title: Reduced cathepsins B and D cause impaired autophagic degradation that can be almost completely restored by overexpression of these two proteases in Sap C-deficient fibroblasts.
abstract: Saposin (Sap) C deficiency, a rare variant form of Gaucher disease, is due to mutations in the Sap C coding region of the prosaposin (PSAP) gene. Sap C is required as an activator of the lysosomal enzyme glucosylceramidase (GCase), which catalyzes glucosylceramide (GC) degradation. Deficit of either GCase or Sap C leads to the accumulation of undegraded GC and other lipids in lysosomes of monocyte/macrophage lineage. Recently, we reported that Sap C mutations affecting a cysteine residue result in increased autophagy. Here, we characterized the basis for the autophagic dysfunction. We analyzed Sap C-deficient and GCase-deficient fibroblasts and observed that autophagic disturbance was only associated with lack of Sap C. By a combined fluorescence microscopy and biochemical studies, we demonstrated that the accumulation of autophagosomes in Sap C-deficient fibroblasts is not due to enhanced autophagosome formation but to delayed degradation of autolysosomes caused, in part, to decreased amount and reduced enzymatic activity of cathepsins B and D. On the contrary, in GCase-deficient fibroblasts, the protein level and enzymatic activity of cathepsin D were comparable with control fibroblasts, whereas those of cathepsin B were almost doubled. Moreover, the enhanced expression of both these lysosomal proteases in Sap C-deficient fibroblasts resulted in close to functional autophagic degradation. Our data provide a novel example of altered autophagy as secondary event resulting from insufficient lysosomal function.
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Tatti
authors.forename: Marialetizia
authors.initials: M
authors.lastname: Motta
authors.forename: Sabrina
authors.initials: S
authors.lastname: Di Bartolomeo
authors.forename: Susanna
authors.initials: S
authors.lastname: Scarpa
authors.forename: Valentina
authors.initials: V
authors.lastname: Cianfanelli
authors.forename: Francesco
authors.initials: F
authors.lastname: Cecconi
authors.forename: Rosa
authors.initials: R
authors.lastname: Salvioli
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects / Q000235:genetics*; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D000071186:Beclin-1; D002401:Cathepsin B / Q000235:genetics* / Q000378:metabolism*; D002402:Cathepsin D / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005776:Gaucher Disease / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D049231:Saposins / Q000172:deficiency* / Q000235:genetics; D020123:Sirolimus / Q000494:pharmacology; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism
chemical_list: C502778:ATG5 protein, human; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D049231:Saposins; D002401:Cathepsin B; D002402:Cathepsin D; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes; D020123:Sirolimus