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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 -
Journal of Bone and Mineral Research :... Jan 2023The plasticity of Schwann cells (SCs) following nerve injury is a critical feature in the regeneration of peripheral nerves as well as surrounding tissues. Here, we show...
The plasticity of Schwann cells (SCs) following nerve injury is a critical feature in the regeneration of peripheral nerves as well as surrounding tissues. Here, we show a pivotal role of Schwann cell-derived cells in alveolar bone regeneration through the specific ablation of proteolipid protein 1 (Plp)-expressing cells and the transplantation of teased nerve fibers and associated cells. With inducible Plp specific genetic tracing, we observe that Plp cells migrate into wounded alveolar defect and dedifferentiate into repair SCs. Notably, these cells barely transdifferentiate into osteogenic cell lineage in both SCs tracing model and transplant model, but secret factors to enhance the proliferation of alveolar skeletal stem cells (aSSCs). As to the mechanism, this effect is associated with the upregulation of extracellular matrix (ECM) receptors and receptor tyrosine kinases (RTKs) signaling and the downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) pathway. Collectively, our data demonstrate that SCs dedifferentiate after neighboring alveolar bone injury and contribute to bone regeneration mainly by a paracrine function. © 2022 American Society for Bone and Mineral Research (ASBMR).
Topics: Phosphatidylinositol 3-Kinases; Schwann Cells; Signal Transduction; Extracellular Signal-Regulated MAP Kinases; Cell Proliferation; Bone Regeneration
PubMed: 36331097
DOI: 10.1002/jbmr.4735 -
PeerJ 2020Excessive fluoride can lead to chronic neurodegeneration characterized by neuron and myelin loss and memory dysfunction. The gut-brain axis hypothesis suggests that gut...
BACKGROUND
Excessive fluoride can lead to chronic neurodegeneration characterized by neuron and myelin loss and memory dysfunction. The gut-brain axis hypothesis suggests that gut microbiota plays a crucial role in regulating brain function. Thus, using probiotics to adjust the gut microenvironment may be a potential therapy for mental diseases.
METHODS
Mice in the prob group were administrated with BS15 for 28 days prior to and throughout a 70-day exposure to sodium fluoride. The drinking water of all groups (F and prob groups) except the control group were replaced by high-fluoride water (100 mg NaF/L) on day 28. Animals in each group were divided into two subsets: one underwent behavioral test, and the other was sacrificed for sampling. The mRNA expression level and protein content related to inflammatory reaction in the ileum and hippocampus were respectively detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of proteins related to myelin structure, apoptosis, and memory in the hippocampus and tight junction proteins in the ileum were determined by RT-qPCR and/or immunohistochemistry. Gut permeability markers (D-lactate and diamine oxidase (DAO)) in the serum were also examined by ELISA.
RESULTS
The results showed that fluoride exposure induced a lower spontaneous exploration ( < 0.05) in T-maze test, which indicated an impairment of memory. Spontaneous exploration of BS15-treated mice was significantly higher ( < 0.05) than that in F group. Fluoride reduced ( < 0.05) levels of myelin structural protein (proteolipid protein) and neurogenesis-associated proteins (brain-derived neurotrophic factor and cAMP/Ca responsive element-binding protein), induced disordered inflammatory cytokines (α, γ, and ; < 0.05), increased pro-apoptotic genes (; < 0.05), and decreased anti-apoptotic genes (; < 0.05) in the hippocampus, of which the influences were reversed by BS15. BS15 treatment exerted significant preventive effects on reversing the gut inflammation induced by excessive fluoride intake by reducing ( < 0.05) the levels of pro-inflammatory cytokines (tumor necrosis factor-alpha (α) and interferon-gamma (γ)) and remarkably increasing ( < 0.05) the level of anti-inflammatory cytokines (). Moreover, the serum DAO activity and D-lactate concentration significantly increased by fluoride were also reduced ( < 0.05) by BS15. This result indicated the profitable effect of BS15 on gut permeability.
CONCLUSION
BS15 intake could benefit the neuroinflammation and demyelination in the hippocampus by improving the gut environment and ameliorating fluorine-induced memory dysfunction.
PubMed: 33083147
DOI: 10.7717/peerj.10125 -
Cell Calcium May 2020Cell membranes spatially define gradients that drive the complexity of biological signals. To guarantee movements and exchanges of solutes between compartments, membrane... (Review)
Review
Cell membranes spatially define gradients that drive the complexity of biological signals. To guarantee movements and exchanges of solutes between compartments, membrane transporters negotiate the passages of ions and other important molecules through lipid bilayers. The Na/Ca exchangers (NCXs) in particular play central roles in balancing Na and Ca fluxes across diverse proteolipid borders in all eukaryotic cells, influencing cellular functions and fate by multiple means. To prevent progression from balance to disease, redundant regulatory mechanisms cooperate at multiple levels (transcriptional, translational, and post-translational) and guarantee that the activities of NCXs are finely-tuned to cell homeostatic requirements. When this regulatory network is disturbed by pathological forces, cells may approach the end of life. In this review, we will discuss the main findings, controversies and open questions about regulatory mechanisms that control NCX functions in health and disease.
Topics: Adenosine Triphosphate; Animals; Humans; Ion Channel Gating; Models, Biological; Sodium-Calcium Exchanger
PubMed: 32070925
DOI: 10.1016/j.ceca.2020.102169 -
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 -
Annals of Clinical and Translational... Nov 2022The mechanisms by which exercise training (ET) elicits beneficial effects on the systemic immune system and the central nervous system (CNS) in autoimmune...
BACKGROUND
The mechanisms by which exercise training (ET) elicits beneficial effects on the systemic immune system and the central nervous system (CNS) in autoimmune neuroinflammation are not fully understood.
OBJECTIVES
To investigate (1) the systemic effects of high-intensity continuous training (HICT) on the migratory potential of autoimmune cells; (2) the direct effects of HICT on blood-brain-barrier (BBB) properties.
METHODS
Healthy mice were subjected to high-intensity continuous training (HICT) by treadmill running. The proteolipid protein (PLP) transfer EAE model was utilized to examine the immunomodulatory effects of training, where PLP-reactive lymph-node cells (LNCs) from HICT and sedentary donor mice were analyzed in vitro and transferred to naïve recipients that developed EAE. To examine neuroprotection, encephalitogenic LNCs from donor mice were transferred into HICT or sedentary recipient mice and the BBB was analyzed.
RESULTS
Transfer of PLP-reactive LNCs obtained from HICT donor mice attenuated EAE severity and inflammation in recipient mice. HICT markedly inhibited very late antigen (VLA)-4 and lymphocyte function-associated antigen (LFA)-1 expression in LNCs. Transfer of encephalitogenic LNCs into HICT recipients resulted in milder EAE and attenuated CNS inflammation. HICT reduced BBB permeability and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in CNS blood vessels.
INTERPRETATION
HICT attenuates EAE development by both immunomodulatory and neuroprotective effects. The reduction in destructive CNS inflammation in EAE is attributed to systemic inhibition of autoreactive cell migratory potential, as well as reduction in BBB permeability, which are associated with reduced VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions.
Topics: Animals; Mice; Encephalomyelitis, Autoimmune, Experimental; Brain; Blood-Brain Barrier; Encephalitis; Inflammation; Encephalomyelitis
PubMed: 36217574
DOI: 10.1002/acn3.51677 -
Transgenic Research Feb 2021Vascular endothelial growth factor (VEGF) is important for lung development and function but ideal mouse models are limited to decipher the quantitative relationship...
Vascular endothelial growth factor (VEGF) is important for lung development and function but ideal mouse models are limited to decipher the quantitative relationship between VEGF expression levels and its proper development and pathogenesis. Human SPC promoter has been used to faithfully express genes or cDNAs in the pulmonary epithelium in many transgenic mouse models. In the study, a mouse model of lung-specific and reversible VEGF repression (hspc-rtTR/Vegf) was generated. Human SPC promoter was used to drive lung-specific rtTR expression, a cDNA coding for doxycycline-regulated transcription repression protein. By crossing with Vegf mice, that has tetracycline operator sequences insertion in 5'-UTR region, it allows us to reversibly inhibit lung VEGF transcription from its endogenous level through doxycycline food, water or injection. The tissue-specific inhibition of VEGF is used to mimic abnormal expression levels of VEGF in lung. Reduced VEGF expression in lung is confirmed by quantitative real time PCR and immunoblotting. Lung development and structure was analyzed by histology analysis and found significantly affected under low VEGF. The pulmonary epithelium and alveolarization are found abnormal with swelling alveolar septum and enlargement of air space. Genome-wide gene expression analysis identified that immune activities are involved in the VEGF-regulated lung functions. The transgenic mouse model can be used to mimic human pulmonary diseases. The mouse model confirms the important regulatory roles of epithelial expressed VEGF in lung development and function. This mouse model is valuable for studying VEGF-regulated lung development, pathogenesis and drug screening under low VEGF expression.
Topics: Animals; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation, Developmental; Humans; Lung; Lung Diseases; Mice; Mice, Transgenic; Organogenesis; Promoter Regions, Genetic; Pulmonary Surfactant-Associated Protein C; Vascular Endothelial Growth Factor A
PubMed: 33394314
DOI: 10.1007/s11248-020-00223-w -
Experimental Cell Research Jan 2023Extracellular vesicles (EVs) are heterogenous populations of proteolipid bi-layered vesicles secreted by cells as an important biological process. EVs cargo can reflect...
Extracellular vesicles (EVs) are heterogenous populations of proteolipid bi-layered vesicles secreted by cells as an important biological process. EVs cargo can reflect the cellular environmental conditions in which cells grow. The use of serum-free conditioned media to harvest EVs leads to stress-mediated cellular changes with longer incubation time and impacts EV production and functionality. This study aims to explore the role of incubation time and temperature on EV production and proteomic cargo. For this purpose, an optimized ultrafiltration-size exclusion chromatography-based technique is developed, which isolates small EVs ranging from 130 to 220 nm. The result shows higher EVs production in cancerous cells (K7M2) compared to noncancerous cells (NIH/3T3), which increases with longer incubation time and elevated temperature. Mass spectrometry-based proteomic characterization of EVs showed incubation time and temperature-dependent proteomic profile. A set of enriched EV proteins were identified in EVs isolated at nutrient-stress (72 h incubation time) and heat-stress (40 °C incubation temperature) environment. Enrichment of Serpinb1a in EVs isolated in heat stress was further validated via immunoblot. Gene enrichment analysis revealed that enriched EV proteins following nutrient stress were involved in negative regulation of transcription, response to oxidative stress, and protein folding. Likewise, enriched EV proteins following heat stress were involved in oxaloacetate and aspartate metabolism, and glutamate catabolic process. EVs isolated under nutrient stress showed pro-proliferative activity whereas EVs isolated under heat stress showed anti-proliferative activity. Our results show that incubation time and temperature can alter EV production, its proteomic cargo, and functionality, which can be used to design need-based standard isolation parameters for reproducible EV research.
Topics: Proteomics; Temperature; Mass Spectrometry; Extracellular Vesicles; Proteins
PubMed: 36584743
DOI: 10.1016/j.yexcr.2022.113454 -
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