mitochondrial function performed in patients' skeletal muscles revealed an imbalance of mitochondrial metabolism, which worsens with age and disease progression.
CONCLUSION
Our results contribute to further knowledge in the field of neuromuscular diseases and mutational mechanisms. This knowledge is important for understanding the molecular nature of human diseases and allows us to improve strategies for identifying disease-causing mutations.
PubMed: 38955476
DOI: 10.1136/jmg-2024-110016 -
Life Science Alliance Sep 2024In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been...
In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.
Topics: Humans; Cytosol; Mitochondria; RNA, Double-Stranded; Prohibitins; RNA, Mitochondrial; Cell Line, Tumor; Repressor Proteins; RNA Transport; Exoribonucleases; Voltage-Dependent Anion Channel 1; Carcinoma, Non-Small-Cell Lung; Mitochondrial Proteins
PubMed: 38955468
DOI: 10.26508/lsa.202302396 -
Microbial Physiology Jul 2024The global poultry industry produces millions of tons of waste feathers every year, which can be degraded to make feed, fertilizer, and daily chemicals. However, feather...
INTRODUCTION
The global poultry industry produces millions of tons of waste feathers every year, which can be degraded to make feed, fertilizer, and daily chemicals. However, feather degradation is a complex process that is not yet fully understood. This results in low degradation efficiency and difficulty in industrial applications. Omics-driven system biology research offers an effective solution to quickly and comprehensively understand the molecules and mechanisms involved in a metabolic pathway.
METHODS
In the early stage of this process, feathers are hydrolyzed into water-soluble keratin monomers. In this study, we used high-throughput RNA-seq technology to analyze the genes involved in the internalization and degradation of keratin monomers in S. maltophilia DHHJ strain cells. Moreover, we used Co-IP with LC-MS/MS technology to search for proteins that interact with recombinant keratin monomers.
RESULTS
We discovered TonB transports and molecular chaperones associating with the keratin monomer, which may play a crucial role in the transmembrane transport of keratin. Meanwhile, multiple proteases belonging to distinct families were identified as binding partners of keratin monomers, among which ATPases associated with diverse cellular activities (AAA+) family proteases are overrepresented. Four genes, including JJL50_15620, JJL50_17955 (TonB-dependent receptors), JJL50_03260 (ABC transporter ATP-binding protein), and JJL50_20035 (ABC transporter substrate-binding protein), were selected as representatives for determining their expressions under different culture conditions using qRT-PCR and they were found to be upregulated in response to keratin degradation consistent with the data from RNA-seq and Co-IP.
CONCLUSION
This study highlights the complexity of keratin biodegradation in S. maltophilia DHHJ, in which multiple pathways are involved such as protein folding, protein transport, and several protease systems. Our findings provide new insights into the mechanism of feather degradation.
PubMed: 38955164
DOI: 10.1159/000540072 -
Theriogenology Jun 2024HT-2 toxin is a type of mycotoxin which is shown to affect gastric and intestinal lesions, hematopoietic and immunosuppressive effects, anorexia, lethargy, nausea....
HT-2 toxin is a type of mycotoxin which is shown to affect gastric and intestinal lesions, hematopoietic and immunosuppressive effects, anorexia, lethargy, nausea. Recently, emerging evidences indicate that HT-2 also disturbs the reproductive system. In this study, we investigated the impact of HT-2 toxin exposure on the organelles of porcine oocytes. Our results found that the abnormal distribution of endoplasmic reticulum increased after HT-2 treatment, with the perturbation of ribosome protein RPS3 and GRP78 expression; Golgi apparatus showed diffused localization pattern and GM130 localization was also impaired, thereby affecting the Rab10-based vesicular transport; Due to the impairment of ribosomes, ER, and Golgi apparatus, the protein supply to lysosomes was hindered, resulting in lysosomal damage, which further disrupted the LC3-based autophagy. Moreover, the results indicated that the function and distribution of mitochondria were also affected by HT-2 toxin, showing with fragments of mitochondria, decreased TMRE and ATP level. Taken together, our study suggested that HT-2 toxin exposure induces damage to the organelles for endomembrane system, which further inhibited the meiotic maturation of porcine oocytes.
PubMed: 38954997
DOI: 10.1016/j.theriogenology.2024.06.019 -
Nature Protocols Jul 2024Here, we present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle assay to... (Review)
Review
Here, we present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle assay to examine their association and fusion with plasma membrane mimic vesicles. This is a Protocol Extension, building on our previous protocol, which used a purely synthetic system comprised of reconstituted proteins in liposomes. We also describe the generation of a peptide based on the vesicular glutamate transporter, which is essential in the isolation process of glutamatergic synaptic vesicles. This method uses easily accessible reagents to generate fusion-competent glutamatergic synaptic vesicles through immunoisolation. The generation of the vGlut peptide can be accomplished in 6 d, while the isolation of the synaptic vesicles by using the peptide can be accomplished in 2 d, with an additional day to fluorescently label the synaptic vesicles for use in a single-vesicle hybrid fusion assay. The single-vesicle fusion assay can be accomplished in 1 d and can unambiguously delineate synaptic vesicle association, dissociation, Ca-independent and Ca-dependent fusion modalities. This assay grants control of the synaptic vesicle environment while retaining the complexity of the synaptic vesicles themselves. This protocol can be adapted to studies of other types of synaptic vesicles or, more generally, different secretory or transport vesicles. The workflow described here requires expertise in biochemistry techniques, in particular, protein purification and fluorescence imaging. We assume that the laboratory has protein-purification equipment, including chromatography systems.
PubMed: 38956381
DOI: 10.1038/s41596-024-01014-x -
International Journal of Surgery... Jul 2024Tibial cortex transverse transport (TTT) surgery has become an ideal treatment for patients with type 2 severe diabetic foot ulcerations (DFUs) while conventional...
BACKGROUND
Tibial cortex transverse transport (TTT) surgery has become an ideal treatment for patients with type 2 severe diabetic foot ulcerations (DFUs) while conventional treatments are ineffective. Based on our clinical practice experience, the protective immune response from TTT surgery may play a role against infections to promote wound healing in patients with DFUs. Therefore, this research aimed to systematically study the specific clinical efficacy and the mechanism of TTT surgery.
MATERIALS AND METHODS
Between June 2022 and September 2023, 68 patients with type 2 severe DFUs were enrolled and therapized by TTT surgery in this cross-sectional and experimental study. Major clinical outcomes including limb salvage rate and antibiotics usage rate were investigated. Ten clinical characteristics and laboratory features of glucose metabolism and kidney function were statistically analyzed. Blood samples from 6 key time points of TTT surgery were collected for label-free proteomics and clinical immune biomarker analysis. Besides, tissue samples from 3 key time points were for spatially resolved metabolomics and transcriptomics analysis, as well as applied to validate the key TTT-regulated molecules by RT-qPCR.
RESULTS
Notably, 64.7% of patients did not use antibiotics during the entire TTT surgery. TTT surgery can achieve a high limb salvage rate of 92.6% in patients with unilateral or bilateral DFUs. Pathway analysis of a total of 252 differentially expressed proteins (DEPs) from the proteomic revealed that the immune response induced by TTT surgery at different stages was first comprehensively verified through multi-omics combined with immune biomarker analysis. The function of upward transport was activating the systemic immune response, and wound healing occurs with downward transport. The spatial metabolic characteristics of skin tissue from patients with DFUs indicated downregulated levels of stearoylcarnitine and the glycerophospholipid metabolism pathway in skin tissue from patients with severe DFUs. Finally, the expressions of PRNP (prion protein) to activate the immune response, PLCB3 (PLCB3, phospholipase C beta 3) and VE-cadherin to play roles in neovascularization, and PPDPF (pancreatic progenitor cell differentiation and proliferation factor), LAMC2 (laminin subunit gamma 2) and SPRR2G (small proline rich protein 2G) to facilitate the developmental process mainly keratinocyte differentiation were statistically significant in skin tissues through transcriptomic and RT-qPCR analysis.
CONCLUSION
Tibial cortex transverse transport (TTT) surgery demonstrates favorable outcomes for patients with severe type 2 DFUs by activating a systemic immune response, contributing to anti-infection, ulcer recurrence, and the limb salvage rate for unilateral or bilateral DFUs. The specific clinical immune responses, candidate proteins, genes, and metabolic characteristics provide directions for in-depth mechanistic research on TTT surgery. Further research and public awareness are needed to optimize TTT surgery in patients with severe type 2 DFUs.
PubMed: 38954658
DOI: 10.1097/JS9.0000000000001897 -
Science Signaling Jul 2024Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of...
Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gα-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-G, a mini-G protein derived from Gα, blocked β-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gα and Gα also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gα:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.
Topics: Humans; Signal Transduction; Glucagon-Like Peptide-1 Receptor; Receptors, G-Protein-Coupled; HEK293 Cells; Protein Engineering; Endocytosis; Protein Transport; Animals
PubMed: 38954638
DOI: 10.1126/scisignal.abq7038