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PLoS Genetics Jun 2021Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II...
Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II cells in mice and hitherto unknown function. In type II pneumocytes, LAMP3 is localized in lamellar bodies, secretory organelles releasing pulmonary surfactant into the extracellular space to lower surface tension at the air/liquid interface. The physiological function of LAMP3, however, remains enigmatic. We generated Lamp3 knockout mice by CRISPR/Cas9. LAMP3 deficient mice are viable with an average life span and display regular lung function under basal conditions. The levels of a major hydrophobic protein component of pulmonary surfactant, SP-C, are strongly increased in the lung of Lamp3 knockout mice, and the lipid composition of the bronchoalveolar lavage shows mild but significant changes, resulting in alterations in surfactant functionality. In ovalbumin-induced experimental allergic asthma, the changes in lipid composition are aggravated, and LAMP3-deficient mice exert an increased airway resistance. Our data suggest a critical role of LAMP3 in the regulation of pulmonary surfactant homeostasis and normal lung function.
Topics: Airway Resistance; Alveolar Epithelial Cells; Animals; Asthma; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Female; Gene Editing; Gene Expression Regulation; Homeostasis; Lipidomics; Lung; Lysosomal-Associated Membrane Protein 3; Mice; Mice, Knockout; Ovalbumin; Protein Isoforms; Pulmonary Alveoli; Pulmonary Surfactant-Associated Protein C; Pulmonary Surfactants; Respiratory Function Tests; Signal Transduction
PubMed: 34161347
DOI: 10.1371/journal.pgen.1009619 -
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.... 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 -
American Journal of Respiratory Cell... Apr 2023Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial lung disease. A barrier to developing more effective therapies for IPF is the dearth...
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial lung disease. A barrier to developing more effective therapies for IPF is the dearth of preclinical models that recapitulate the early pathobiology of this disease. Intratracheal bleomycin, the conventional preclinical murine model of IPF, fails to reproduce the intrinsic dysfunction to the alveolar epithelial type 2 cell (AEC2) that is believed to be a proximal event in the pathogenesis of IPF. Murine fibrosis models based on (Surfactant Protein C gene) mutations identified in patients with interstitial lung disease cause activation of the AEC2 unfolded protein response and endoplasmic reticulum stress-an AEC2 dysfunction phenotype observed in IPF. Although these models achieve spontaneous fibrosis, they do so with precedent lung injury and thus are challenged to phenocopy the general clinical course of patients with IPF-gradual progressive fibrosis and loss of lung function. Here, we report a refinement of a murine mutation model to recapitulate the clinical course, physiological impairment, parenchymal cellular composition, and biomarkers associated with IPF. This platform provides the field with an innovative model to understand IPF pathogenesis and index preclinical therapeutic candidates.
Topics: Animals; Mice; Alveolar Epithelial Cells; Disease Progression; Idiopathic Pulmonary Fibrosis; Lung; Mutation; Pulmonary Surfactant-Associated Protein C
PubMed: 36473455
DOI: 10.1165/rcmb.2022-0203MA -
Developmental and Comparative Immunology Mar 2021Nk-lysin (Nkl), an antimicrobial peptide (AMP) product of natural killer cells and cytotoxic T cells in mammals, has recently been characterized in a number of finfish...
Nk-lysin (Nkl), an antimicrobial peptide (AMP) product of natural killer cells and cytotoxic T cells in mammals, has recently been characterized in a number of finfish species. In this study, we identified six genes with sequence homology to Nkl and characterized their patterns of mRNA expression and abundances in rainbow trout (Oncorhynchus mykiss). The cDNA sequences for the six Nkls encoded precursor peptides of 128-133 aa in length, and mature peptides of 109-111 aa in length. Genomic DNA of the nkl1-4 genes consisted of five exons and four introns, whereas the nkl-like a & b genes consisted of four exons and three introns. Chromosomal locations of these peptides show that nkl1 was located on chromosome arm 25q, whereas the other five nkl genes were clustered on chromosome arm 19q. Phylogenetic analysis revealed a conserved structure of Nkls among the teleosts and further protein sequence analyses suggests that all six nkl genes fall within the Nkl sub-family of the Saposin family of proteins. Patterns of tissue-specific mRNA expression were asymmetric among the six trout Nkl homologues, with nkl1, nkl3, and nkl-like a & b occurring in immune competent organs such as spleen, gill, intestine and kidney, as well as pineal gland, brain and oocytes. However, nkl2 and nkl4, showed primary abundances in brain, pineal gland and oocyte tissues. Using mRNA sequencing, in whole-body pools of juvenile trout fry (1 g bw) exposed to Flavobacterium psychrophilum infection, we observed modest up-regulation (2-3 fold) of five (nkl 2-4 and nkl-like a & b) of the six nkl mRNAs over the five-day post-challenge time-course. However, no upregulation could be recorded in spleen tissue measured by qPCR in juvenile trout (270 g bw). Using mRNA sequencing again, mRNA abundances were determined in gill of juvenile trout (~57.7 g bw) exposed to various aquaculture stressors. The results indicated that all six nkls (nkl1-4 and nkl-like a and nkl-like b) were downregulated when exposed to high temperature, and that nkl1 was significantly downregulated following salinity challenge. Overall, these newly characterized AMPs may contribute to host innate immunity as they are modulated following pathogen challenge and by physiological stressors.
Topics: Amino Acid Sequence; Animals; Antimicrobial Peptides; Aquaculture; Chromosome Mapping; Fish Proteins; Flavobacterium; Gene Expression; Gills; Immunity, Innate; Oncorhynchus mykiss; Phylogeny; Proteolipids; RNA, Messenger; Sequence Alignment; Spleen; Stress, Physiological; Tissue Distribution
PubMed: 33301795
DOI: 10.1016/j.dci.2020.103961 -
Cellular and Molecular Life Sciences :... Jul 2022The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin...
The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.
Topics: Axons; Central Nervous System; Humans; Lipid Bilayers; Myelin Proteolipid Protein; Myelin Sheath; Protein Isoforms
PubMed: 35829923
DOI: 10.1007/s00018-022-04428-6 -
Frontiers in Cellular and Infection... 2020Outer membrane vesicles (OMVs) are nanoscale proteoliposomes secreted from the cell envelope of all Gram-negative bacteria. Originally considered as an artifact of the... (Review)
Review
Outer membrane vesicles (OMVs) are nanoscale proteoliposomes secreted from the cell envelope of all Gram-negative bacteria. Originally considered as an artifact of the cell wall, OMVs are now recognized as a general secretion system, which serves to improve the fitness of bacteria and facilitate bacterial interactions in polymicrobial communities as well as interactions between the microbe and the host. In general, OMVs are released in increased amounts from pathogenic bacteria and have been found to harbor much of the contents of the parental bacterium. They mainly encompass components of the outer membrane and the periplasm including various virulence factors such as toxins, adhesins, and immunomodulatory molecules. Numerous studies have clearly shown that the delivery of toxins and other virulence factors via OMVs essentially influences their interactions with host cells. Here, we review the OMV-mediated intracellular deployment of toxins and other virulence factors with a special focus on intestinal pathogenic . Especially, OMVs ubiquitously produced and secreted by enterohemorrhagic (EHEC) appear as a highly advanced mechanism for secretion and simultaneous, coordinated and direct delivery of bacterial virulence factors into host cells. OMV-associated virulence factors are not only stabilized by the association with OMVs, but can also often target previously unknown target structures and perform novel activities. The toxins are released by OMVs in their active forms and are transported via cell sorting processes to their specific cell compartments, where they can develop their detrimental effects. OMVs can be considered as bacterial "long distance weapons" that attack host tissues and help bacterial pathogens to establish the colonization of their biological niche(s), impair host cell function, and modulate the defense of the host. Thus, OMVs contribute significantly to the virulence of the pathogenic bacteria.
Topics: Animals; Bacterial Outer Membrane; Bacterial Secretion Systems; Bacterial Toxins; Enterohemorrhagic Escherichia coli; Enterotoxigenic Escherichia coli; Enterotoxins; Escherichia coli Infections; Humans; Intestines; Protein Transport; Proteolipids; Stress, Physiological; Virulence; Virulence Factors
PubMed: 32211344
DOI: 10.3389/fcimb.2020.00091 -
International Journal of Molecular... Aug 2021The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from... (Review)
Review
The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the "regulins". Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are newly discovered tissue-specific regulators of SERCA. Herein, we compare the functional properties of the regulin family of SERCA transmembrane peptide subunits and consider their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. We present new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insights into how differential binding to the so-called inhibitory groove of SERCA-formed by transmembrane helices M2, M6, and M9-can result in distinct functional outcomes.
Topics: Animals; Calcium; Calcium-Binding Proteins; Humans; Models, Molecular; Muscle Proteins; Proteolipids; Sarcoplasmic Reticulum Calcium-Transporting ATPases
PubMed: 34445594
DOI: 10.3390/ijms22168891 -
NeuroImage Jan 2023To investigate the association of ihMT (inhom signals with the demyelination and remyelination phases of the acute cuprizone mouse model in comparison with histology,...
BACKGROUND
To investigate the association of ihMT (inhom signals with the demyelination and remyelination phases of the acute cuprizone mouse model in comparison with histology, and to assess the extent of tissue damage and repair from MRI data.
METHODS
Acute demyelination by feeding 0.2% cuprizone for five weeks, followed by a four-week remyelination period was applied on genetically modified plp-GFP mice. Animals were scanned at different time points of the demyelination and remyelination phases of the cuprizone model using a multimodal MRI protocol, including ihMT T-filters, MPF (Macromolecular Proton Fraction) and R (longitudinal relaxation rate). For histology, plp-GFP (proteolipid protein - Green Fluorescent Protein) microscopy and LFB (Luxol Fast Blue) staining were employed as references for the myelin content. Comparison of MRI with histology was performed in the medial corpus callosum (mCC) and cerebral cortex (CTX) at two brain levels whereas ROI-wise and voxel-based analyses of the MRI metrics allowed investigating in vivo the spatial extent of myelin alterations.
RESULTS
IhMT high-pass T-filters, targeted toward long T components, showed significant temporal variations in the mCC consistent with the effects induced by the cuprizone toxin. In addition, the corresponding signals correlated strongly and significantly with the myelin content assessed by GFP fluorescence and LFB staining over the demyelination and the remyelination phases. The signal of the band-pass T-filter, which isolates short T components, showed changes over time that were poorly correlated with histology, hence suggesting a sensitivity to pathological processes possibly not related to myelin. Although MPF was also highly correlated to histology, ihMT high-pass T-filters showed better capability to characterize the spatial-temporal patterns during the demyelination and remyelination phases of the acute cuprizone model (e.g., rostro-caudal gradient of demyelination in the mCC previously described in the literature).
CONCLUSIONS
IhMT sequences selective for long T components are specific and sensitive in vivo markers of demyelination and remyelination and have successfully captured the spatially heterogeneous pattern of the demyelination and remyelination mechanisms in the cuprizone model. Interestingly, differences in signal variations between the ihMT high-pass and band-pass T-filter, suggest a sensitivity of the ihMT sequences targeted to short Ts to alterations other than those of myelin. Future studies will need to further address these differences by examining more closely the origin of the short T components and the variation of each T component in pathology.
Topics: Animals; Mice; Cuprizone; Demyelinating Diseases; Remyelination; Magnetic Resonance Imaging; Myelin Sheath; Mice, Inbred C57BL; Disease Models, Animal
PubMed: 36464096
DOI: 10.1016/j.neuroimage.2022.119785 -
Advances in Cancer Research 2022Disruption of the native membrane organization of Ras by the farnesyltransferase inhibitor tipifarnib in the late 1990s constituted the first indirect approach to drug... (Review)
Review
Disruption of the native membrane organization of Ras by the farnesyltransferase inhibitor tipifarnib in the late 1990s constituted the first indirect approach to drug target Ras. Since then, our understanding of how dynamically Ras shuttles between subcellular locations has changed significantly. Ras proteins have to arrive at the plasma membrane for efficient MAPK-signal propagation. On the plasma membrane Ras proteins are organized into isoform specific proteo-lipid assemblies called nanocluster. Recent evidence suggests that Ras nanocluster have a specific lipid composition, which supports the recruitment of effectors such as Raf. Conversely, effectors possess lipid-recognition motifs, which appear to serve as co-incidence detectors for the lipid domain of a given Ras isoform. Evidence suggests that dimeric Raf proteins then co-assemble dimeric Ras in an immobile complex, thus forming the minimal unit of an active nanocluster. Here we review established and novel trafficking chaperones and trafficking factors of Ras, along with the set of lipid and protein modulators of Ras nanoclustering. We highlight drug targeting approaches and opportunities against these determinants of functional Ras membrane organization. Finally, we reflect on implications for Ras signaling in polarized cells, such as epithelia, which are a common origin of tumorigenesis.
Topics: Cell Membrane; Drug Delivery Systems; Humans; Molecular Targeted Therapy; Neoplasms; Signal Transduction; ras Proteins
PubMed: 35101236
DOI: 10.1016/bs.acr.2021.07.005