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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 -
Journal of Autoimmunity Nov 2023Sensitization to self-peptides induces various immunological responses, from autoimmunity to tumor immunity, depending on the peptide sequence; however, the underlying...
Harnessing autoimmunity with dominant self-peptide: Modulating the sustainability of tissue-preferential antigen-specific Tregs by governing the binding stability via peptide flanking residues.
Sensitization to self-peptides induces various immunological responses, from autoimmunity to tumor immunity, depending on the peptide sequence; however, the underlying mechanisms remain unclear, and thus, curative therapeutic options considering immunity balance are limited. Herein, two overlapping dominant peptides of myelin proteolipid protein, PLP136-150 and PLP139-151, which induce different forms of experimental autoimmune encephalomyelitis (EAE), monophasic and relapsing EAE, respectively, were investigated. Mice with monophasic EAE exhibited highly resistant to EAE re-induction with any encephalitogenic peptides, whereas mice with relapsing EAE were susceptible, and progressed, to EAE re-induction. This resistance to relapse and re-induction in monophasic EAE mice was associated with the maintenance of potent CD69CD103CD4CD25 regulatory T-cells (Tregs) enriched with antigen specificity, which expanded preferentially in the central nervous system with sustained suppressive activity. This tissue-preferential sustainability of potent antigen-specific Tregs was correlated with the antigenicity of PLP136-150, depending on its flanking residues. That is, the flanking residues of PLP136-150 enable to form pivotally arranged strong hydrogen bonds that secured its binding stability to MHC-class II. These potent Tregs acting tissue-preferentially were induced only by sensitization of PLP136-150, not by its tolerance induction, independent of EAE development. These findings suggest that, for optimal therapy, "benign autoimmunity" can be critically achieved through inverse vaccination with self-peptides by manipulating their flanking residues.
Topics: Animals; T-Lymphocytes, Regulatory; Encephalomyelitis, Autoimmune, Experimental; Mice; Myelin Proteolipid Protein; Autoimmunity; Autoantigens; Female; Peptide Fragments; Protein Binding; Peptides; Disease Models, Animal; Mice, Inbred C57BL
PubMed: 37716077
DOI: 10.1016/j.jaut.2023.103094 -
Frontiers in Bioscience (Landmark... Aug 2023Plasmolipin (PLLP) is a membrane protein located in lipid rafts that participates in the formation of myelin. It is also implicated in many pathologies, such as...
BACKGROUND
Plasmolipin (PLLP) is a membrane protein located in lipid rafts that participates in the formation of myelin. It is also implicated in many pathologies, such as neurological disorders, type 2 diabetes, and cancer metastasis. To better understand how PLLP interacts with raft components (gangliosides and cholesterol), we undertook a global study combining simulations and physicochemical measurements of molecular interactions in various PLLP-ganglioside systems.
METHODS
studies consisted of molecular dynamics simulations in reconstructed membrane environments. PLLP-ganglioside interaction measurements were performed by microtensiometry at the water-air interface on ganglioside monolayers.
RESULTS
We have elucidated the mode of interaction of PLLP with ganglioside GM1 and characterized this interaction at the molecular level. We showed that GM1 induces the structuring of the extracellular loops of PLLP and that this interaction propagates a conformational signal through the plasma membrane, involving a cholesterol molecule located between transmembrane domains. This conformational wave is finally transmitted to the intracellular domain of the protein, consistent with the role of PLLP in signal transduction.
CONCLUSIONS
This study is a typical example of the epigenetic dimension of protein structure, a concept developed by our team to describe the chaperone effect of gangliosides on disordered protein motifs which associate with lipid rafts. From a physiological point of view, these data shed light on the role of gangliosides in myelin formation. From a pathological point of view, this study will help to design innovative therapeutic strategies focused on ganglioside-PLLP interactions in various PLLP-associated diseases.
Topics: Humans; G(M1) Ganglioside; Gangliosides; Membrane Microdomains; Myelin Sheath; Proteolipids; Myelin and Lymphocyte-Associated Proteolipid Proteins
PubMed: 37664934
DOI: 10.31083/j.fbl2808157 -
PeerJ 2023Multiple sclerosis (MS) is a chronic inflammatory neurologic disease characterized by the demyelinating injury of the central nervous system (CNS). It was reported that...
Multiple sclerosis (MS) is a chronic inflammatory neurologic disease characterized by the demyelinating injury of the central nervous system (CNS). It was reported that the mutant peptide came from myelin proteolipid protein (PLP) and myelin basic protein (MBP) might play a critical role in immunotherapy function of MS. However, endogenous peptides in demyelinating brain tissue of MS and their role in the pathologic process of MS have not been revealed. Here, we performed peptidomic analysis of freshly isolated callosum (CC) from the brains of CPZ-treated mice and normal diet controls of male C57BL/6 mice by LC-MS/MS. Identified a total of 217 peptides were expressed at different levels in MS mice model compared with controls. By performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, we found that the precursor protein of these differently expressed peptides (DEPs) were associated with myelin sheath and oxidative phosphorylation. Our study is the first brain peptidomic of MS mice model, revealing the distinct features of DEPs in demyelination brain tissue. These DPEs may provide further insight into the pathogenesis and complexity of MS, which would facilitate the discovery of the potential novel and effective strategy for the treatment of MS.
Topics: Male; Animals; Mice; Mice, Inbred C57BL; Chromatography, Liquid; Tandem Mass Spectrometry; Central Nervous System; Multiple Sclerosis; Disease Models, Animal; Peptides
PubMed: 37637167
DOI: 10.7717/peerj.15846 -
Clinical Case Reports Sep 2023This study aimed to characterize the clinical features, developmental milestones, and the natural history of Pelizaeus-Merzbacher disease (PMD) associated with gene...
This study aimed to characterize the clinical features, developmental milestones, and the natural history of Pelizaeus-Merzbacher disease (PMD) associated with gene duplications. The study examined 16 PMD Patients ranging in age from 7 to 48 years, who had a documented gene duplication. The study examined and analyzed the medical and developmental histories of the subjects utilizing a combination of resources that included medical history questionnaires, medical record reviews, and a 31-point functional disability scale that had been previously validated. The data extracted from the medical records and questionnaires for analysis included information related to medical and developmental histories, level of ambulation and cognition, and degree of functional disability. The summation of findings among the study population demonstrated that the presenting symptoms, developmental milestones achieved, and progression of symptoms reported are consistent with many previous studies of patients with duplications. All patients exhibited onset within the first year of life, with nystagmus predominating as the first symptom noticed. All patients exhibited delays in both motor and language development; however, many individuals were able to meet several developmental milestones. They exhibited some degree of continued motor impairment with none having the ability to walk independently. All patients were able to complete at least some of the cognition achievements and although not all were verbal, a number were able to use communication devices to complete these tasks. A critical tool of the study was the functional disability scale which provided a major advantage in helping quantify the clinical course of PMD, and for several, we were able to gather this information at more than one point in time. These reported findings in our cohort contribute important insight into the clinical heterogeneity and potential underlying mechanisms that define the molecular pathogenesis of the disease. This is one of only a small number of natural history studies examining the clinical course of a cohort of patients with duplications within the context of a validated functional disability scoring system. This study is unique in that it is limited to subjects with gene duplications. This study demonstrated many commonalities to other studies that have characterized the features of PMD and other PLP1-related disorders but also provide significant new insights into the evolving story that marks the natural history.
PubMed: 37636890
DOI: 10.1002/ccr3.7814 -
Nutrients Aug 2023Human genetic studies have associated Neuronatin gene variants with anorexia nervosa (AN) and obesity. Studies on the expression of the Neuronatin gene product, a...
Human genetic studies have associated Neuronatin gene variants with anorexia nervosa (AN) and obesity. Studies on the expression of the Neuronatin gene product, a proteolipid, are lacking. We investigated the relationship between circulating Neuronatin, body mass index (BMI), body composition (BC), physical activity (PA), and psychometric outcomes in patients with AN, normal weight, and obesity. Plasma Neuronatin was measured by ELISA in (1) 79 subjects of five BMI categories (AN/BMI < 17.5 kg/m; normal weight/BMI 18.5-25 kg/m; obesity/BMI 30-40 kg/m; obesity/BMI 40-50 kg/m; obesity/BMI > 50 kg/m) with assessment of BC (bioimpedance analysis; BIA); (2) 49 women with AN (BMI 14.5 ± 1.8 kg/m) with measurements of BC (BIA) and PA (accelerometry); (3) 79 women with obesity (BMI 48.8 ± 7.8 kg/m) with measurements of anxiety (GAD-7), stress (PSQ-20), depression (PHQ-9) and eating behavior (EDI-2). Overall, a positive correlation was found between Neuronatin and BMI ( = 0.006) as well as total fat mass (FM; = 0.036). In AN, Neuronatin did not correlate with BMI, FM, or PA ( > 0.05); no correlations were found between Neuronatin and psychometric outcomes in obesity ( > 0.05). The findings suggest an FM-dependent peripheral Neuronatin expression. The decreased Neuronatin expression in AN provides evidence that Neuronatin is implicated in the pathogenesis of eating disorders.
Topics: Humans; Female; Body Mass Index; Accelerometry; Anorexia Nervosa; Obesity; Adipose Tissue
PubMed: 37630847
DOI: 10.3390/nu15163657 -
International Journal of Molecular... Aug 2023Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain-Barré syndrome. On the other hand, the...
Anti-Glycolipid Antibody Examination in Five EAE Models and Theiler's Virus Model of Multiple Sclerosis: Detection of Anti-GM1, GM3, GM4, and Sulfatide Antibodies in Relapsing-Remitting EAE.
Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain-Barré syndrome. On the other hand, the role in multiple sclerosis (MS), inflammatory demyelinating disease in the central nervous system (CNS), is largely unknown, although the presence of anti-glycolipid antibodies was reported to differ among MS patients with relapsing-remitting (RR), primary progressive (PP), and secondary progressive (SP) disease courses. We investigated whether the induction of anti-glycolipid antibodies could differ among experimental MS models with distinct clinical courses, depending on induction methods. Using three mouse strains, SJL/J, C57BL/6, and A.SW mice, we induced five distinct experimental autoimmune encephalomyelitis (EAE) models with myelin oligodendrocyte glycoprotein (MOG), MOG, or myelin proteolipid protein (PLP), with or without an additional adjuvant curdlan injection. We also induced a viral model of MS, using Theiler's murine encephalomyelitis virus (TMEV). Each MS model had an RR, SP, PP, hyperacute, or chronic clinical course. Using the sera from the MS models, we quantified antibodies against 11 glycolipids: GM1, GM2, GM3, GM4, GD3, galactocerebroside, GD1a, GD1b, GT1b, GQ1b, and sulfatide. Among the MS models, we detected significant increases in four anti-glycolipid antibodies, GM1, GM3, GM4, and sulfatide, in PLP-induced EAE with an RR disease course. We also tested cellular immune responses to the glycolipids and found CD1d-independent lymphoproliferative responses only to sulfatide with decreased interleukin (IL)-10 production. Although these results implied that anti-glycolipid antibodies might play a role in remissions or relapses in RR-EAE, their functional roles need to be determined by mechanistic experiments, such as injections of monoclonal anti-glycolipid antibodies.
Topics: Animals; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Encephalomyelitis, Autoimmune, Experimental; Theilovirus; Sulfoglycosphingolipids; Neoplasm Recurrence, Local; Antibodies; Myelin-Oligodendrocyte Glycoprotein; Glycolipids
PubMed: 37629117
DOI: 10.3390/ijms241612937 -
International Journal of Molecular... Aug 2023The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying...
The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood-brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice.
Topics: Animals; Mice; Cuprizone; Blood-Brain Barrier; Diffusion Tensor Imaging; Evans Blue; Gadolinium; Antibodies; Immunoconjugates; Coloring Agents; Demyelinating Diseases
PubMed: 37628867
DOI: 10.3390/ijms241612688 -
The Journal of Biological Chemistry Sep 2023Termination codon readthrough (TCR) is a process in which ribosomes continue to translate an mRNA beyond a stop codon generating a C-terminally extended protein isoform....
Termination codon readthrough (TCR) is a process in which ribosomes continue to translate an mRNA beyond a stop codon generating a C-terminally extended protein isoform. Here, we demonstrate TCR in mammalian NNAT mRNA, which encodes NNAT, a proteolipid important for neuronal differentiation. This is a programmed event driven by cis-acting RNA sequences present immediately upstream and downstream of the canonical stop codon and is negatively regulated by NONO, an RNA-binding protein known to promote neuronal differentiation. Unlike the canonical isoform NNAT, we determined that the TCR product (NNATx) does not show detectable interaction with the sarco/endoplasmic reticulum Ca-ATPase isoform 2 Ca pump, cannot increase cytoplasmic Ca levels, and therefore does not enhance neuronal differentiation in Neuro-2a cells. Additionally, an antisense oligonucleotide that targets a region downstream of the canonical stop codon reduced TCR of NNAT and enhanced the differentiation of Neuro-2a cells to cholinergic neurons. Furthermore, NNATx-deficient Neuro-2a cells, generated using CRISPR-Cas9, showed increased cytoplasmic Ca levels and enhanced neuronal differentiation. Overall, these results demonstrate regulation of neuronal differentiation by TCR of NNAT. Importantly, this process can be modulated using a synthetic antisense oligonucleotide.
Topics: Animals; Calcium; Cell Differentiation; Codon, Terminator; Mammals; Oligonucleotides, Antisense; Protein Biosynthesis; Receptors, Antigen, T-Cell; RNA, Messenger; Neurons
PubMed: 37611826
DOI: 10.1016/j.jbc.2023.105184 -
The Journal of Clinical Investigation Oct 2023B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific...
B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific recombinant monoclonal IgG1 Abs (rAbs) derived from MS patient CSF plasmablasts bound to conformational proteolipid protein 1 (PLP1) membrane complexes and, when injected into mouse brain with human complement, recapitulated histologic features of MS pathology: oligodendrocyte cell loss, complement deposition, and CD68+ phagocyte infiltration. Conformational PLP1 membrane epitopes were complex and governed by the local cholesterol and glycolipid microenvironment. Abs against conformational PLP1 membrane complexes targeted multiple surface epitopes, were enriched within the CSF compartment, and were detected in most MS patients, but not in inflammatory and noninflammatory neurologic controls. CSF PLP1 complex Abs provide a pathogenic autoantibody biomarker specific for MS.
Topics: Mice; Animals; Humans; Multiple Sclerosis; Myelin Sheath; Immunoglobulin G; Epitopes; Proteolipids
PubMed: 37561592
DOI: 10.1172/JCI162731