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Scientific Reports Jan 2020CorA proteins belong to 2-TM-GxN family of membrane proteins, and play a major role in Mg transport in prokaryotes and eukaryotic mitochondria. The selection of...
CorA proteins belong to 2-TM-GxN family of membrane proteins, and play a major role in Mg transport in prokaryotes and eukaryotic mitochondria. The selection of substrate is believed to occur via the signature motif GxN, however there is no consensus how strict this selection within the family. To answer this question, we employed fluorescence-based transport assays on three different family members, namely CorA from bacterium Thermotoga maritima, CorA from the archeon Methanocaldococcus jannaschii and ZntB from bacterium Escherichia coli, reconstituted into proteoliposomes. Our results show that all three proteins readily transport Mg, Co, Ni and Zn, but not Al. Despite the similarity in cation specificity, ZntB differs from the CorA proteins, as in the former transport is stimulated by a proton gradient, but in the latter by the membrane potential, confirming the hypothesis that CorA and ZntB proteins diverged to different transport mechanisms within the same protein scaffold.
Topics: Bacterial Proteins; Biological Transport; Cation Transport Proteins; Cations; Copper; Escherichia coli; Magnesium; Methanocaldococcus; Nickel; Proteolipids; Thermotoga maritima; Zinc
PubMed: 31965019
DOI: 10.1038/s41598-020-57869-z -
Nature Sep 2020Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked...
Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). Most PLP1 mutations, including point mutations and supernumerary copy variants, lead to severe and fatal disease. Patients who lack PLP1 expression, and Plp1-null mice, can display comparatively mild phenotypes, suggesting that PLP1 suppression might provide a general therapeutic strategy for PMD. Here we show, using CRISPR-Cas9 to suppress Plp1 expression in the jimpy (Plp1) point-mutation mouse model of severe PMD, increased myelination and restored nerve conduction velocity, motor function and lifespan of the mice to wild-type levels. To evaluate the translational potential of this strategy, we identified antisense oligonucleotides that stably decrease the levels of Plp1 mRNA and PLP protein throughout the neuraxis in vivo. Administration of a single dose of Plp1-targeting antisense oligonucleotides in postnatal jimpy mice fully restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function and extended lifespan up to an eight-month end point. These results suggest that PLP1 suppression could be developed as a treatment for PMD in humans. More broadly, we demonstrate that oligonucleotide-based therapeutic agents can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, establishing a new pharmaceutical modality for myelin disorders.
Topics: Animals; CRISPR-Cas Systems; Disease Models, Animal; Female; Gene Editing; Hypoxia; Male; Mice; Mice, Mutant Strains; Motor Activity; Myelin Proteolipid Protein; Myelin Sheath; Oligodendroglia; Oligonucleotides, Antisense; Pelizaeus-Merzbacher Disease; Point Mutation; Respiratory Function Tests; Survival Analysis
PubMed: 32610343
DOI: 10.1038/s41586-020-2494-3 -
Theranostics 2021Estrogen-dependent cancers (e.g., breast, endometrial, and ovarian cancers) are among the leading causes of morbidity and mortality in women worldwide. Recently,...
Estrogen-dependent cancers (e.g., breast, endometrial, and ovarian cancers) are among the leading causes of morbidity and mortality in women worldwide. Recently, exosomes released by tumor-infiltrating CD8 T cells have been under the spotlight in the field of cancer immunotherapy. Our study aims at elucidating the underlying mechanisms of the crosstalk between estrogen signaling and CD8 T cells, and possible intervention values in uterine corpus endometrial cancer (UCEC). Micro RNA-seq was conducted to screen differentially expressed micro RNA in UCEC. Bioinformatic analysis was processed to predict the target of miR-765. RNA silencing or overexpressing and pharmacologic inhibitors were used to assess the functions of ERβ/miR-765/PLP2/Notch axis in UCEC cell proliferation and invasion and . imaging was performed to evaluate the metastasis of tumor in mice. Combined fluorescent hybridization for miR-765 and immunofluorescent labeling for CD8 was carried out to prove the co-localization between miR-765 and CD8 T cells. Exosomes derived from CD45ROCD8 T cells were isolated to detect the regulatory effects on UCEC. miR-765 is characterized as the most downregulated miRNA in UCEC, and there is a negative correlation between miR-765 and Proteolipid protein 2 (PLP2) in UCEC lesion. Estrogen significantly down-regulates miR-765 level, and facilitates the development of UCEC by estrogen receptor (ER) β. Mechanistically, this process is mediated through the miRNAs (e.g., miR-3584-5p, miR-7-5p, miR-150-5p, and miR-124-3p) cluster-controlled regulation of the PLP2, which further regulates Ki-67 and multiple epithelial-mesenchymal transition (EMT)-related molecules (e.g, E-cadherin and Vimentin) in a Notch signaling pathway-dependent manner. Interestingly, the selective ER degrader Fulvestrant alleviates estrogen-mediated miR-765/PLP2 expression regulation and UCEC development in ERβ-dependent and -independent manners. Additionally, CD45ROCD8 T cell-derived exosomes release more miR-765 than that from CD45ROCD8 T cells. In therapeutic studies, these exosomes limit estrogen-driven disease development via regulation of the miR-765/PLP2 axis. This observation reveals novel molecular mechanisms underlying estrogen signaling and CD8 T cell-released exosomes in UCEC development, and provides a potential therapeutic strategy for UCEC patients with aberrant ERβ/miR-765/PLP2/Notch signaling axis.
Topics: Adult; Animals; CD8-Positive T-Lymphocytes; Cadherins; Cell Proliferation; Cells, Cultured; Down-Regulation; Endometrial Neoplasms; Epithelial-Mesenchymal Transition; Estrogen Receptor beta; Estrogens; Exosomes; Female; Humans; Leukocyte Common Antigens; MARVEL Domain-Containing Proteins; Mice; Mice, Nude; MicroRNAs; Middle Aged; Proteolipids; RNA Interference; Receptors, Notch; Signal Transduction
PubMed: 33859750
DOI: 10.7150/thno.58337 -
ACS Applied Bio Materials Jan 2021Multiple sclerosis is complex and heterogeneous. Better tools are needed to be able to monitor this disease among individuals, but blood-based biomarkers are often too...
Multiple sclerosis is complex and heterogeneous. Better tools are needed to be able to monitor this disease among individuals, but blood-based biomarkers are often too rare to profile. In this work, we developed antigen-specific biomaterials to replicate the central nervous system niche where multiple sclerosis biomarkers are amplified. We incorporated mouse brain homogenate into a microporous gelatin methacrylate network. Homogenate-containing biomaterials differentially stimulated cells and led to the marked amplification of disease-relevant, antigen-specific B cells. These results demonstrate that biomaterials containing primary tissue homogenate retain antigen specificity and may be a useful tool for decoding human autoimmunity.
Topics: Animals; Antigens; Autoimmunity; B-Lymphocytes; B7-2 Antigen; Biocompatible Materials; Brain; Encephalomyelitis, Autoimmune, Experimental; Gelatin; Mice; Myelin Proteolipid Protein; Peptide Fragments; Spleen
PubMed: 35014289
DOI: 10.1021/acsabm.0c01048 -
Clinics (Sao Paulo, Brazil) 2021Leukoaraiosis is described as white matter lesions that are associated with cognitive dysfunction, neurodegenerative disorders, etc. Myelin depletion is a salient...
OBJECTIVES
Leukoaraiosis is described as white matter lesions that are associated with cognitive dysfunction, neurodegenerative disorders, etc. Myelin depletion is a salient pathological feature of, and the loss of oligodendrocytes is one of the most robust alterations evident in, white matter degeneration. Recent studies have revealed that claudin proteins are aberrantly expressed in leukoaraiosis and regulate oligodendrocyte activity. However, the roles of claudin-1 and claudin-3 in oligodendrocytes and leukoaraiosis are still not well-defined.
METHODS
Quantitative polymerase chain reaction was used to measure the expression of claudin-1 (CLDN1), claudin-3 (CLDN3), and myelinogenesis-related genes such as myelin basic protein (MBP), proteolipid protein (PLP), oligodendrocyte transcription factor 2 (OLIG2), and SRY-box transcription factor 10 (SOX10) in leukoaraiosis patients (n=122) and healthy controls (n=122). The expression of claudin-1 and claudin-3 was either ectopically silenced or augmented in Oli-neu oligodendrocytes, and colony formation, apoptosis, and migration assays were performed. Finally, the expression of myelin proteins was evaluated by western blotting.
RESULTS
Our results revealed that in addition to SOX10, the expression levels of claudin-1, claudin-3, and myelinogenesis-related proteins were prominently downregulated in leukoaraiosis patients, compared to those in healthy controls. Furthermore, the growth and migration of Oli-neu cells were downregulated upon silencing claudin-1 or claudin-3. However, the overexpression of claudin-1 or claudin-3 resulted in the reduction of the degree of apoptosis in Oli-neu cells. In addition, claudin-1 and claudin-3 promoted the expression of MBP, OLIG2, PLP, and SOX10 at the translational level.
CONCLUSION
Our data has demonstrated that the abnormal expression of claudin-1 and claudin-3 regulates the pathological progression of leukoaraiosis by governing the viability and myelination of oligodendrocytes. These findings provide novel insights into the regulatory mechanisms underlying the roles of claudin-1 and claudin-3 in leukoaraiosis.
Topics: Claudin-1; Claudin-3; Humans; Leukoaraiosis; Myelin Sheath; Oligodendroglia
PubMed: 34008771
DOI: 10.6061/clinics/2021/e2167 -
Cellular and Molecular Life Sciences :... Jan 2022Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic progressive pulmonary fibrosis and a poor prognosis. Genetic studies,...
Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic progressive pulmonary fibrosis and a poor prognosis. Genetic studies, including transcriptomic and proteomics, have provided new insight into revealing mechanisms of IPF. Herein we provided a novel strategy to identify biomarkers by integrative analysis of transcriptomic and proteomic profiles of IPF patients. We examined the landscape of IPF patients' gene expression in the transcription and translation phases and investigated the expression and functions of two new potential biomarkers. Differentially expressed (DE) mRNAs were mainly enriched in pathways associated with immune system activities and inflammatory responses, while DE proteins are related to extracellular matrix production and wound repair. The upregulated genes in both phases are associated with wound repair and cell differentiation, while the downregulated genes in both phases are associated with reduced immune activities and the damage of the alveolar tissues. On this basis, we identified thirteen potential marker genes. Among them, we validated the expression changes of butyrophilin-like 9 (BTNL9) and plasmolipin (PLLP) and investigated their functional pathways in the IPF mechanism. Both genes are downregulated in the tissues of IPF patients and Bleomycin-induced mice, and co-expression analysis indicates that they have a protective effect by inhibiting extracellular matrix production and promoting wound repair in alveolar epithelial cells.
Topics: Animals; Biomarkers; Bleomycin; Butyrophilins; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Extracellular Matrix; Female; Gene Expression Profiling; Humans; Idiopathic Pulmonary Fibrosis; Male; Mice; Mice, Inbred C57BL; Middle Aged; Myelin and Lymphocyte-Associated Proteolipid Proteins; Proteome; Proteomics; RNA-Seq; Transcriptome; Wound Healing
PubMed: 35015148
DOI: 10.1007/s00018-021-04094-0 -
Journal of Neurochemistry Jun 2024During myelination, large quantities of proteins are synthesized and transported from the endoplasmic reticulum (ER)-trans-Golgi network (TGN) to their appropriate... (Review)
Review
During myelination, large quantities of proteins are synthesized and transported from the endoplasmic reticulum (ER)-trans-Golgi network (TGN) to their appropriate locations within the intracellular region and/or plasma membrane. It is widely believed that oligodendrocytes uptake neuronal signals from neurons to regulate the endocytosis- and exocytosis-mediated intracellular trafficking of major myelin proteins such as myelin-associated glycoprotein (MAG) and proteolipid protein 1 (PLP1). The small GTPases of the adenosine diphosphate (ADP) ribosylation factor (Arf) family constitute a large group of signal transduction molecules that act as regulators for intracellular signaling, vesicle sorting, or membrane trafficking in cells. Studies on mice deficient in Schwann cell-specific Arfs-related genes have revealed abnormal myelination formation in peripheral nerves, indicating that Arfs-mediated signaling transduction is required for myelination in Schwann cells. However, the complex roles in these events remain poorly understood. This review aims to provide an update on signal transduction, focusing on Arf and its activator ArfGEF (guanine nucleotide exchange factor for Arf) in oligodendrocytes and Schwann cells. Future studies are expected to provide important information regarding the cellular and physiological processes underlying the myelination of oligodendrocytes and Schwann cells and their function in modulating neural activity.
PubMed: 38894552
DOI: 10.1111/jnc.16141 -
Microbiology Spectrum Sep 2021Candida albicans is a prevalent opportunistic human fungal pathogen for which treatment is limited to only four main classes of antifungal drugs, with the azole and...
Candida albicans is a prevalent opportunistic human fungal pathogen for which treatment is limited to only four main classes of antifungal drugs, with the azole and echinocandin classes being used most frequently. Drug tolerance, the ability of some cells to grow slowly in supra-MIC drug concentrations, decreases the number of available treatment options. Here, we investigated factors affecting tolerance and resistance to ketoconazole in C. albicans. We found both temperature and the composition of growth medium significantly affected tolerance with little effect on resistance. In deletion analysis of known efflux pump genes, was partially required for azole tolerance, while and were dispensable. Tolerance also required Hsp90 and calcineurin components; , which encodes a transcription factor downstream of calcineurin, was required only partially. Deletion of , which encodes a vacuolar ATPase subunit, and concanamycin A, a V-ATPase inhibitor, abolished tolerance, indicating the importance of vacuolar energy transactions in tolerance. Thus, tolerance to ketoconazole is regulated by multiple factors, including physiological and genetic mechanisms. Due to the ever-expanding range of invasive medical procedures and treatments, invasive fungal infections now pose a serious global threat to many people living in an immunocompromised status. Like humans, fungi are eukaryotic, which significantly limits the number of unique antifungal targets; the current arsenal of antifungal agents is limited to just three frontline drug classes. Additional treatment complexities result from the development of drug tolerance and resistance, which further narrows therapeutic options; however, the difference between tolerance and resistance remains largely unknown. This study demonstrates that tolerance and resistance are regulated by multiple genetic and physiological factors. It is prudent to note that some factors affect tolerance only, while other factors affect both tolerance and resistance. The complex underlying mechanisms of these drug responses are highlighted by the fact that there are both shared and distinct mechanisms that regulate tolerance and resistance.
Topics: ATP Binding Cassette Transporter, Subfamily B; Antifungal Agents; Candida albicans; Candidiasis; Drug Resistance, Fungal; Drug Tolerance; Fungal Proteins; HSP90 Heat-Shock Proteins; Humans; Ketoconazole; Membrane Transport Proteins; Microbial Sensitivity Tests; Nerve Tissue Proteins; Proteolipids; Proton-Translocating ATPases; Temperature
PubMed: 34160280
DOI: 10.1128/Spectrum.00321-21 -
Methods in Molecular Biology (Clifton,... 2021On the plasma membrane, Ras is organized into laterally segregated proteo-lipid complexes called nanoclusters. The extent of Ras nanoclustering correlates with its...
On the plasma membrane, Ras is organized into laterally segregated proteo-lipid complexes called nanoclusters. The extent of Ras nanoclustering correlates with its signaling output, positioning nanocluster as dynamic signaling gain modulators. Recent evidence suggests that stacked dimers of Ras and Raf are elemental units at least of one type of Ras nanocluster. However, it is still incompletely understood, in which physiological contexts nanoclustering is regulated and which constituents are parts of nanocluster. Nonetheless, disruption of nanoclustering faithfully diminishes Ras activity in cells, suggesting Ras nanocluster as potential drug targets.While there are several methods available to study Ras nanocluster , fluorescence or Förster resonance energy transfer (FRET ) between fluorescently labeled, nanoclustered Ras proteins is a relatively simple readout. FRET measurements using fluorescence lifetime imaging microscopy (FLIM ) have proven to be robust and sensitive to determine Ras nanoclustering changes. Loss of FRET that emerges due to nanoclustering reports on all processes upstream of Ras nanoclustering, i.e., also on proper trafficking or lipid modification of Ras. Here we report our standard FLIM-FRET protocol to measure nanoclustering-dependent FRET of Ras in mammalian cells. Importantly, nanoclustering-dependent FRET is one of the few methods that can detect differences between the Ras isoforms.
Topics: Cell Membrane; Fluorescence Resonance Energy Transfer; Humans; Image Processing, Computer-Assisted; Membrane Lipids; Microscopy, Fluorescence; Nanoparticles; Signal Transduction; ras Proteins
PubMed: 33977480
DOI: 10.1007/978-1-0716-1190-6_13 -
JCI Insight Apr 2024Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD cardiomyopathy is characterized by abnormal intracellular...
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD cardiomyopathy is characterized by abnormal intracellular Ca2+ homeostasis and mitochondrial dysfunction. We used dystrophin and utrophin double-knockout (mdx:utrn-/-) mice in a sarcolipin (SLN) heterozygous-knockout (sln+/-) background to examine the effect of SLN reduction on mitochondrial function in the dystrophic myocardium. Germline reduction of SLN expression in mdx:utrn-/- mice improved cardiac sarco/endoplasmic reticulum (SR) Ca2+ cycling, reduced cardiac fibrosis, and improved cardiac function. At the cellular level, reducing SLN expression prevented mitochondrial Ca2+ overload, reduced mitochondrial membrane potential loss, and improved mitochondrial function. Transmission electron microscopy of myocardial tissues and proteomic analysis of mitochondria-associated membranes showed that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiomyocytes. These findings indicate that SLN upregulation plays a substantial role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD cardiomyopathy.
Topics: Animals; Male; Mice; Calcium; Cardiomyopathies; Disease Models, Animal; Dystrophin; Mice, Inbred mdx; Mice, Knockout; Mitochondria, Heart; Muscle Proteins; Muscular Dystrophy, Duchenne; Myocardium; Myocytes, Cardiac; Proteolipids; Utrophin
PubMed: 38564291
DOI: 10.1172/jci.insight.170185