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Biochemical Society Transactions Feb 2017The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a... (Review)
Review
The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with.
Topics: Biological Transport; Biotechnology; Lipid Bilayers; Membrane Lipids; Membrane Proteins; Models, Chemical; Models, Molecular; Polymers; Proteolipids; Unilamellar Liposomes
PubMed: 28202656
DOI: 10.1042/BST20160019 -
American Journal of Physiology. Cell... Feb 2022Duchenne muscular dystrophy (DMD) is an inherited muscle wasting disease. Metabolic impairments and oxidative stress are major secondary mechanisms that severely worsen...
Duchenne muscular dystrophy (DMD) is an inherited muscle wasting disease. Metabolic impairments and oxidative stress are major secondary mechanisms that severely worsen muscle function in DMD. Here, we sought to determine whether germline reduction or ablation of sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum (SR) Ca ATPase (SERCA), improves muscle metabolism and ameliorates muscle pathology in the mouse model of DMD. Glucose and insulin tolerance tests show that glucose clearance rate and insulin sensitivity were improved in the SLN haploinsufficient () and SLN-deficient () mice. The histopathological analysis shows that fibrosis and necrosis were significantly reduced in muscles of and mice. SR Ca uptake, mitochondrial complex protein levels, complex activities, mitochondrial Ca uptake and release, and mitochondrial metabolism were significantly improved, and lipid peroxidation and protein carbonylation were reduced in the muscles of and mice. These data demonstrate that reduction or ablation of SLN expression can improve muscle metabolism, reduce oxidative stress, decrease muscle pathology, and protects the mice from glucose intolerance.
Topics: Animals; Blood Glucose; Female; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Mice, Knockout; Muscle Proteins; Muscle, Skeletal; Oxidative Stress; Proteolipids
PubMed: 34986021
DOI: 10.1152/ajpcell.00125.2021 -
Proceedings of the National Academy of... Nov 2017EmrE is a small multidrug resistance transporter found in that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates....
EmrE is a small multidrug resistance transporter found in that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.
Topics: Antiporters; Binding Sites; Biological Transport; Dicyclohexylcarbodiimide; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Proteins; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Molecular Dynamics Simulation; Onium Compounds; Organophosphorus Compounds; Phosphatidylcholines; Phosphatidylglycerols; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Proteolipids; Protons; Recombinant Proteins; Substrate Specificity; Thermodynamics; Xenobiotics
PubMed: 29114048
DOI: 10.1073/pnas.1708671114 -
Pharmacological Research Dec 2015Obesity is increasing at an alarming rate, both in adults and adolescents, across the globe due to increased consumption of caloric rich diet. Obesity and its associated... (Review)
Review
Obesity is increasing at an alarming rate, both in adults and adolescents, across the globe due to increased consumption of caloric rich diet. Obesity and its associated complications appear to be major contributing factors not only to diabetes/heart disease but also to cancer, and neurological diseases causing a huge burden on the health care system. To date, there are no effective treatments to reduce weight gain, other than caloric restriction and exercise which are often difficult to enforce. There are very few drugs available for treating obesity and those that are available only reduce obesity by ∼ 10%. Identifying mechanisms to increase energy expenditure, on top of the increase elicited by exercise, would be more beneficial to control weight gain. The purpose of this review is to highlight the role of sarcolipin (SLN), a regulator of SERCA pump, in muscle thermogenesis and metabolism. We will further discuss if enhancing SLN activity could be an effective mechanism to increase energy expenditure and control weight gain. We will also discuss the merits of adaptive thermogenesis in muscle and brown fat as potential mechanisms to increase energy expenditure during caloric overload. That said, there is still a great need for further research into the mechanism of diet induced thermogenesis and its relevance to overall metabolism and obesity.
Topics: Animals; Body Weight; Energy Metabolism; Humans; Muscle Proteins; Muscle, Skeletal; Obesity; Proteolipids; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thermogenesis
PubMed: 26521759
DOI: 10.1016/j.phrs.2015.10.020 -
American Journal of Physiology. Heart... Jan 2021Sarcolipin (SLN) is an inhibitor of sarco/endoplasmic reticulum (SR) Ca-ATPase (SERCA) and expressed at high levels in the ventricles of animal models for and patients...
Sarcolipin (SLN) is an inhibitor of sarco/endoplasmic reticulum (SR) Ca-ATPase (SERCA) and expressed at high levels in the ventricles of animal models for and patients with Duchenne muscular dystrophy (DMD). The goal of this study was to determine whether the germline ablation of SLN expression improves cardiac SERCA function and intracellular Ca (Ca) handling and prevents cardiomyopathy in the mouse model of DMD. Wild-type, , SLN-haploinsufficient (), and SLN-deficient () mice were used for this study. SERCA function and Ca handling were determined by Ca uptake assays and by measuring single-cell Ca transients, respectively. Age-dependent disease progression was determined by histopathological examinations and by echocardiography in 6-, 12-, and 20-mo-old mice. Gene expression changes in the ventricles of mice were determined by RNA-Seq analysis. SERCA function and Ca cycling were improved in the ventricles of mice. Fibrosis and necrosis were significantly decreased, and cardiac function was enhanced in the mice until the study endpoint. The mice also exhibited similar beneficial effects. RNA-Seq analysis identified distinct gene expression changes including the activation of the apelin pathway in the ventricles of mice. Our findings suggest that reducing SLN expression is sufficient to improve cardiac SERCA function and Ca cycling and prevent cardiomyopathy in mice. First, reducing sarcopolin (SLN) expression improves sarco/endoplasmic reticulum Ca uptake and intracellular Ca handling and prevents cardiomyopathy in mice. Second, reducing SLN expression prevents diastolic dysfunction and improves cardiac contractility in mice Third, reducing SLN expression activates apelin-mediated cardioprotective signaling pathways in heart.
Topics: Animals; Apelin; Calcium; Calcium Signaling; Cardiomyopathies; Disease Models, Animal; Female; Fibrosis; Gene Expression Regulation; Haploinsufficiency; Male; Mice, Inbred C57BL; Mice, Inbred mdx; Mice, Knockout; Muscle Proteins; Muscular Dystrophy, Duchenne; Myocardium; Necrosis; Proteolipids; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Ventricular Function, Left; Mice
PubMed: 33216625
DOI: 10.1152/ajpheart.00601.2020 -
European Journal of Pharmaceutical... Feb 2017Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic...
Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories.
Topics: Erythrocytes; Extracellular Vesicles; Flow Cytometry; Humans; Microscopy, Electron, Transmission; Nanostructures; Proteolipids; Reference Standards
PubMed: 27622921
DOI: 10.1016/j.ejps.2016.09.008 -
Frontiers in Immunology 2022Cuproptosis is a newly discovered programmed cell death dependent on overload copper-induced mitochondrial respiration dysregulation. The positive response to...
BACKGROUND
Cuproptosis is a newly discovered programmed cell death dependent on overload copper-induced mitochondrial respiration dysregulation. The positive response to immunotherapy, one of the most important treatments for invasive breast cancer, depends on the dynamic balance between tumor cells and infiltrating lymphocytes in the tumor microenvironment (TME). However, cuproptosis-related genes (CRGs) in clinical prognosis, immune cell infiltration, and immunotherapy response remain unclear in breast cancer progression.
METHODS
The expression and mutation patterns of 12 cuproptosis-related genes were systematically evaluated in the BRCA training group. Through unsupervised clustering analysis and developing a cuproptosis-related scoring system, we further explored the relationship between cuproptosis and breast cancer progression, prognosis, immune cell infiltration, and immunotherapy.
RESULTS
We identified two distinct CuproptosisClusters, which were correlated with the different patterns between clinicopathological features, prognosis, and immune cell infiltration. Moreover, the differences of the three cuproptosis-related gene subtypes were evaluated based on the CuproptosisCluster-related DEGs. Then, a cuproptosis-related gene signature (PGK1, SLC52A2, SEC14L2, RAD23B, SLC16A6, CCL5, and MAL2) and the scoring system were constructed to quantify the cuproptosis pattern of BRCA patients in the training cohort, and the testing cohorts validated them. Specifically, patients from the low-CRG_score group were characterized by higher immune cell infiltration, immune checkpoint expression, immune checkpoint inhibitor (ICI) scores, and greater sensitivity to immunotherapy. Finally, we screened out RAD23B as a favorable target and indicated its expression was associated with breast cancer progression, drug resistance, and poor prognosis in BRCA patients by performing real-time RT-PCR, cell viability, and IC50 assay.
CONCLUSIONS
Our results confirmed the essential function of cuproptosis in regulating the progression, prognosis, immune cell infiltration, and response to breast cancer immunotherapy. Quantifying cuproptosis patterns and constructing a CRG_score could help explore the potential molecular mechanisms of cuproptosis regulating BRCA advancement and provide more effective immunotherapy and chemotherapy targets.
Topics: Female; Humans; Breast Neoplasms; Immune Checkpoint Inhibitors; Immunotherapy; Myelin and Lymphocyte-Associated Proteolipid Proteins; Prognosis; Tumor Microenvironment; Copper; Apoptosis
PubMed: 36341328
DOI: 10.3389/fimmu.2022.978909 -
International Journal of Molecular... Aug 2021Myelin is of vital importance to the central nervous system and its disruption is related to a large number of both neurodevelopmental and neurodegenerative diseases....
Myelin is of vital importance to the central nervous system and its disruption is related to a large number of both neurodevelopmental and neurodegenerative diseases. The differences observed between human and rodent oligodendrocytes make animals inadequate for modeling these diseases. Although developing human in vitro models for oligodendrocytes and myelinated axons has been a great challenge, 3D cell cultures derived from iPSC are now available and able to partially reproduce the myelination process. We have previously developed a human iPSC-derived 3D brain organoid model (also called BrainSpheres) that contains a high percentage of myelinated axons and is highly reproducible. Here, we have further refined this technology by applying multiple readouts to study myelination disruption. Myelin was assessed by quantifying immunostaining/confocal microscopy of co-localized myelin basic protein (MBP) with neurofilament proteins as well as proteolipid protein 1 (PLP1). Levels of PLP1 were also assessed by Western blot. We identified compounds capable of inducing developmental neurotoxicity by disrupting myelin in a systematic review to evaluate the relevance of our BrainSphere model for the study of the myelination/demyelination processes. Results demonstrated that the positive reference compound (cuprizone) and two of the three potential myelin disruptors tested (Bisphenol A, Tris(1,3-dichloro-2-propyl) phosphate, but not methyl mercury) decreased myelination, while ibuprofen (negative control) had no effect. Here, we define a methodology that allows quantification of myelin disruption and provides reference compounds for chemical-induced myelin disruption.
Topics: Axons; Brain; Cell Culture Techniques; Central Nervous System; Humans; Induced Pluripotent Stem Cells; Models, Biological; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Nerve Fibers, Myelinated; Neurotoxicity Syndromes; Oligodendroglia; Organoids
PubMed: 34502381
DOI: 10.3390/ijms22179473 -
Biochimica Et Biophysica Acta Jan 2015The motional averaging of powder pattern line shapes is one of the most fundamental aspects of sold-state NMR. Since membrane proteins in liquid crystalline phospholipid... (Review)
Review
The motional averaging of powder pattern line shapes is one of the most fundamental aspects of sold-state NMR. Since membrane proteins in liquid crystalline phospholipid bilayers undergo fast rotational diffusion, all of the signals reflect the angles of the principal axes of their dipole-dipole and chemical shift tensors with respect to the axis defined by the bilayer normal. The frequency span and sign of the axially symmetric powder patterns that result from motional averaging about a common axis provide sufficient structural restraints for the calculation of the three-dimensional structure of a membrane protein in a phospholipid bilayer environment. The method is referred to as rotationally aligned (RA) solid-state NMR and demonstrated with results on full-length, unmodified membrane proteins with one, two, and seven trans-membrane helices. RA solid-state NMR is complementary to other solid-state NMR methods, in particular oriented sample (OS) solid-state NMR of stationary, aligned samples. Structural distortions of membrane proteins from the truncations of terminal residues and other sequence modifications, and the use of detergent micelles instead of phospholipid bilayers have also been demonstrated. Thus, it is highly advantageous to determine the structures of unmodified membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. RA solid-state NMR provides a general method for obtaining accurate and precise structures of membrane proteins under near-native conditions.
Topics: Amino Acid Sequence; Diffusion; Magnetic Resonance Spectroscopy; Membrane Proteins; Molecular Sequence Data; Protein Folding; Proteolipids; Rotation
PubMed: 24747039
DOI: 10.1016/j.bbamem.2014.04.002 -
Biochimica Et Biophysica Acta.... Apr 2024The proteolipid code determines how cytosolic proteins find and remodel membrane surfaces. Here, we investigate how this process works with sorting nexins Snx1 and Snx3.... (Review)
Review
The proteolipid code determines how cytosolic proteins find and remodel membrane surfaces. Here, we investigate how this process works with sorting nexins Snx1 and Snx3. Both proteins form sorting machines by recognizing membrane zones enriched in phosphatidylinositol 3-phosphate (PI3P), phosphatidylserine (PS) and cholesterol. This co-localized combination forms a unique "lipid codon" or lipidon that we propose is responsible for endosomal targeting, as revealed by structures and interactions of their PX domain-based readers. We outline a membrane recognition and remodeling mechanism for Snx1 and Snx3 involving this code element alongside transmembrane pH gradients, dipole moment-guided docking and specific protein-protein interactions. This generates an initial membrane-protein assembly (memtein) that then recruits retromer and additional PX proteins to recruit cell surface receptors for sorting to the trans-Golgi network (TGN), lysosome and plasma membranes. Post-translational modification (PTM) networks appear to regulate how the sorting machines form and operate at each level. The commonalities and differences between these sorting nexins show how the proteolipid code orchestrates parallel flows of molecular information from ribosome emergence to organelle genesis, and illuminates a universally applicable model of the membrane.
Topics: Carrier Proteins; Vesicular Transport Proteins; Sorting Nexins; Protein Transport; Proteolipids
PubMed: 38408696
DOI: 10.1016/j.bbamem.2024.184305