-
Biophysical Journal Jul 2018G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports...
G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports have arrived at disparate conclusions regarding the existence, stability, and stoichiometry of GPCR oligomers, partly because of cellular complexity and ensemble averaging of intrareconstitution heterogeneities that complicate the interpretation of oligomerization data. To overcome these limitations, we exploited fluorescence-microscopy-based high-content analysis of single proteoliposomes. This allowed multidimensional quantification of intrinsic monomer-monomer interactions of three class A GPCRs (β-adrenergic receptor, cannabinoid receptor type 1, and opsin). Using a billion-fold less protein than conventional assays, we quantified oligomer stoichiometries, association constants, and the influence of two ligands and membrane curvature on oligomerization, revealing key similarities and differences for three GPCRs with decidedly different physiological functions. The assays introduced here will assist with the quantitative experimental observation of oligomerization for transmembrane proteins in general.
Topics: Ligands; Protein Multimerization; Protein Structure, Quaternary; Proteolipids; Receptors, G-Protein-Coupled; Signal Transduction; Solubility
PubMed: 30021106
DOI: 10.1016/j.bpj.2018.05.036 -
Frontiers in Immunology 2024NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member...
NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of from Atlantic salmon () using EST sequences. In the present study, we reported and characterized two additional transcripts of from . Besides, we evaluated the tissue distribution of three from and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against (LF-89) and . These peptides induced the expression of immune genes related to innate and adaptive immune responses and . The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.
Topics: Animals; Antimicrobial Peptides; Fish Diseases; Fish Proteins; Immunity, Innate; Proteolipids; Salmo salar; Signal Transduction
PubMed: 38655253
DOI: 10.3389/fimmu.2024.1191966 -
Canadian Journal of Veterinary Research... Jan 1990The pathogenesis of the ceroid-lipofuscinoses, inherited storage diseases of children, was studied in an ovine model. This was shown to have clinical and pathological...
The pathogenesis of the ceroid-lipofuscinoses, inherited storage diseases of children, was studied in an ovine model. This was shown to have clinical and pathological features most in common with the late infantile and juvenile human forms of the disease. The ability to study sequential changes allowed the retinal lesions to be described as a dystrophy of photoreceptor outer segments which preceded loss of the photoreceptor cells. An early decrease in amplitude of the c-wave electroretinograph was attributed to a decrease in the transpigment epithelial component. The decreased a- and b-wave amplitudes were attributed to the changes in and loss of, photoreceptor cells. The chemical components of isolated storage cytosomes were analyzed and shown to consist mostly of protein. Sequence analysis of the dominantly stored protein showed that it was identical to the DCCD reactive proteolipid or subunit c of mitochondrial adenosine triphosphate synthase and that it comprised approximately 50% of storage material. Based on the adage that the dominantly stored species should reflect the underlying biochemical anomaly, it was concluded that it was of pathogenic significance. This highly hydrophobic protein tends to extract with lipids in chloroform/methanol and is thus known as a proteolipid. Some of the remainder of the stored proteins also had this characteristic. It was concluded that ovine ceroid-lipofuscinosis was a proteinosis, more specifically a proteolipid proteinosis and as such it forms the prototype of a new class of storage diseases. Recognition of the nature of the dominantly stored chemical species has helped understanding of a variety of chemical and physical characteristics attributed to the whole pigment.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Amino Acid Sequence; Animals; Disease Models, Animal; Humans; Molecular Sequence Data; Neuronal Ceroid-Lipofuscinoses; Proteolipids; Sheep; Sheep Diseases
PubMed: 2306665
DOI: No ID Found -
Fish & Shellfish Immunology Nov 2023The NK-lysin antimicrobial peptide, first identified in mammals, possesses both antibacterial and cytotoxic activity against cancer cell lines. Homologue peptides...
The NK-lysin antimicrobial peptide, first identified in mammals, possesses both antibacterial and cytotoxic activity against cancer cell lines. Homologue peptides isolated from different fish species have been examined for their functional characteristics in the last few years. In this study, a NK-lysin transcript was identified in silico from the head kidney transcriptome of the Antarctic teleost Trematomus bernacchii. The corresponding amino acid sequence, slightly longer than NK-lysins of other fish species, contains six cysteine residues that in mammalian counterparts form three disulphide bridges. Real time-PCR analysis indicated its predominant expression in T. bernacchii immune-related organs and tissues, with greatest mRNA abundance detected in gills and spleen. Instead of focusing on the full T. bernacchii derived NK-lysin mature molecule, we selected a 27 amino acid residue peptide (named NKL-WT), corresponding to the potent antibiotic NK-2 sequence found in human NK-lysin. Moreover, we designed a mutant peptide (named NKL-MUT) in which two alanine residues substitute the two cysteines found in the NKL-WT. The two peptides were obtained by solid phase organic synthesis to investigate their functional features. NKL-WT and NKL-MUT displayed antibacterial activity against the human pathogenic bacterium Enterococcus faecalis and the ESKAPE pathogen Acinetobacter baumannii, respectively. Moreover, at the determined Minimum Inhibitory Concentration and Minimum Bactericidal Concentration values against these pathogens, both peptides showed high selectivity as they did not exhibit any haemolytic activity on erythrocytes or cytotoxic activity against mammalian primary cell lines. Finally, the NKL-MUT selectively triggers the killing of the melanoma cell line B16F10 by means of a pro-apoptotic pathway at a concentration range in which no effects were found in normal mammalian cell lines. In conclusion, the two peptides could be considered as promising candidates in the fight against antibiotic resistance and tumour proliferative action, and also be used as innovative adjuvants, either to decrease chemotherapy side effects or to enhance anticancer drug activity.
Topics: Humans; Animals; Antarctic Regions; Fish Proteins; Peptides; Anti-Bacterial Agents; Perciformes; Proteolipids; Fishes; Mammals
PubMed: 37734650
DOI: 10.1016/j.fsi.2023.109099 -
The Journal of Biological Chemistry Jun 2010Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1...
Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1 receptor-associated kinases IRAK1 and IRAK4. We have found that both IRAK1 and IRAK4 can directly phosphorylate Mal. In addition, co-expression of Mal with either IRAK resulted in depletion of Mal from cell lysates. This is likely to be due to Mal phosphorylation by the IRAKs because kinase-inactive forms of either IRAK had no effect. Furthermore, lipopolysaccharide stimulation resulted in ubiquitination and degradation of Mal, which was inhibited using an IRAK1/4 inhibitor or by knocking down expression of IRAK1 and IRAK4. MyD88 is not a substrate for either IRAK and did not undergo degradation. We therefore conclude that Mal is a substrate for IRAK1 and IRAK4 with phosphorylation promoting ubiquitination and degradation of Mal. This process may serve to negatively regulate signaling by TLR2 and TLR4.
Topics: Cell Line; Gene Expression Regulation; Humans; Interleukin-1 Receptor-Associated Kinases; Lipopolysaccharides; Mass Spectrometry; Membrane Transport Proteins; Models, Biological; Myelin Proteins; Myelin and Lymphocyte-Associated Proteolipid Proteins; Myeloid Differentiation Factor 88; Phosphorylation; Proteolipids; Signal Transduction; Toll-Like Receptor 2; Toll-Like Receptor 4; Ubiquitin
PubMed: 20400509
DOI: 10.1074/jbc.M109.098137 -
Scientific Reports Mar 2017Chicken NK-lysin (cNK-lysin), the chicken homologue of human granulysin, is a cationic amphiphilic antimicrobial peptide (AMP) that is produced by cytotoxic T cells and...
Chicken NK-lysin (cNK-lysin), the chicken homologue of human granulysin, is a cationic amphiphilic antimicrobial peptide (AMP) that is produced by cytotoxic T cells and natural killer cells. We previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core α-helical region of cNK-lysin, have antimicrobial activity against apicomplexan parasites such as Eimeria spp., via membrane disruption. In addition to the antimicrobial activity of AMPs, the immunomodulatory activity of AMPs mediated by their interactions with host cells is increasingly recognized. Thus, in this study, we investigated whether cNK-lysin derived peptides modulate the immune response in the chicken macrophage cell line HD11 and in chicken primary monocytes by evaluating the induction of chemokines, anti-inflammatory properties, and activation of signalling pathways. cNK-2 induced the expression of CCL4, CCL5 and interleukin(IL)-1β in HD11 cells and CCL4 and CCL5 in primary monocytes. We also determined that cNK-2 suppresses the lipopolysaccharide-induced inflammatory response by abrogating IL-1β expression. The immunomodulatory activity of cNK-2 involves the mitogen-activated protein kinases-mediated signalling pathway, including p38, extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinases, as well as the internalization of cNK-2 into the cells. These results indicate that cNK-2 is a potential novel immunomodulating agent rather than an antimicrobial agent.
Topics: Animals; Anti-Bacterial Agents; Cell Survival; Chemokines; Chickens; Cytokines; Humans; Immunomodulation; Inflammation Mediators; MAP Kinase Signaling System; Peptides; Proteolipids; Signal Transduction
PubMed: 28332637
DOI: 10.1038/srep45099 -
Swiss Medical Weekly Feb 2001Human surfactant protein A (SP-A) exhibits extensive complexity at several levels: genetic, transcript (splicing), protein, and composition and size of protein... (Review)
Review
Human surfactant protein A (SP-A) exhibits extensive complexity at several levels: genetic, transcript (splicing), protein, and composition and size of protein oligomers. Its multiple and important roles in innate host defense, regulation of inflammation, and in aspects of pulmonary surfactant may have necessitated such a complexity from an evolutionary point of view. Moreover, understanding of such a complexity may be useful in the study of disease pathogenesis and the development of disease diagnostics and/or therapeutics.
Topics: Alleles; Humans; Lung Diseases; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Sensitivity and Specificity
PubMed: 11416882
DOI: 10.4414/smw.2001.06136 -
The Journal of Biological Chemistry Dec 2019Posttranslational modifications of proteins, such as phosphorylation and dephosphorylation, play critical roles in cellular functions through diverse cell signaling...
Posttranslational modifications of proteins, such as phosphorylation and dephosphorylation, play critical roles in cellular functions through diverse cell signaling pathways. Protein kinases and phosphatases have been described early on as key regulatory elements of the phosphorylated state of proteins. Tight spatial and temporal regulation of protein kinase and phosphatase activities has to be achieved in the cell to ensure accurate signal transduction. We demonstrated previously that phosphorylation of a membrane protein can lead to its topological rearrangement. Additionally, we found that both the rate and extent of topological rearrangement upon phosphorylation are lipid charge- and lipid environment-dependent. Here, using a model membrane protein (the bacterial lactose permease LacY reconstituted in proteoliposomes) and a combination of real-time measurements and steady-state assessments of protein topology, we established a set of experimental conditions to dissect the effects of phosphorylation and dephosphorylation of a membrane protein on its topological orientation. We also demonstrate that the phosphorylation-induced topological switch of a membrane protein can be reversed upon protein dephosphorylation, revealing a new regulatory role for phosphorylation/dephosphorylation cycles. Furthermore, we determined that the rate of topological rearrangement reversal is correlated with phosphatase activity and is influenced by the membrane's lipid composition, presenting new insights into the spatiotemporal control of the protein phosphorylation state. Together, our results highlight the importance of the compartmentalization of phosphorylation/dephosphorylation cycles in controlling membrane protein topology and, therefore, function, which are influenced by the local lipid environment of the membrane protein.
Topics: Membrane Lipids; Phospholipids; Protein Folding; Protein Processing, Post-Translational; Proteolipids
PubMed: 31645436
DOI: 10.1074/jbc.RA119.010785 -
The Journal of Biological Chemistry Apr 1994In this study, we show that two biochemical markers of neuronal ceroid lipofuscinoses (NCLs) are present in a mutant mouse (mnd/mnd) that exhibits symptoms of the... (Comparative Study)
Comparative Study
In this study, we show that two biochemical markers of neuronal ceroid lipofuscinoses (NCLs) are present in a mutant mouse (mnd/mnd) that exhibits symptoms of the disease. Subunit c of the mitochondrial F1F0-ATP synthase, a proteolipid that accumulates in storage bodies of most forms of NCL and several animal models, is dramatically increased in mnd/mnd mouse brain, kidney, liver, heart, and pancreas. Interestingly, another related proteolipid, subunit c of the vacuolar H(+)-ATPase, also accumulates in several mnd/mnd tissues. The molar ratio of the vacuolar subunit c to the F1F0 subunit c is approximately one to two in enriched storage bodies from brain. The relative accumulation of the vacuolar subunit c correlates with its abundance in normal tissues. It appears in decreasing amounts in brain, kidney, and liver and is not detected in heart or pancreas. Aged mice and two mutant mouse lines, juvenile bare (jb) and mucopolysaccharidosis, type VII (gusmps), did not accumulate either of these proteolipids. Dolichol-linked oligosaccharides also accumulate in NCLs and are increased 17-fold in mnd/mnd mouse brain. Thus, mnd/mnd mice seem to be an excellent model for NCLs since they not only share clinical signs and histopathology, but also two biochemical markers. The accumulation of the vacuolar subunit c in this model may prove to be a marker for distinguishing different forms of NCLs.
Topics: Amino Acid Sequence; Animals; Brain; Carbohydrate Sequence; Cattle; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Humans; Kidney; Liver; Macromolecular Substances; Mice; Mice, Neurologic Mutants; Molecular Sequence Data; Motor Neurons; Mucopolysaccharidosis VII; Myocardium; Neuronal Ceroid-Lipofuscinoses; Organ Specificity; Organelles; Pancreas; Polyisoprenyl Phosphate Oligosaccharides; Proteolipids; Proton-Translocating ATPases; Sequence Homology, Amino Acid; Sheep; Vacuoles
PubMed: 8144516
DOI: No ID Found -
Analytical Chemistry Jan 2016Investigation of intramembranal protease catalysis demands the generation of intact biomembrane assemblies with structural integrity and lateral mobility. Here, we...
Investigation of intramembranal protease catalysis demands the generation of intact biomembrane assemblies with structural integrity and lateral mobility. Here, we report the development of a microsphere supported-biomembrane platform enabling characterization of γ-secretase and substrate within proteolipobead assemblies via microscopy and flow cytometry. The active enzyme loading levels were tracked using an activity-based probe, with the biomembranes delineated by carbocyanine lipid reporters. Proteolipobeads formed from HeLa proteoliposomes gave rise to homogeneous distributions of active γ-secretase within supported biomembranes with native-like fluidity. The substrate loading into supported biomembranes was detergent-dependent, as evidenced by even colocalization of substrate and lipid tracers in confocal 3D imaging of individual proteolipobeads. Moreover, the loading level was tunable with bulk substrate concentration. γ-Secretase substrate cleavage and its inhibition within γ-secretase proteolipobeads were observed. This platform offers a means to visualize enzyme and substrate loading, activity, and inhibition in a controllable biomembrane microenvironment.
Topics: Amyloid Precursor Protein Secretases; Detergents; Enzyme Activation; HeLa Cells; Humans; Lipid Bilayers; Models, Molecular; Molecular Probes; Molecular Structure; Proteolipids; Substrate Specificity
PubMed: 26699370
DOI: 10.1021/acs.analchem.5b03762