Lysosomal proteome analysis reveals that CLN3-defective cells have multiple enzyme deficiencies associated with changes in intracellular trafficking.

Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The CLN3 gene encodes a lysosomal membrane protein of unknown function, and CLN3 mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.

Lysosomal Dysfunction Caused by Cellular Accumulation of Silica Nanoparticles.

Nanoparticles (NPs) are widely used as components of drugs or cosmetics and hold great promise for biomedicine, yet their effects on cell physiology remain poorly understood. Here we demonstrate that clathrin-independent dynamin 2-mediated caveolar uptake of surface-functionalized silica nanoparticles (SiNPs) impairs cell viability due to lysosomal dysfunction. We show that internalized SiNPs accumulate in lysosomes resulting in inhibition of autophagy-mediated protein turnover and impaired degradation of internalized epidermal growth factor, whereas endosomal recycling proceeds unperturbed. This phenotype is caused by perturbed delivery of cargo via autophagosomes and late endosomes to SiNP-filled cathepsin B/L-containing lysosomes rather than elevated lysosomal pH or altered mTOR activity. Given the importance of autophagy and lysosomal protein degradation for cellular proteostasis and clearance of aggregated proteins, these results raise the question of beneficial use of NPs in biomedicine and beyond.

Transport activity and presence of ClC-7/Ostm1 complex account for different cellular functions.

Loss of the lysosomal ClC-7/Ostm1 2Cl(-)/H(+) exchanger causes lysosomal storage disease and osteopetrosis in humans and additionally changes fur colour in mice. Its conversion into a Cl(-) conductance in Clcn7(unc/unc) mice entails similarly severe lysosomal storage, but less severe osteopetrosis and no change in fur colour. To elucidate the basis for these phenotypical differences, we generated Clcn7(td/td) mice expressing an ion transport-deficient mutant. Their osteopetrosis was as severe as in Clcn7(-/-) mice, suggesting that the electric shunt provided by ClC-7(unc) can partially rescue osteoclast function. The normal coat colour of Clcn7(td/td) mice and their less severe neurodegeneration suggested that the ClC-7 protein, even when lacking measurable ion transport activity, is sufficient for hair pigmentation and that the conductance of ClC-7(unc) is harmful for neurons. Our in vivo structure-function analysis of ClC-7 reveals that both protein-protein interactions and ion transport must be considered in the pathogenesis of ClC-7-related diseases.

LYSET/TMEM251- a novel key component of the mannose 6-phosphate pathway.

Degradation of macromolecules delivered to lysosomes by processes such as autophagy or endocytosis is crucial for cellular function. Lysosomes require more than 60 soluble hydrolases in order to catabolize such macromolecules. These soluble hydrolases are tagged with mannose6-phosphate (M6P) moieties in sequential reactions by the Golgi-resident GlcNAc-1-phosphotransferase complex and NAGPA/UCE/uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase), which allows their delivery to endosomal/lysosomal compartments through trafficking mediated by cation-dependent and -independent mannose 6-phosphate receptors (MPRs). We and others recently identified TMEM251 as a novel regulator of the M6P pathway via independent genome-wide genetic screening strategies. We renamed TMEM251 to LYSET (lysosomal enzyme trafficking factor) to establish nomenclature reflective to this gene's function. LYSET is a Golgi-localized transmembrane protein important for the retention of the GlcNAc-1-phosphotransferase complex in the Golgi-apparatus. The current understanding of LYSET's importance regarding human biology is 3-fold: 1) highly pathogenic viruses that depend on lysosomal hydrolase activity require LYSET for infection. 2) The presence of LYSET is critical for cancer cell proliferation in nutrient-deprived environments in which extracellular proteins must be catabolized. 3) Inherited pathogenic alleles of LYSET can cause a severe inherited disease which resembles GlcNAc-1-phosphotransferase deficiency (i.e., mucolipidosis type II).Abbreviations: GlcNAc-1-PT: GlcNAc-1-phosphotransferase; KO: knockout; LSD: lysosomal storage disorder; LYSET: lysosomal enzyme trafficking factor; M6P: mannose 6-phosphate; MPRs: mannose-6-phosphate receptors, cation-dependent or -independent; MBTPS1/site-1 protease: membrane bound transcription factor peptidase, site 1; MLII: mucolipidosis type II; WT: wild-type.

Lysosomal enzyme trafficking factor LYSET enables nutritional usage of extracellular proteins.

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

Impact of the gut microbiota on the m6A epitranscriptome of mouse cecum and liver.

The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. Here we examine whether host epitranscriptomic marks are affected by the gut microbiota. We use methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to identify N6-methyladenosine (m6A) modifications in mRNA of mice carrying conventional, modified, or no microbiota. We find that variations in the gut microbiota correlate with m6A modifications in the cecum, and to a lesser extent in the liver, affecting pathways related to metabolism, inflammation and antimicrobial responses. We analyze expression levels of several known writer and eraser enzymes, and find that the methyltransferase Mettl16 is downregulated in absence of a microbiota, and one of its target mRNAs, encoding S-adenosylmethionine synthase Mat2a, is less methylated. We furthermore show that Akkermansia muciniphila and Lactobacillus plantarum affect specific m6A modifications in mono-associated mice. Our results highlight epitranscriptomic modifications as an additional level of interaction between commensal bacteria and their host.

Accumulation of bis(monoacylglycero)phosphate and gangliosides in mouse models of neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses comprise a group of inherited severe neurodegenerative lysosomal disorders characterized by lysosomal dysfunction and massive accumulation of fluorescent lipopigments and aggregated proteins. To examine the role of lipids in neurodegenerative processes of these diseases, we analysed phospho- and glycolipids in the brains of ctsd-/- and nclf mice, disease models of cathepsin D and CLN6 deficiency, respectively. Both ctsd-/- and nclf mice exhibited increased levels of GM2 and GM3 gangliosides. Immunohistochemically GM2 and GM3 staining was found preferentially in neurons and glial cells, respectively, of ctsd-/- mice. Of particular note, a 20-fold elevation of the unusual lysophospholipid bis(monoacylglycero)phosphate was specifically detected in the brain of ctsd-/- mice accompanied with sporadic accumulation of unesterified cholesterol in distinct cells. The impaired processing of the sphingolipid activator protein precursor, an in vitro cathepsin D substrate, in the brain of ctsd-/- mice may provide the mechanistic link to the storage of lipids. These studies show for the first time that cathepsin D regulates the lysosomal phospho- and glycosphingolipid metabolism suggesting that defects in the composition, trafficking and/or recycling of membrane components along the late endocytic pathway may be critical for the pathogenesis of early onset neuronal ceroid lipofuscinoses.

3-Hydroxyglutaric acid is transported via the sodium-dependent dicarboxylate transporter NaDC3.

Patients with glutaryl-CoA dehydrogenase (GCDH) deficiency accumulate glutaric acid (GA) and 3-hydroxyglutaric acid (3OH-GA) in their blood and urine. To identify the transporter mediating the translocation of 3OH-GA through membranes, kidney tissue of Gcdh-/- mice have been investigated because of its central role in urinary excretion of this metabolite. Using microarray analyses of kidney-expressed genes in Gcdh-/- mice, several differentially expressed genes encoding transporter proteins were identified. Real-time polymerase chain reaction analysis confirmed the upregulation of the sodium-dependent dicarboxylate cotransporter 3 (NaDC3) and the organic cation transporter 2 (OCT2). Expression analysis of NaDC3 in Xenopus laevis oocytes by the two-electrode-voltage-clamp technique demonstrated the sodium-dependent translocation of 3OH-GA with a K (M) value of 0.95 mM. Furthermore, tracer flux measurements in Chinese hamster ovary cells overexpressing OCT2 showed that 3OH-GA inhibited significantly the uptake of methyl-4-phenylpyridinium, whereas 3OH-GA is not transported by OCT2. The data demonstrate for the first time the membrane translocation of 3OH-GA mediated by NaDC3 and the cis-inhibitory effect on OCT2-mediated transport of cations.

Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.

In vitro sulfation of N-acetyllactosaminide by soluble recombinant human beta-Gal-3'-sulfotransferase.

Membrane-bound beta-Gal-3'-sulfotransferase (GP3ST) was expressed and used for in vitro sulfation of Tamm-Horsfall glycoprotein. Further, the regioselective transfer of sulfate to an N-acetyllactosamine derivative could be realised with soluble chimeric GP3ST, also in combination with Lac transglycosylation by means of beta-galactosidase. Two alternative straightforward chemical syntheses for the target compound could be elaborated.

A cation counterflux supports lysosomal acidification.

The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl(-) with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7-deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes.

Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl- accumulation.

During lysosomal acidification, proton-pump currents are thought to be shunted by a chloride ion (Cl-) channel, tentatively identified as ClC-7. Surprisingly, recent data suggest that ClC-7 instead mediates Cl-/proton (H+) exchange. We generated mice carrying a point mutation converting ClC-7 into an uncoupled (unc) Cl- conductor. Despite maintaining lysosomal conductance and normal lysosomal pH, these Clcn7(unc/unc) mice showed lysosomal storage disease like mice lacking ClC-7. However, their osteopetrosis was milder, and they lacked a coat color phenotype. Thus, only some roles of ClC-7 Cl-/H+ exchange can be taken over by a Cl- conductance. This conductance was even deleterious in Clcn7(+/unc) mice. Clcn7(-/-) and Clcn7(unc/unc) mice accumulated less Cl- in lysosomes than did wild-type mice. Thus, lowered lysosomal chloride may underlie their common phenotypes.