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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 -
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 -
Cell Death & Disease Jul 2024Metastasis is the major culprit of treatment failure in nasopharyngeal carcinoma (NPC). Aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2), a core circadian...
Metastasis is the major culprit of treatment failure in nasopharyngeal carcinoma (NPC). Aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2), a core circadian gene, plays a crucial role in the development of various tumors. Nevertheless, the biological role and mechanism of ARNTL2 are not fully elucidated in NPC. In this study, ARNTL2 expression was significantly upregulated in NPC tissues and cells. Overexpression of ARNTL2 facilitated NPC cell migration and invasion abilities, while inhibition of ARNTL2 in similarly treated cells blunted migration and invasion abilities in vitro. Consistently, in vivo xenograft tumor models revealed that ARNTL2 silencing reduced nude mice inguinal lymph node and lung metastases, as well as tumor growth. Mechanistically, ARNTL2 negatively regulated the transcription expression of AMOTL2 by directly binding to the AMOTL2 promoter, thus reducing the recruitment and stabilization of AMOTL2 to LATS1/2 kinases, which strengthened YAP nuclear translocation by suppressing LATS-dependent YAP phosphorylation. Inhibition of AMOTL2 counteracted the effects of ARNTL2 knockdown on NPC cell migration and invasion abilities. These findings suggest that ARNTL2 may be a promising therapeutic target to combat NPC metastasis and further supports the crucial roles of circadian genes in cancer development.
Topics: Humans; Animals; Nasopharyngeal Carcinoma; Neoplasm Invasiveness; Cell Line, Tumor; Adaptor Proteins, Signal Transducing; Mice, Nude; YAP-Signaling Proteins; Angiomotins; Cell Movement; Mice; Transcription Factors; ARNTL Transcription Factors; Nasopharyngeal Neoplasms; Basic Helix-Loop-Helix Transcription Factors; Signal Transduction; Gene Expression Regulation, Neoplastic; Mice, Inbred BALB C; Protein Serine-Threonine Kinases; Male; Neoplasm Metastasis; Female; Tumor Suppressor Proteins
PubMed: 38956029
DOI: 10.1038/s41419-024-06860-x -
Molecular Diversity Jul 2024Alpha-fetoprotein (AFP) is a glycoprotein primarily expressed during embryogenesis, with declining levels postnatally. Elevated AFP levels correlate with pathological...
Alpha-fetoprotein (AFP) is a glycoprotein primarily expressed during embryogenesis, with declining levels postnatally. Elevated AFP levels correlate with pathological conditions such as liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Recent investigations underscore AFP's intracellular role in HCC progression, wherein it forms complexes with proteins like Phosphatase and tensin homolog (PTEN), Caspase 3 (CASP3), and Retinoic acid receptors and Retinoid X receptors (RAR/RXR). RAR and RXR regulate gene expression linked to cell death and tumorigenesis in normal physiology. AFP impedes RAR/RXR dimerization, nuclear translocation, and function, promoting gene expression favoring cancer progression in HCC that provoked us to target AFP as a drug candidate. Despite extensive studies, inhibitors targeting AFP to disrupt complex formation and activities remain scarce. In this study, employing protein-protein docking, amino acid residues involved in AFP-RARβ interaction were identified, guiding the definition of AFP's active site for potential inhibitor screening. Currently, kinase inhibitors play a significant role in cancer treatment and, the present study explores the potential of repurposing FDA-approved protein kinase inhibitors to target AFP. Molecular docking with kinase inhibitors revealed Lapatinib as a candidate drug of the AFP-RARβ complex. Molecular dynamics simulations and binding energy calculations, employing Mechanic/Poisson-Boltzmann Surface Area (MM-PBSA), confirmed Lapatinib's stability with AFP. The study suggests Lapatinib's potential in disrupting the AFP-RARβ complex, providing a promising avenue for treating molecularly stratified AFP-positive HCC or its early stages.
PubMed: 38955977
DOI: 10.1007/s11030-024-10915-8 -
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 -
Biochimica Et Biophysica Acta.... Jun 2024Many bacterial processes are powered by the sodium motive force (smf) and in case of pathogens, the smf contributes to virulence. Vibrio cholerae, the causative agent of...
Many bacterial processes are powered by the sodium motive force (smf) and in case of pathogens, the smf contributes to virulence. Vibrio cholerae, the causative agent of Cholera disease, possesses a Na-translocating NADH:quinone oxidoreductase (NQR), a six-subunit membrane protein assembly. The 3D structure of NQR revealed the arrangement of the six subunits NqrABCDEF, the position of all redox cofactors (four flavins, two [2Fe-2S] centers) and the binding sites for the substrates NADH (in NqrF) and ubiquinone (in NqrB). Upon oxidation of NADH, electrons are shuttled twice across the membrane, starting with cytoplasmic FAD and electron transfer to the [2Fe2S] cluster and from there to an intra-membranous [2Fe-2S] cluster, periplasmic FMN, FMN and from there to riboflavin. This riboflavin is located at the cytoplasmic entry site of the sodium channel in NqrB, and it donates an electron to ubiquinone-8 positioned at the cytoplasmic aspect of NqrB. Targeting the substrate binding sites of NQR is a promising strategy to identify new inhibitors against many bacterial pathogens. Detailed structural information on the binding mode of natural inhibitors and small molecules in the active sites of NQR is now available, paving the way for the development of new antibiotics. The NQR shows different conformations as revealed in recent cryo-EM and crystallographic studies combined with spectroscopic analyses. These conformations represent distinct steps in the catalytic cycle. Considering the structural and functional data available, we propose a mechanism of Na-NQR based on conformational coupling of electron transfer and Na translocation reaction steps.
PubMed: 38955304
DOI: 10.1016/j.bbabio.2024.149485 -
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