Dynamic communication between intracellular organelles often takes place at specialized membrane contact sites that form between their membranes. Here we detail a procedure for the purification of endoplasmic reticulum-plasma membrane (ER-PM) junctions from the mouse brain. We describe steps for homogenizing isolated brain hemispheres and sequential centrifugation to remove the nuclear fraction from the other membrane fractions.
View Article and Find Full Text PDFNeuraminidase 1 (Neu1) cleaves terminal sialic acids from sialoglycoproteins in endolysosomes and at the plasma membrane. As such, Neu1 regulates immune cells, primarily those of the monocytic lineage. Here we examined how Neu1 influences microglia by modulating the sialylation of full-length Trem2 (Trem2-FL), a multifunctional receptor that regulates microglial survival, phagocytosis, and cytokine production.
View Article and Find Full Text PDFEndoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca signaling.
View Article and Find Full Text PDFEndoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca signaling.
View Article and Find Full Text PDFHigh energy-demanding tissues, such as skeletal muscle, require mitochondrial proteostasis to function properly. Two quality-control mechanisms, the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles, safeguard mitochondrial proteostasis. However, whether these processes interact is unknown.
View Article and Find Full Text PDFIntracellular organelles carry out many of their functions by engaging in extensive interorganellar communication through specialized membrane contact sites (MCSs) formed where two organelles tether to each other or to the plasma membrane (PM) without fusing. In recent years, these ubiquitous membrane structures have emerged as central signaling hubs that control a multitude of cellular pathways, ranging from lipid metabolism/transport to the exchange of metabolites and ions (i.e.
View Article and Find Full Text PDFRhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1/ETV7 mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1.
View Article and Find Full Text PDFAlix is a ubiquitously expressed scaffold protein that participates in numerous cellular processes related to the remodeling/repair of membranes and the actin cytoskeleton. Alix exists in monomeric and dimeric/multimeric configurations, but how dimer formation occurs and what role the dimer has in Alix-mediated processes are still largely elusive. Here, we reveal a mechanism for Alix homodimerization mediated by disulfide bonds under physiological conditions and demonstrate that the Alix dimer is enriched in exosomes and F-actin cytoskeleton subcellular fractions.
View Article and Find Full Text PDFGM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal β-galactosidase (β-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death.
View Article and Find Full Text PDFMol Ther Methods Clin Dev
December 2021
During primary tumorigenesis isolated cancer cells may undergo genetic or epigenetic changes that render them responsive to additional intrinsic or extrinsic cues, so that they enter a transitional state and eventually acquire an aggressive, metastatic phenotype. Among these changes is the alteration of the cell metabolic/catabolic machinery that creates the most permissive conditions for invasion, dissemination, and survival. The lysosomal system has emerged as a crucial player in this malignant transformation, making this system a potential therapeutic target in cancer.
View Article and Find Full Text PDFExosomes are dynamic nanovesicles secreted by virtually all cells and are present in all biological fluids. Given their highly heterogeneous content exosomes have been implicated in many physiological and pathological processes that they exert by influencing cell-cell and cell-ECM communication. In recent years an increasing number of methods have been established for the purification and characterization of exosomes.
View Article and Find Full Text PDFExpansion of fibrous connective tissue and abnormal deposition of extracellular matrix (ECM) are at the basis of many fibrotic diseases. Fibrosis can occur in response to both physiological and pathological cues, including wound healing, tissue remodeling/repair and inflammation. Chronic fibrosis can lead to severe tissue damage, organ failure and death.
View Article and Find Full Text PDFGalactosialidosis is a rare lysosomal storage disease caused by a congenital defect of protective protein/cathepsin A (PPCA) and secondary deficiency of neuraminidase-1 and β-galactosidase. PPCA is a lysosomal serine carboxypeptidase that functions as a chaperone for neuraminidase-1 and β-galactosidase within a lysosomal multi-protein complex. Combined deficiency of the three enzymes leads to accumulation of sialylated glycoproteins and oligosaccharides in tissues and body fluids and manifests in a systemic disease pathology with severity mostly correlating with the type of mutation(s) and age of onset of the symptoms.
View Article and Find Full Text PDFExosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization.
View Article and Find Full Text PDFCongenital deficiency of the lysosomal sialidase neuraminidase 1 (NEU1) causes the lysosomal storage disease, sialidosis, characterized by impaired processing/degradation of sialo-glycoproteins and sialo-oligosaccharides, and accumulation of sialylated metabolites in tissues and body fluids. Sialidosis is considered an ultra-rare clinical condition and falls into the category of the so-called orphan diseases, for which no therapy is currently available. In this study we aimed to identify potential therapeutic modalities, targeting primarily patients affected by type I sialidosis, the attenuated form of the disease.
View Article and Find Full Text PDFCoordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1.
View Article and Find Full Text PDFLysosomal exocytosis is a ubiquitous process negatively regulated by neuraminidase 1 (NEU1), a sialidase mutated in the glycoprotein storage disease sialidosis. In mice, excessive lysosomal exocytosis is at the basis of disease pathogenesis. Yet, the tissue-specific molecular consequences of this deregulated pathway are still unfolding.
View Article and Find Full Text PDFUnderstanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1.
View Article and Find Full Text PDFNeuraminidase-1 (NEU1) is the sialidase responsible for the catabolism of sialoglycoconjugates in lysosomes. Congenital NEU1 deficiency causes sialidosis, a severe lysosomal storage disease associated with a broad spectrum of clinical manifestations, which also include skeletal deformities, skeletal muscle hypotonia and weakness. Neu1(-/-) mice, a model of sialidosis, develop an atypical form of muscle degeneration caused by progressive expansion of the connective tissue that infiltrates the muscle bed, leading to fiber degeneration and atrophy.
View Article and Find Full Text PDFBoth pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F.
View Article and Find Full Text PDFThe lysosomal storage disease sialidosis is caused by a primary deficiency of the sialidase N-acetyl-α-neuraminidase-1 (NEU1). Patients with type I sialidosis develop an attenuated, non-neuropathic form of the disease also named cherry red spot myoclonus syndrome, with symptoms arising during juvenile/ adult age. NEU1 requires binding to its chaperone, protective protein/cathepsin A (PPCA), for lysosomal compartmentalization, stability and catalytic activation.
View Article and Find Full Text PDFRemoval of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1-4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs.
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