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Scientific Reports Feb 2022Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown...
Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown of PLP2 inhibited the growth and metastasis of melanoma cells. In the present work, we studied the antitumor activity of peptide Rb4 derived from protein PLP2. In vitro, Rb4 induced F-actin polymerization, prevented F-actin depolymerization and increased the ER-derived cytosolic calcium. Such effects were associated with necrosis of murine melanoma B16F10-Nex2 cells and with inhibition of the viability of human cancer cell lines. Loss of plasma membrane integrity, dilation of mitochondria, cytoplasm vacuolation and absence of chromatin condensation characterized tumor cell necrosis. Cleavage of PARP-1 and inhibition of RIP1 expression were also observed. In vivo, peptide Rb4 reduced the lung metastasis of tumor cells and delayed the subcutaneous melanoma growth in a syngeneic model. Rb4 induced the expression of two DAMPs molecules, HMGB1 and calreticulin, in B16F10-Nex2. Our results suggest that peptide Rb4 acts directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. We suggest that peptide Rb4 is a promising compound to be developed as an anticancer drug.
Topics: Animals; Antineoplastic Agents; Calreticulin; Cell Death; Cell Line, Tumor; Gene Expression; HMGB1 Protein; Humans; MARVEL Domain-Containing Proteins; Melanoma; Mice; Necrosis; Nuclear Pore Complex Proteins; Peptides; Poly (ADP-Ribose) Polymerase-1; Proteolipids; RNA-Binding Proteins; Skin Neoplasms
PubMed: 35190586
DOI: 10.1038/s41598-022-06429-8 -
Biochimica Et Biophysica Acta.... Dec 2017The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion...
The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active K1.1 and K1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using K1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of K1.1 and K1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.
Topics: Elapid Venoms; Escherichia coli; Fluorescent Dyes; Gene Expression; Genetic Vectors; Humans; Isoxazoles; Kv1.1 Potassium Channel; Kv1.3 Potassium Channel; Membrane Potentials; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Proteolipids; Recombinant Proteins; Subcellular Fractions; Valinomycin
PubMed: 28888365
DOI: 10.1016/j.bbamem.2017.09.002 -
BMC Biology Feb 2024Membranes are protein and lipid structures that surround cells and other biological compartments. We present a conceptual model wherein all membranes are organized into...
Membranes are protein and lipid structures that surround cells and other biological compartments. We present a conceptual model wherein all membranes are organized into structural and functional zones. The assembly of zones such as receptor clusters, protein-coated pits, lamellipodia, cell junctions, and membrane fusion sites is explained to occur through a protein-lipid code. This challenges the theory that lipids sort proteins after forming stable membrane subregions independently of proteins.
Topics: Proteolipids; Membranes; Carrier Proteins; Cell Membrane
PubMed: 38414038
DOI: 10.1186/s12915-024-01849-6 -
Molecular Biology Reports Jul 2022Recent studies have highlighted that uncoupling of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by sarcolipin (SLN) increases ATP consumption and contributes to heat...
BACKGROUND
Recent studies have highlighted that uncoupling of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by sarcolipin (SLN) increases ATP consumption and contributes to heat liberation. Exploiting this thermogenic mechanism in skeletal muscle may provide an attractive strategy to counteract obesity and associated metabolic disorders. In the present study, we have investigated the role of SLN on substrate metabolism in human skeletal muscle cells.
METHODS AND RESULTS
After generation of skeletal muscle cells with stable SLN knockdown (SLN-KD), cell viability, glucose and oleic acid (OA) metabolism, mitochondrial function, as well as gene expressions were determined. Depletion of SLN did not influence cell viability. However, glucose and OA oxidation were diminished in SLN-KD cells compared to control myotubes. Basal respiration measured by respirometry was also observed to be reduced in cells with SLN-KD. The metabolic perturbation in SLN-KD cells was reflected by reduced gene expression levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and forkhead box O1 (FOXO1). Furthermore, accumulation of OA was increased in cells with SLN-KD compared to control cells. These effects were accompanied by increased lipid formation and incorporation of OA into complex lipids. Additionally, formation of complex lipids and free fatty acid from de novo lipogenesis with acetate as substrate was enhanced in SLN-KD cells. Detection of lipid droplets using Oil red O staining also showed increased lipid accumulation in SLN-KD cells.
CONCLUSIONS
Overall, our study sheds light on the importance of SLN in maintaining metabolic homeostasis in human skeletal muscle. Findings from the current study suggest that therapeutic strategies involving SLN-mediated futile cycling of SERCA might have significant implications in the treatment of obesity and associated metabolic disorders.
Topics: Glucose; Humans; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Obesity; Proteolipids; Sarcoplasmic Reticulum Calcium-Transporting ATPases
PubMed: 35364719
DOI: 10.1007/s11033-022-07387-0 -
The Journal of Neuroscience : the... Apr 2020During differentiation, oligodendrocyte precursor cells (OPCs) extend a network of processes that make contact with axons and initiate myelination. Recent studies...
During differentiation, oligodendrocyte precursor cells (OPCs) extend a network of processes that make contact with axons and initiate myelination. Recent studies revealed that actin polymerization is required for initiation of myelination whereas actin depolymerization promotes myelin wrapping. Here, we used primary OPCs in culture isolated from neonatal rat cortices of both sexes and young male and female mice with oligodendrocyte-specific deletion of mechanistic target of rapamycin (mTOR) to demonstrate that mTOR regulates expression of specific cytoskeletal targets and actin reorganization in oligodendrocytes during developmental myelination. Loss or inhibition of mTOR reduced expression of profilin2 and ARPC3, actin polymerizing factors, and elevated levels of active cofilin, which mediates actin depolymerization. The deficits in actin polymerization were revealed in reduced phalloidin and deficits in oligodendrocyte cellular branching complexity at the peak of morphologic differentiation and a delay in initiation of myelination. We further show a critical role for mTOR in expression and localization of myelin basic protein () mRNA and MBP protein to the cellular processes where it is necessary at the myelin membrane for axon wrapping. mRNA transport deficits were confirmed by single molecule RNA FISH. Moreover, expression of the kinesin family member 1B, an mRNA transport protein, was reduced in CC1+ cells in the and in mTOR inhibited oligodendrocytes undergoing differentiation These data support the conclusion that mTOR regulates both initiation of myelination and axon wrapping by targeting cytoskeletal reorganization and MBP localization to oligodendrocyte processes. Myelination is essential for normal CNS development and adult axon preservation and function. The mechanistic target of rapamycin (mTOR) signaling pathway has been implicated in promoting CNS myelination; however, there is a gap in our understanding of the mechanisms by which mTOR promotes developmental myelination through regulating specific downstream targets. Here, we present evidence that mTOR promotes the initiation of myelination through regulating specific cytoskeletal targets and cellular process expansion by oligodendrocyte precursor cells as well as expression and cellular localization of myelin basic protein.
Topics: Actin-Related Protein 2-3 Complex; Actins; Animals; Axons; Cell Differentiation; Cytoskeleton; Kinesins; Mice; Mice, Knockout; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Oligodendroglia; Rats; Rats, Sprague-Dawley; Stem Cells; TOR Serine-Threonine Kinases; Zebrafish
PubMed: 32139584
DOI: 10.1523/JNEUROSCI.1434-18.2020 -
Critical Reviews in Biochemistry and... 2015Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized... (Review)
Review
Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed.
Topics: Animals; Cytological Techniques; Fluorescence Resonance Energy Transfer; Humans; In Vitro Techniques; Membrane Fusion; Proteolipids; SNARE Proteins; Synaptic Vesicles
PubMed: 25788028
DOI: 10.3109/10409238.2015.1023252 -
Biophysical Journal Jun 2016In cell proliferation, stem cell differentiation, chemoresistance, and tissue organization, the ubiquitous role of YAP/TAZ continues to impact our fundamental...
In cell proliferation, stem cell differentiation, chemoresistance, and tissue organization, the ubiquitous role of YAP/TAZ continues to impact our fundamental understanding in numerous physiological and disease systems. YAP/TAZ is an important signaling nexus integrating diverse mechanical and biochemical signals, such as ECM stiffness, adhesion ligand density, or cell-cell contacts, and thus strongly influences cell fate. Recent studies show that YAP/TAZ mechanical sensing is dependent on RhoA-regulated stress fibers. However, current understanding of YAP/TAZ remains limited due to the unknown interaction between the canonical Hippo pathway and cell tension. Furthermore, the multiscale relationship connecting adhesion signaling to YAP/TAZ activity through cytoskeleton dynamics remains poorly understood. To identify the roles of key signaling molecules in mechanical signal sensing and transduction, we present a, to our knowledge, novel computational model of the YAP/TAZ signaling pathway. This model converts extracellular-matrix mechanical properties to biochemical signals via adhesion, and integrates intracellular signaling cascades associated with cytoskeleton dynamics. We perform perturbations of molecular levels and sensitivity analyses to predict how various signaling molecules affect YAP/TAZ activity. Adhesion molecules, such as FAK, are predicted to rescue YAP/TAZ activity in soft environments via the RhoA pathway. We also found that changes of molecule concentrations result in different patterns of YAP/TAZ stiffness response. We also investigate the sensitivity of YAP/TAZ activity to ECM stiffness, and compare with that of SRF/MAL, which is another important regulator of differentiation. In addition, the model shows that the unresolved synergistic effect of YAP/TAZ activity between the mechanosensing and the Hippo pathways can be explained by the interaction of LIM-kinase and LATS. Overall, our model provides a, to our knowledge, novel platform for studying YAP/TAZ activity in the context of integrating different signaling pathways. This platform can be used to gain, to our knowledge, new fundamental insights into roles of key molecular and mechanical regulators on development, tissue engineering, or tumor progression.
Topics: Actins; Adaptor Proteins, Signal Transducing; Algorithms; Computer Simulation; Cytoskeleton; Elasticity; Extracellular Matrix; Intracellular Signaling Peptides and Proteins; Mechanotransduction, Cellular; Models, Biological; Myelin and Lymphocyte-Associated Proteolipid Proteins; rho-Associated Kinases; rhoA GTP-Binding Protein
PubMed: 27276271
DOI: 10.1016/j.bpj.2016.04.040 -
Cellular and Molecular Life Sciences :... Apr 2022Proteolipids are proteins with unusual lipid-like properties. It has long been established that PLP and plasmolipin, which are two unrelated membrane-tetra-spanning...
Proteolipids are proteins with unusual lipid-like properties. It has long been established that PLP and plasmolipin, which are two unrelated membrane-tetra-spanning myelin proteolipids, can be converted in vitro into a water-soluble form with a distinct conformation, raising the question of whether these, or other similar proteolipids, can adopt two different conformations in the cell to adapt their structure to distinct environments. Here, we show that MALL, another proteolipid with a membrane-tetra-spanning structure, distributes in membranes outside the nucleus and, within the nucleus, in membrane-less, liquid-like PML body biomolecular condensates. Detection of MALL in one or other environment was strictly dependent on the method of cell fixation used, suggesting that MALL adopts different conformations depending on its physical environment -lipidic or aqueous- in the cell. The acquisition of the condensate-compatible conformation requires PML expression. Excess MALL perturbed the distribution of the inner nuclear membrane proteins emerin and LAP2β, and that of the DNA-binding protein BAF, leading to the formation of aberrant nuclei. This effect, which is consistent with studies identifying overexpressed MALL as an unfavorable prognostic factor in cancer, could contribute to cell malignancy. Our study establishes a link between proteolipids, membranes and biomolecular condensates, with potential biomedical implications.
Topics: Biomolecular Condensates; Cell Nucleus; Humans; Molecular Conformation; Neoplasms; Proteolipids
PubMed: 35399121
DOI: 10.1007/s00018-022-04270-w -
Protein Expression and Purification Jul 2019Human P-glycoprotein (P-gp) is an ATP-binding cassette transporter that has been implicated in altering the pharmacokinetics of anticancer drugs in normal tissues and...
Human P-glycoprotein (P-gp) is an ATP-binding cassette transporter that has been implicated in altering the pharmacokinetics of anticancer drugs in normal tissues and development of multidrug resistance in tumor cells via drug efflux. There is still no definitive explanation of the mechanism by which P-gp effluxes drugs. One of the challenges of large-scale purification of membrane transporters is the selection of a suitable detergent for its optimal extraction from cell membranes. In addition, further steps of purification can often lead to inactivation and aggregation, decreasing the yield of purified protein. Here we report the large-scale purification of human P-gp expressed in High-Five insect cells using recombinant baculovirus. The purification strategies we present yield homogeneous functionally active wild type P-gp and its E556Q/E1201Q mutant, which is defective in carrying out ATP hydrolysis. Three detergents (1,2-diheptanoyol-sn-glycero-3-phosphocholine, dodecyl maltoside and n-octyl-β-d-glucopyranoside) were used to solubilize and purify P-gp from insect cell membranes. P-gp purification was performed first using immobilized metal affinity chromatography, then followed by a second step of either anion exchange chromatography or size exclusion chromatography to yield protein in concentrations of 2-12 mg/mL. Size exclusion chromatography was the preferred method, as it allows separation of monomeric transporters from aggregates. We show that the purified protein, when reconstituted in proteoliposomes and nanodiscs, exhibits both basal and substrate or inhibitor-modulated ATPase activity. This report thus provides a convenient and robust method to obtain large amounts of active homogeneously purified human P-gp that is suitable for biochemical, biophysical and structural characterization.
Topics: ATP Binding Cassette Transporter, Subfamily B; Baculoviridae; Cell Extracts; Chromatography, Liquid; Detergents; Escherichia coli; Glucosides; Humans; Phospholipids; Proteolipids; Recombinant Proteins
PubMed: 30851394
DOI: 10.1016/j.pep.2019.03.002 -
Aging Sep 2021Ovarian carcinoma (OC) is the deadliest gynecologic malignancy in females worldwide. Circular RNA Foxo3 (Foxo3) plays essential roles in various cancers. However, the...
BACKGROUND
Ovarian carcinoma (OC) is the deadliest gynecologic malignancy in females worldwide. Circular RNA Foxo3 (Foxo3) plays essential roles in various cancers. However, the detailed function of Foxo3 in OC remains unclear. This study aimed to investigate the role of Foxo3 in OC and the underlying molecular mechanism.
METHODS
The abundance of Foxo3 was detected in OC cell lines by qPCR. Lentivirus transduction, CCK-8, wound healing assays, transwell migration and invasion assays, luciferase reporter assay, western blotting, fluorescence hybridization (FISH), transmission electron microscopy, nanoparticle tracking analysis, and bioinformatics analysis were performed to investigate the underlying mechanism.
RESULTS
The results demonstrated that Foxo3 was significantly upregulated in OC cell lines. Overexpression and knockdown of Foxo3 promoted and inhibited the proliferation, migration, and invasion of OC cells, respectively. Foxo3 could bind to miR-422a to negatively regulate miR-422a expression. Also, proteolipid protein 2 (PLP2) was a targeting gene of miR-422a. Additionally, Foxo3 was highly expressed in exosomes derived from OC cells. Furthermore, Foxo3 could be shuttled to OC cells by exosomes and promoted OC progression.
CONCLUSIONS
Foxo3 promoted OC progression through exosome-mediated intercellular interaction to target miR-422a/PLP2 axis. Foxo3 may serve as a potential biomarker for OC.
Topics: Carcinoma, Ovarian Epithelial; Computational Biology; Exosomes; Female; Forkhead Box Protein O3; Humans; In Situ Hybridization, Fluorescence; MARVEL Domain-Containing Proteins; MicroRNAs; Ovarian Neoplasms; Proteolipids; RNA, Circular; Up-Regulation
PubMed: 34555810
DOI: 10.18632/aging.203550