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PLoS Neglected Tropical Diseases Feb 2021In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal,...
In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.
Topics: Life Cycle Stages; Microbodies; Nucleotides; Sugars; Trypanosoma brucei brucei
PubMed: 33592041
DOI: 10.1371/journal.pntd.0009132 -
Journal of Fungi (Basel, Switzerland) Jan 2021Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in...
Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in under diverse energy conditions. Supercomplexes were obtained by solubilization of mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I:IV), 1400 kDa (I:III), 1600 kDa (I:III:IV), and 1800 kDa (I:III:IV). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.
PubMed: 33440829
DOI: 10.3390/jof7010042 -
The Journal of Biological Chemistry 2021Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. αS has been described to exist in both...
Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200-1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.
Topics: Amino Acid Sequence; Animals; Cell Membrane; Humans; Lentivirus; Neurons; Parkinson Disease; Primary Cell Culture; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Rats; Temperature; alpha-Synuclein; beta-Synuclein
PubMed: 33428933
DOI: 10.1016/j.jbc.2021.100271 -
Journal of Immunological Methods Feb 2021Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the...
Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 to 75% to 0-25%, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.
Topics: Animals; Antigens; Cytokines; Detergents; Digitonin; Lymph Nodes; Mice; Mice, Inbred C57BL; Vaccination
PubMed: 33333059
DOI: 10.1016/j.jim.2020.112943 -
Virologica Sinica Jun 2021Zika virus (ZIKV) is associated with severe birth defects and Guillain-Barré syndrome and no approved vaccines or specific therapies to combat ZIKV infection are...
Zika virus (ZIKV) is associated with severe birth defects and Guillain-Barré syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine (HHT), bruceine D (BD), dihydroartemisinin (DHA) and digitonin (DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549. The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.
Topics: Animals; Antiviral Agents; Chlorocebus aethiops; High-Throughput Screening Assays; Humans; Vero Cells; Virus Replication; Zika Virus; Zika Virus Infection
PubMed: 33231855
DOI: 10.1007/s12250-020-00316-0 -
Journal of Pharmaceutical Sciences Jan 2021The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing...
Differential Detergent Fractionation of Membrane Protein From Small Samples of Hepatocytes and Liver Tissue for Quantitative Proteomic Analysis of Drug Metabolizing Enzymes and Transporters.
The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing enzymes (DMEs) and transporters. This study evaluated differential detergent fractionation (DDF) of membrane protein from progressively smaller numbers of primary mouse hepatocytes (5 million down to 50,000 cells) and limited liver tissue (25-50 mg) in quantifying select DMEs and transporters by QTAP. Two non-ionic detergents, digitonin and Triton-X-100, were applied in sequence to permeabilize cells and extract membrane proteins. Comparison was made with a membrane protein extraction kit and with homogenization in hypotonic buffer and subsequent differential centrifugation (DC). DDF produced linear membrane protein yields with increasing hepatocyte numbers and better permeabilization evidenced by the higher ratio of cytosolic to membrane protein yields. DDF produced 5-times more membrane protein from liver tissue than DC. The concentration of DMEs and transporters remained consistent in the fractions prepared by DDF from progressively smaller numbers of hepatocytes, but declined in kit fractions. In liver tissue, the concentrations were comparatively higher in DDF versus kit and DC. In conclusion, sequential digitonin and Triton-X-100 fractionation of membrane protein from limited samples is efficient, reproducible and cost-effective for QTAP of DMEs and transporters.
Topics: Animals; Detergents; Hepatocytes; Liver; Membrane Proteins; Mice; Pharmaceutical Preparations; Proteomics
PubMed: 33148403
DOI: 10.1016/j.xphs.2020.10.037 -
Digitonin concentration is determinant for mitochondrial supercomplexes analysis by BlueNative page.Biochimica Et Biophysica Acta.... Jan 2021The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although...
The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although the migration of supercomplexes has been demonstrated being real, there are still several concerns about its ability to reveal genuine interactions between respiratory complexes. Moreover, the use of different solubilization conditions generates conflicting interpretations. Here, we thoroughly compare the impact of different digitonin concentrations on the liquid dispersions' physical properties and correlate with the respiratory complexes' migration pattern and supercomplexes. Our results demonstrate that digitonin concentration generates liquid dispersions with specific size and variability critical to distinguish between a real association of complexes from being trapped in the same micelle.
Topics: Animals; Digitonin; Electron Transport Complex I; Mice; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial Proteins; Native Polyacrylamide Gel Electrophoresis
PubMed: 33129827
DOI: 10.1016/j.bbabio.2020.148332 -
International Journal of Molecular... Oct 2020Conformational conversion of the cellular prion protein, PrP, into the abnormally folded isoform, PrP, is a key pathogenic event in prion diseases. However, the exact...
Conformational conversion of the cellular prion protein, PrP, into the abnormally folded isoform, PrP, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91-106 were generated in the absence of endogenous PrP, designated Tg(PrP∆91-106)/ mice and intracerebrally inoculated with various prions. Tg(PrP∆91-106)/ mice were resistant to RML, 22L and FK-1 prions, neither producing PrP∆91-106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrP∆91-106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrP∆91-104 after incubation with BSE-PrP-prions but not with RML- and 22L-PrP-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91-104 into PrP∆91-104 even after incubation with RML- and 22L-PrP-prions. These results suggest that residues 91-106 or 91-104 of PrP are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrP into PrP.
Topics: Animals; Baculoviridae; Base Sequence; Brain; Cattle; Cloning, Molecular; Disease Susceptibility; Encephalopathy, Bovine Spongiform; Gene Expression; Injections, Intraventricular; Mice; Mice, Transgenic; PrPC Proteins; PrPSc Proteins; Proteostasis Deficiencies; Recombinant Proteins; Scrapie; Sequence Deletion; Species Specificity
PubMed: 33019549
DOI: 10.3390/ijms21197260 -
Scientific Reports Aug 2020Cancer cells release small extracellular vesicles, exosomes, that have been shown to contribute to various aspects of cancer development and progression. Differential...
Cancer cells release small extracellular vesicles, exosomes, that have been shown to contribute to various aspects of cancer development and progression. Differential analysis of exosomal proteomes from cancerous and non-tumorigenic breast cell lines can provide valuable information related to breast cancer progression and metastasis. Moreover, such a comparison can be explored to find potentially new protein biomarkers for early disease detection. In this study, exosomal proteomes of MDA-MB-231, a metastatic breast cancer cell line, and MCF-10A, a non-cancerous epithelial breast cell line, were identified by nano-liquid chromatography coupled to tandem mass spectrometry. We also tested three exosomes isolation methods (ExoQuick, Ultracentrifugation (UC), and Ultrafiltration-Ultracentrifugation) and detergents (n-dodecyl β-D-maltoside, Triton X-100, and Digitonin) for solubilization of exosomal proteins and enhanced detection by mass spectrometry. A total of 1,107 exosomal proteins were identified in both cell lines, 726 of which were unique to the MDA-MB-231 breast cancer cell line. Among them, 87 proteins were predicted to be relevant to breast cancer and 16 proteins to cancer metastasis. Three exosomal membrane/surface proteins, glucose transporter 1 (GLUT-1), glypican 1 (GPC-1), and disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), were identified as potential breast cancer biomarkers and validated with Western blotting and high-resolution flow cytometry. We demonstrated that exosomes are a rich source of breast cancer-related proteins and surface biomarkers that may be used for disease diagnosis and prognosis.
Topics: Biomarkers, Tumor; Breast Neoplasms; Exosomes; Female; Humans; Mass Spectrometry; Proteome; Proteomics; Tumor Cells, Cultured; Ultracentrifugation
PubMed: 32782317
DOI: 10.1038/s41598-020-70393-4 -
Scientific Reports Aug 2020Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control....
Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control. VCP binds various cofactors, which determine pathway selectivity and substrate processing. Here, we used co-immunoprecipitation and mass spectrometry studies coupled to in silico analyses to identify the Leishmania infantum VCP (LiVCP) interactome and to predict molecular interactions between LiVCP and its major cofactors. Our data support a largely conserved VCP protein network in Leishmania including known but also novel interaction partners. Network proteomics analysis confirmed LiVCP-cofactor interactions and provided novel insights into cofactor-specific partners and the diversity of LiVCP complexes, including the well-characterized VCP-UFD1-NPL4 complex. Gene Ontology analysis coupled with digitonin fractionation and immunofluorescence studies support cofactor subcellular compartmentalization with either cytoplasmic or organellar or vacuolar localization. Furthermore, in silico models based on 3D homology modeling and protein-protein docking indicated that the conserved binding modules of LiVCP cofactors, except for NPL4, interact with specific binding sites in the hexameric LiVCP protein, similarly to their eukaryotic orthologs. Altogether, these results allowed us to build the first VCP protein interaction network in parasitic protozoa through the identification of known and novel interacting partners potentially associated with distinct VCP complexes.
Topics: Computer Simulation; Leishmania infantum; Multiprotein Complexes; Protein Structure, Quaternary; Protozoan Proteins; Valosin Containing Protein
PubMed: 32753747
DOI: 10.1038/s41598-020-70010-4