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Biochimica Et Biophysica Acta.... Nov 2018Human MATE1 (multidrug and toxin extrusion 1, hMATE1) is a H/organic cation (OC) exchanger responsible for the final step of toxic organic cation excretion in the kidney...
Human MATE1 (multidrug and toxin extrusion 1, hMATE1) is a H/organic cation (OC) exchanger responsible for the final step of toxic organic cation excretion in the kidney and liver. To investigate the mechanism of transport, we have established an in vitro assay procedure that includes its expression in insect cells, solubilization with octyl glucoside, purification, and reconstitution into liposomes. The resultant proteoliposomes containing hMATE1 as the sole protein component took up radiolabeled tetraethylammonium (TEA) in a ∆pH-dependent and electroneutral fashion. Furthermore, lipid-detergent micelle containing hMATE1 showed ∆pH-dependent TEA binding similar to transport. Mutated hMATE1 with replacement E273Q completely lacked these TEA binding and transport. In the case of divalent substrates, transport was electrogenic. These observations indicate that the stoichiometry of OC/H exchange is independent of substrate charge. Purification and reconstitution of hMATE1 is considered to be suitable for understanding the detailed molecular mechanisms of hMATE1. The results suggest that Glu273 of hMATE1 plays essential roles in substrate binding and transport.
Topics: Cations; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Mutagenesis, Site-Directed; Organic Cation Transport Proteins; Protein Binding; Proteolipids; Recombinant Proteins; Substrate Specificity; Tetraethylammonium
PubMed: 30028956
DOI: 10.1016/j.bbamem.2018.07.005 -
Poultry Science Aug 2020This study aimed to determine the transcription profile of NK-lysin gene in native chickens. Moreover, it was targeted toward determining the primary, three-dimensional,...
This study aimed to determine the transcription profile of NK-lysin gene in native chickens. Moreover, it was targeted toward determining the primary, three-dimensional, and molecular dynamic structures of NK-lysin and granulysin peptides to understand their mode of action and intracellular transduction pathways using in silico analysis. The results revealed that NK-lysin gene in native chickens and Gallus gallus were closely related to those of other avian species. However, there was a low sequence homology when aligned with the mammalian peptides. The coding region of NK-lysin peptide in native chickens encoded 140 amino acids as found in G. gallus with a homology of 98% that declined to 20%, particularly in mammalian species. The results revealed that the NK-lysin in native chickens was closely related to that in avian species at a range of 71-76%. However, it was different from that of other mammalians in terms of nucleotide and amino acid identities. The mRNA transcripts of NK-lysin had high and moderate expression levels in the testis and pancreas, respectively. Nonetheless, the small intestine, kidney, spleen, and liver had a low expression level. The NK-lysin peptides contained more than 50% of the total AA with a nonpolar feature, whereas polar AA constituted up to 30% of AA. The results also indicated that the hydrophilic regions and positively charged amino acids were predominant on the surface of the investigated peptides. The NK-lysin was folded in 4-5 helical units and 3-4 loop structures in their saposin domain. The third helical peptide was long in both avian and bovine species (104-123 residues). However, the fourth helical peptide was short in humans, pigs, and chimpanzees (101-123, 104-123, and 102-124 residues, respectively), with the helical unit residues of 95-97, 96-99, and 96-98, respectively. The obtained results can be helpful in developing novel approaches that could be used as alternatives or adjuncts to the existing means of control.
Topics: Amino Acid Sequence; Animals; Cattle; Chickens; Gene Expression Profiling; Molecular Dynamics Simulation; Protein Domains; Proteolipids; Structure-Activity Relationship; Swine
PubMed: 32731965
DOI: 10.1016/j.psj.2020.04.005 -
Genes Oct 2022As an antimicrobial peptide, NK-lysin () plays an important role in the innate immune system of organisms. In this study, 300 piglets (68 Landrace pigs, 158 Large White...
As an antimicrobial peptide, NK-lysin () plays an important role in the innate immune system of organisms. In this study, 300 piglets (68 Landrace pigs, 158 Large White pigs and 74 Songliao Black pigs) were used to further explore the function of gene in porcine immune system. The quantitative real-time PCR analysis detected the gene's expression, and the result demonstrated that mRNA was expressed in lung, spleen, stomach, kidney, liver and heart, and the expression level decreased sequentially. A single-nucleotide polymorphism (SNP, g.59070355 G > A) in intron 3 of the gene was detected by PCR amplification and sequencing. The results of the Chi-square (χ2) test showed that the genotype of the SNP was consistent with the Hardy-Weinberg equilibrium. What's more, association analysis results showed the SNP in gene was significantly associated with T lymphocyte subpopulations. Different genotypes had significant effects on the proportion of CD4CD8, CD4CD8, CD4CD8, CD8, CD4/CD8 in peripheral blood ( < 0.05). These results further suggested that could be recognized as a promising immune gene for swine disease resistance breeding.
Topics: Swine; Animals; Proteolipids; Lymphocyte Subsets; Genomics
PubMed: 36360222
DOI: 10.3390/genes13111985 -
ASN Neuro 2017Alterations in the myelin proteolipid protein gene ( PLP1) may result in rare X-linked disorders in humans such as Pelizaeus-Merzbacher disease and spastic paraplegia... (Review)
Review
Alterations in the myelin proteolipid protein gene ( PLP1) may result in rare X-linked disorders in humans such as Pelizaeus-Merzbacher disease and spastic paraplegia type 2. PLP1 expression must be tightly regulated since null mutations, as well as elevated PLP1 copy number, both lead to disease. Previous studies with Plp1-lacZ transgenic mice have demonstrated that mouse Plp1 ( mPlp1) intron 1 DNA (which accounts for slightly more than half of the gene) is required for the mPlp1 promoter to drive significant levels of reporter gene expression in brain. However not much is known about the mechanisms that control expression of the human PLP1 gene ( hPLP1). Therefore this review will focus on sequences in hPLP1 intron 1 DNA deemed important for hPLP1 gene activity as well as a couple of "human-specific" supplementary exons within the first intron which are utilized to generate novel splice variants, and the potential role that these sequences may play in PLP1-linked disorders.
Topics: Animals; Humans; Introns; Myelin Proteolipid Protein; Nervous System Diseases
PubMed: 28735559
DOI: 10.1177/1759091417720583 -
FEBS Letters Sep 1997Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling... (Review)
Review
Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer.
Topics: ATP Synthetase Complexes; Binding Sites; Models, Molecular; Multienzyme Complexes; Phosphotransferases (Phosphate Group Acceptor); Protein Conformation; Proteolipids; Proton-Translocating ATPases
PubMed: 9323021
DOI: 10.1016/s0014-5793(97)00997-6 -
Scientific Reports Sep 2021Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasures to control MDR Salmonella outbreaks....
Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasures to control MDR Salmonella outbreaks. In this study, antimicrobial activity of cationic α-helical bovine NK-lysin-derived antimicrobial peptides was evaluated against MDR Salmonella outbreak isolates. NK2A and NK2B strongly inhibited MDR Salmonella growth while NK1 and NK2C showed minimum-to-no growth inhibition. Scrambled-NK2A, which is devoid of α-helicity but has the same net positive charge as NK2A, also failed to inhibit bacterial growth. Incubation of negatively charged MDR Salmonella with NK2A showed increased Zeta potential, indicating bacterial-peptide electrostatic attraction. Confocal and transmission electron microscopy studies revealed NK2A-mediated damage to MDR Salmonella membranes. LPS inhibited NK2A-mediated growth suppression in a dose-dependent response, suggesting irreversible NK2A-LPS binding. LPS-NK2A binding and bacterial membrane disruption was also confirmed via electron microscopy using gold nanoparticle-NK2A conjugates. Finally, NK2A-loaded polyanhydride nanoparticles showed sustained peptide delivery and anti-bacterial activity. Together, these findings indicate that NK2A α-helicity and positive charge are prerequisites for antimicrobial activity and that MDR Salmonella killing is mediated by direct interaction of NK2A with LPS and the inner membrane, leading to bacterial membrane permeabilization. With further optimization using nano-carriers, NK2A has the potential to become a potent anti-MDR Salmonella agent.
Topics: Animals; Antimicrobial Peptides; Cattle; Disease Models, Animal; Disease Outbreaks; Drug Evaluation, Preclinical; Drug Resistance, Multiple, Bacterial; Female; Humans; Injections, Intraperitoneal; Mice; Microbial Sensitivity Tests; Proteolipids; Salmonella; Salmonella Infections
PubMed: 34588573
DOI: 10.1038/s41598-021-98860-6 -
STAR Protocols Jun 2022Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water...
Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux. The consequent effects on proteoliposome size can be tracked using the fluorescence of an internalized fluorophore. This enables controlled characterization of water flux by AQPs. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020).
Topics: Aquaporins; Permeability; Proteolipids; Water
PubMed: 35496800
DOI: 10.1016/j.xpro.2022.101312 -
The Biochemical Journal Jan 1991
Review
Topics: Animals; Base Sequence; Bronchoalveolar Lavage Fluid; Gene Expression Regulation; Glycoproteins; Humans; Molecular Sequence Data; Proteolipids; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; RNA, Messenger
PubMed: 1991023
DOI: 10.1042/bj2730249 -
The Journal of Biological Chemistry Nov 1987Two newly described surfactant proteolipids (SPL), Phe and pVal, are produced by proteolytic processing of distinct precursors of Mr = 40,000 and 22,000, respectively....
Two newly described surfactant proteolipids (SPL), Phe and pVal, are produced by proteolytic processing of distinct precursors of Mr = 40,000 and 22,000, respectively. These proteins are structurally related and intimately associated with surfactant phospholipids. We now demonstrate the expression of both SPL(Phe) and SPL(pVal) in explants of human fetal lung from 16-24 weeks of gestation. Content, synthesis, and mRNA for the proteolipids were low prior to organ culture of fetal lung. Induction of synthesis of the proteolipids occurred rapidly in explant culture in the absence of exogenous hormones and was enhanced by addition of dexamethasone. Increased synthesis of the proteolipids was detected by enzyme-linked immunosorbent assay and by [35S]methionine incorporation into the glycosylated Mr = 40,000-43,000 SPL (Phe) precursor. The response to dexamethasone occurred rapidly and contrasted with effects of dexamethasone on the expression of surfactant-associated protein- (SAP) 35, a distinct surfactant glycoprotein. 8-Br-cAMP did not significantly increase proteolipid content but markedly increased synthesis of SAP-35 in identical cultures. Increased proteolipid content was associated with increased mRNA for each protein as determined by the Northern blot analysis. Proteolipid RNA was also increased by 8-Br-cAMP, however, not to the extent observed with the glucocorticoid. Immunohistochemical analysis of fetal lung with anti-proteolipid antiserum confirmed that the dexamethasone-enhanced synthesis of the proteins by Type II epithelial cells. The time and hormone dependence of the regulation of expression of both SPL(Phe) and SPL(pVal) precursors were distinct from that of SAP-35. Expression of the surfactant proteolipids increased during explant culture of human fetal lung and was further enhanced by glucocorticoid. Developmental and hormonal regulation of the surfactant proteolipids may be important factors in surfactant function at birth.
Topics: Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Glucocorticoids; Humans; Immunohistochemistry; Lung; Methionine; Molecular Weight; Peptides; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Protein C; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; RNA
PubMed: 2445738
DOI: No ID Found -
PloS One 2018This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this...
This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this efflux pump in individual liposomes using optical microscopy. Pgp, a member of ABC (ATP-binding cassette) transporter family, is known to contribute to the cellular multidrug resistance (MDR) against variety of drugs. The efficacy of many therapeutics is, thus, hampered by this efflux pump, leading to a high demand for simple and effective strategies to monitor the interactions of candidate drugs with this protein. Here, we applied small Pgp proteoliposomes to prepare giant Pgp-bearing liposomes via modified electroformation techniques. The presence of Pgp in the membrane of giant proteoliposomes was confirmed using immunohistochemistry. Assessment of Pgp ATPase activity suggested that this transporter retained its activity upon reconstitution into giant liposomes, with an ATPase specific activity of 439 ± 103 nmol/mg protein/min. For further confirmation, we assessed the transport activity of Pgp in these proteoliposomes by monitoring the translocation of rhodamine 123 (Rho123) across the membrane using confocal microscopy at various ATP concentrations (0-2 mM) and in the presence of Pgp inhibitors. Rate of change in Rho123 concentration inside the liposomal lumen was used to estimate the Rho123 transport rates (1/s) for various ATP concentrations, which were then applied to retrieve the Michaelis-Menten constant (Km) of ATP in Rho123 transport (0.42 ± 0.75 mM). Similarly, inhibitory effects of verapamil, colchicine, and cyclosporin A on Pgp were studied in this system and the IC50 values for these Pgp inhibitors were found 26.6 ± 6.1 μM, 94.6 ± 47.6 μM, and 0.21 ± 0.07 μM, respectively. We further analyzed the transport data using a kinetic model that enabled dissecting the passive diffusion of Rho123 from its Pgp-mediated transport across the membrane. Based on this model, the permeability coefficient of Rho123 across the liposomal membrane was approximately 1.25×10-7 cm/s. Comparing the membrane permeability in liposomes with and without Pgp revealed that the presence of this protein did not have a significant impact on membrane integrity and permeability. Furthermore, we used this model to obtain transport rate constants for the Pgp-mediated transport of Rho123 (m3/mol/s) at various ATP and inhibitor concentrations, which were then applied to estimate values of 0.53 ± 0.66 mM for Km of ATP and 25.2 ± 5.0 μM for verapamil IC50, 61.8 ± 34.8 μM for colchicine IC50, and 0.23 ± 0.09 μM for cyclosporin A IC50. The kinetic parameters obtained from the two analyses were comparable, suggesting a minimal contribution from the passive Rho123 diffusion across the membrane. This approach may, therefore, be applied for screening the transport activity of Pgp against potential drug candidates.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Biological Transport; Cricetinae; Drug Resistance, Multiple; Proteolipids; Rhodamine 123
PubMed: 29912971
DOI: 10.1371/journal.pone.0199279