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Biophysical Journal Jan 2020Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a...
Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na/H antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via ζ-potential (ζ) measurements after lipid exchange and after scrambling of asymmetry. ζ-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na/H antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23°C and thus enabled characterization of the Na/H antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments.
Topics: Lipids; Proteolipids; Salmonella typhimurium; Unilamellar Liposomes
PubMed: 31843262
DOI: 10.1016/j.bpj.2019.10.043 -
Open Biology Dec 2021MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with...
MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.
Topics: Amino Acid Sequence; Animals; COS Cells; Chlorocebus aethiops; Cytoplasm; HEK293 Cells; Humans; Membrane Proteins; Micelles; Protein Domains; Protein Multimerization; Proteolipids; Single Molecule Imaging
PubMed: 34847774
DOI: 10.1098/rsob.210103 -
Cell Stem Cell Oct 2019Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in...
Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.
Topics: Animals; Cell Differentiation; Cells, Cultured; Deferiprone; Ferroptosis; Humans; Induced Pluripotent Stem Cells; Iron; Iron Chelating Agents; Lipid Peroxidation; Mice; Mice, Mutant Strains; Mutation; Myelin Proteolipid Protein; Oligodendroglia; Pelizaeus-Merzbacher Disease; Stem Cell Transplantation; Targeted Gene Repair
PubMed: 31585094
DOI: 10.1016/j.stem.2019.09.003 -
Biochemical and Biophysical Research... Mar 2022L-enantiomers of antimicrobial peptides (AMPs) are sensitive to proteolytic degradation; however, D-enantiomers of AMPs are expected to provide improved proteolytic... (Comparative Study)
Comparative Study
L-enantiomers of antimicrobial peptides (AMPs) are sensitive to proteolytic degradation; however, D-enantiomers of AMPs are expected to provide improved proteolytic resistance. The present study aimed to comparatively investigate the in vitro antibacterial activity, trypsin and serum stability, toxicity, and in vivo antibacterial activity of L-enantiomeric bovine NK2A (L-NK2A) and its D-enantiomeric NK2A (D-NK2A). Circular dichroism spectroscopy of D-NK2A and L-NK2A in anionic liposomes showed α-helical structures and the α-helical conformation of D-NK2A was a mirror image of L-NK2A. Both D-NK2A and L-NK2A displayed minimal in vitro and in vivo toxicities. RP-HPLC and mass spectrometry analyses revealed that D-NK2A, but not L-NK2A, was resistant to trypsin digestion. D-NK2A and L-NK2A showed similar in vitro bacterial killing activities against Histophilus somni. Slightly reduced antibacterial activity was observed when D-NK2A and L-NK2A were pre-incubated with serum. Confocal and transmission electron microscopic findings confirmed that both peptides induced disruption of bacterial inner- and outer-membranes. Improved survivals with D-NK2A treatment were observed when compared to L-NK2A in a murine model of acute H. somni septicemia. We conclude that antibacterial activity and mode of action of NK2A are not chiral specific. With further optimization, D-NK2A may be a viable AMP candidate to combat bacterial infections.
Topics: Animals; Anti-Bacterial Agents; Antimicrobial Peptides; Cattle; Circular Dichroism; Kaplan-Meier Estimate; Mice; Microscopy, Electron, Transmission; Pasteurellaceae; Pasteurellaceae Infections; Protein Stability; Protein Structure, Secondary; Proteolipids; Stereoisomerism
PubMed: 35101666
DOI: 10.1016/j.bbrc.2022.01.071 -
Clinical and Translational Medicine Feb 2021Nearly a half million people around the world are diagnosed with bladder cancer each year, and an incomplete understanding of its pathogenicity and lack of efficient...
BACKGROUND
Nearly a half million people around the world are diagnosed with bladder cancer each year, and an incomplete understanding of its pathogenicity and lack of efficient biomarkers having been discovered lead to poor clinical management of bladder cancer. Fat mass and obesity-associated protein (FTO) is a critical player in carcinogenesis. We, here, explored the role of FTO and unraveled the mechanism of its function in bladder cancer.
METHODS
Identification of the correlation of FTO with bladder cancer was based on both bioinformatics and clinical analysis of tissue samples collected from a cohort of patients at a hospital and microarray data. Gain-of-function and loss-of-function assays were conducted in vivo and in vitro to assess the effect of FTO on bladder carcinoma tumor growth and its impact on the bladder carcinoma cell viability. Moreover, the interactions of intermediate products were also investigated to elucidate the mechanisms of FTO function.
RESULTS
Bladder tumor tissues had increased FTO expression which correlated with clinical bladder cancer prognosis and outcomes. Both in vivo and in vitro, it played the function of an oncogene in stimulating the cell viability and tumorigenicity of bladder cancer. Furthermore, FTO catalyzed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) demethylation, regulated microRNA miR-384 and mal T cell differentiation protein 2 (MAL2) expression, and modulated the interactions among these processes.
CONCLUSIONS
The interplay of these four clinically relevant factors contributes to the oncogenesis of bladder cancer. FTO facilitates the tumorigenesis of bladder cancer through regulating the MALAT/miR-384/MAL2 axis in m6A RNA modification manner, which ensures the potential of FTO for serving as a diagnostic or prognostic biomarker in bladder cancer.
Topics: Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Cohort Studies; Disease Progression; Female; Humans; Male; Middle Aged; Myelin and Lymphocyte-Associated Proteolipid Proteins; Prognosis; Urinary Bladder; Urinary Bladder Neoplasms
PubMed: 33634966
DOI: 10.1002/ctm2.310 -
Cells Apr 2021The gene encodes a 17-kDa protein containing four putative transmembrane segments whose expression is restricted to human T cells, polarized epithelial cells and... (Review)
Review
The gene encodes a 17-kDa protein containing four putative transmembrane segments whose expression is restricted to human T cells, polarized epithelial cells and myelin-forming cells. The MAL protein has two unusual biochemical features. First, it has lipid-like properties that qualify it as a member of the group of proteolipid proteins. Second, it partitions selectively into detergent-insoluble membranes, which are known to be enriched in condensed cell membranes, consistent with MAL being distributed in highly ordered membranes in the cell. Since its original description more than thirty years ago, a large body of evidence has accumulated supporting a role of MAL in specialized membranes in all the cell types in which it is expressed. Here, we review the structure, expression and biochemical characteristics of MAL, and discuss the association of MAL with raft membranes and the function of MAL in polarized epithelial cells, T lymphocytes, and myelin-forming cells. The evidence that MAL is a putative receptor of the epsilon toxin of , the expression of MAL in lymphomas, the hypermethylation of the gene and subsequent loss of MAL expression in carcinomas are also presented. We propose a model of MAL as the organizer of specialized condensed membranes to make them functional, discuss the role of MAL as a tumor suppressor in carcinomas, consider its potential use as a cancer biomarker, and summarize the directions for future research.
Topics: Animals; Cell Membrane; Epithelial Cells; Humans; Lymphocytes; Myelin and Lymphocyte-Associated Proteolipid Proteins; Neoplasms; Schwann Cells
PubMed: 33946345
DOI: 10.3390/cells10051065 -
Proceedings of the National Academy of... Sep 2017Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease...
Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease diagnosis. However, existing assays for ZnT8 autoantibody (ZnT8A) are limited to detection by soluble domains of ZnT8, owing to difficulties in maintaining proper folding of a full-length ZnT8 protein outside its native membrane environment. Through a combined bioengineering and nanotechnology approach, we have developed a proteoliposome-based full-length ZnT8 self-antigen (full-length ZnT8 proteoliposomes; PLR-ZnT8) for efficient detection of ZnT8A on a plasmonic gold chip (pGOLD). The protective lipid matrix of proteoliposomes improved the proper folding and structural stability of full-length ZnT8, helping PLR-ZnT8 immobilized on pGOLD (PLR-ZnT8/pGOLD) achieve high-affinity capture of ZnT8A from T1D sera. Our PLR-ZnT8/pGOLD exhibited efficient ZnT8A detection for T1D diagnosis with ∼76% sensitivity and ∼97% specificity ( = 307), superior to assays based on detergent-solubilized full-length ZnT8 and the C-terminal domain of ZnT8. Multiplexed assays using pGOLD were also developed for simultaneous detection of ZnT8A, islet antigen 2 autoantibody, and glutamic acid decarboxylase autoantibody for diagnosing T1D.
Topics: Diabetes Mellitus, Type 1; HEK293 Cells; Humans; Protein Array Analysis; Proteolipids; Zinc Transporter 8
PubMed: 28874568
DOI: 10.1073/pnas.1711169114 -
Cancer Biology & Therapy May 2018This study investigated miR-422a and PLP2 expressions in breast cancer cells and breast cancer stem cells (BCSCs). Besides, their influences on polymorphism changes were...
OBJECTIVE
This study investigated miR-422a and PLP2 expressions in breast cancer cells and breast cancer stem cells (BCSCs). Besides, their influences on polymorphism changes were observed.
METHODS
Flow cytometry and fluorescence-activated cell sorting was performed and CD24/CD44 cells were sorted from breast cancer cells and recognized as BCSCs. Microarray was applied to search for the differentially expressed miRNAs and mRNAs between MCF7 and BCSCs. The aberrant expression of miR-422a and PLP2 was further confirmed by RT-qPCR and the direct targeted relationship was verified by dual-luciferase reporter assay. After in vitro transfection, the expression of miR-422a and PLP2 were manipulated and biological functions of BMSCs were compared with CCK-8, colony formation and sphere formation assay. The tumorigenesis ability of transfected BMSCs was also investigated in NOD/SCID tumor mice models.
RESULTS
BMSCs were successfully established from MCF7 cells and miR-422a expression was downregulated while PLP2 level decreased in BMSCs. MiR-422a directly targets the 3'UTR of PLP2 and suppressed its expression. Besides, the up-regulation of miR-422a contributed to weakened ability of proliferation and microsphere formation of BMSCs, while PLP2 overexpression facilitated those biological abilities. Tumorigenesis of BMSCs in mice models was impaired by either overexpression of miR-442a or silencing of PLP2.
CONCLUSION
Up-regulation of miR-422a attenuated microsphere formation, proliferation and tumor formation of breast cancer stem cells via suppressing the PLP2 expression.
Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Female; Heterografts; Humans; MARVEL Domain-Containing Proteins; MCF-7 Cells; Mice, Inbred NOD; Mice, SCID; MicroRNAs; Neoplastic Stem Cells; Proteolipids; Transfection
PubMed: 29509055
DOI: 10.1080/15384047.2018.1433497 -
Frontiers in Immunology 2022Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS. The etiology of MS is complex, and results from the interaction of multiple...
BACKGROUND
Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS. The etiology of MS is complex, and results from the interaction of multiple environmental and genetic factors. Although human leukocyte antigen-HLA alleles such as HLA-DR2 and -DR3 are considered the strongest genetic factors, the environmental factors responsible for disease predisposition are not well understood. Recently, diet and gut microbiota have emerged as an important environmental factors linked to the increased incidence of MS. Especially, western diets rich inprotein and fat have been linked to the increased incidence of obesity. Numerous clinical data indicate a role of obesity and gut microbiota in MS; however, the mechanistic link between gut microbiota and obesity in the pathobiology of MS remains unclear. The present study determines the mechanisms driving MS severity in the context of obesity utilizing a high-fat diet (HFD) induced obese HLA-DR3 class-II transgenic mouse model of MS.
METHODS
HLA-DR3 transgenic mice were kept on a standard HFD diet or Normal Chow (NC) for eight weeks. Gut microbiota composition and functional analysis were performed from the fecal DNA of mice. Experimental autoimmune encephalomyelitis-EAE (an animal model of MS) was induced by immunization with the proteolipid protein-PLP peptide in complete Freud's Adjuvant (CFA) and pertussis toxin.
RESULTS
We observed that HFD-induced obesity caused gut dysbiosis and severe disease compared to mice on NC. Amelioration of disease severity in mice depleted of gut microbiota suggested an important role of gut bacteria in severe EAE in obese mice. Fecal microbiota analysis in HFD mice shows gut microbiota alterations with an increase in the abundance of and bacteria and modulation of various bacterial metabolic pathways including bacterial hydrogen sulfide biosynthetic pathways. Finally, mice on HFD showed increased gut permeability and systemic inflammation suggesting a role gut barrier modulation in obesity induced disease severity.
CONCLUSIONS
This study provides evidence for the involvement of the gut microbiome and associated metabolic pathways plus gut permeability in obesity-induced modulation of EAE disease severity. A better understanding of the same will be helpful to identify novel therapeutic targets to reduce disease severity in obese MS patients.
Topics: Animals; Diet, High-Fat; Disease Models, Animal; Dysbiosis; Encephalomyelitis, Autoimmune, Experimental; HLA-DR2 Antigen; HLA-DR3 Antigen; Humans; Hydrogen Sulfide; Mice; Mice, Obese; Mice, Transgenic; Multiple Sclerosis; Obesity; Pertussis Toxin; Proteolipids; Severity of Illness Index
PubMed: 36164343
DOI: 10.3389/fimmu.2022.966417 -
Toxins Dec 2021Zearalenone (ZEA), a common mycotoxin in grains and animal feeds, has been associated with male reproductive disorders. However, the potential toxicity mechanism of ZEA... (Comparative Study)
Comparative Study
Zearalenone (ZEA), a common mycotoxin in grains and animal feeds, has been associated with male reproductive disorders. However, the potential toxicity mechanism of ZEA is not fully understood. In this study, in vivo and in vitro models were used to explore the effects of ZEA on the blood-testis barrier (BTB) and related molecular mechanisms. First, male BALB/C mice were administered ZEA orally (40 mg/kg·bw) for 5-7 d. Sperm motility, testicular morphology, and expressions of BTB junction proteins and autophagy-related proteins were evaluated. In addition, TM4 cells (mouse Sertoli cells line) were used to delineate the molecular mechanisms that mediate the effects of ZEA on BTB. Our results demonstrated that ZEA exposure induced severe testicular damage in histomorphology and an ultrastructural, time-dependent decrease in the expression of blood-testis barrier junction-related proteins, accompanied by an increase in the expression of autophagy-related proteins. Additionally, similar to the in vitro results, the dose-dependent treatment of ZEA increased the level of cytoplasmic Ca and the levels of the autophagy markers LC3-II and p62, in conjunction with a decrease in the BTB junction proteins occludin, claudin-11, and Cx43, with the dislocation of the gap junction protein Cx43. Meanwhile, inhibition of autophagy by CQ and 3-MA or inhibition of cytoplasmic Ca by BAPTA-AM was sufficient to reduce the effects of ZEA on the TM4 cell BTB. To summarize, this study emphasizes the role of Ca-mediated autophagy in ZEA-induced BTB destruction, which deepens our understanding of the molecular mechanism of ZEA-induced male reproductive disorders.
Topics: Animals; Autophagy; Blood-Testis Barrier; Male; Mice; Mice, Inbred BALB C; Mycotoxins; Myelin and Lymphocyte-Associated Proteolipid Proteins; Sertoli Cells; Sperm Motility; Testis; Zearalenone
PubMed: 34941713
DOI: 10.3390/toxins13120875