BCM-95 and (2-hydroxypropyl)-β-cyclodextrin reverse autophagy dysfunction and deplete stored lipids in Sap C-deficient fibroblasts.

Saposin (Sap) C deficiency is a rare variant form of Gaucher disease caused by impaired Sap C expression or accelerated degradation, and associated with accumulation of glucosylceramide and other lipids in the endo/lysosomal compartment. No effective therapies are currently available for the treatment of Sap C deficiency. We previously reported that a reduced amount and enzymatic activity of cathepsin (Cath) B and Cath D, and defective autophagy occur in Sap C-deficient fibroblasts.

Gaucher disease due to saposin C deficiency is an inherited lysosomal disease caused by rapidly degraded mutant proteins.

Saposin (Sap) C is an essential cofactor for the lysosomal degradation of glucosylceramide (GC) by glucosylceramidase (GCase) and its functional impairment underlies a rare variant form of Gaucher disease (GD). Sap C promotes rearrangement of lipid organization in lysosomal membranes favoring substrate accessibility to GCase. It is characterized by six invariantly conserved cysteine residues involved in three intramolecular disulfide bonds, which make the protein remarkably stable to acid environment and degradation.

Cathepsin-mediated regulation of autophagy in saposin C deficiency.

Saposin C deficiency, a rare variant form of Gaucher disease, is due to mutations in the prosaposin gene (PSAP) affecting saposin C expression and/or function. We previously reported that saposin C mutations affecting one cysteine residue result in autophagy dysfunction. We further demonstrated that the accumulation of autophagosomes, observed in saposin C-deficient fibroblasts, is due to an impairment of autolysosome degradation, partially caused by the reduced amount and enzymatic activity of CTSB (cathepsin B) and CTSD (cathepsin D).

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding.

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.

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.

Autophagy in Gaucher disease due to saposin C deficiency.

Gaucher disease, due to a deficit of glucosylceramidase or, rarely, of its activator saposin C, is characterized by accumulation of glucosylceramide in the lysosomes of monocyte/macrophage lineage. In our study we demonstrate that saposin C deficiency due to mutations involving a cysteine residue results in increased autophagy. Autophagy was monitored by LC3 analysis and confirmed by electron microscopy; we observed a correlation among saposin C mutation, Gaucher phenotype and increased autophagy.

Saposin C mutations in Gaucher disease patients resulting in lysosomal lipid accumulation, saposin C deficiency, but normal prosaposin processing and sorting.

Gaucher disease (GD) is characterized by accumulation of glucosylceramide (GC) in the cells of monocyte/macrophage system. The degradation of GC is controlled by glucosylceramidase (GCase) and saposin (Sap) C, a member of a family of four small glycoproteins (Saps A, B, C and D), all derived by proteolytic processing of a common precursor, prosaposin (PSAP). Saps contain six cysteine residues, forming three disulfide bridges, that affect their structure and function.

The potential action of galactose as a "chemical chaperone": increase of beta galactosidase activity in fibroblasts from an adult GM1-gangliosidosis patient.

Background: The glycosphingolipid storage disorder GM1-gangliosidosis is a severe neurodegenerative condition for which no therapy is currently available. Protein misfolding in lysosomal defects may have the potential to be corrected by chemical chaperones: in vitro and clinical approaches are being investigated.

Aims: We investigated the in vitro effect of galactose on some lysosomal hydrolases, and its in vitro efficacy as a chemical chaperone in GM1-gangliosidosis.

The secretion and maturation of prosaposin and procathepsin D are blocked in embryonic neural progenitor cells.

The notion that prosaposin (Prosap) is likely involved in brain development and regeneration led us to explore its expression in stem/progenitor neural cells and its fate after cell differentiation. The expression of procathepsin-cathepsin D (proCath-Cath D), an endoprotease that plays an important role in the processing and sorting of Prosap, has been concomitantly examined. Our data evidenced that in embryonic human neural progenitor cells (eHNPCs) intact and high molecular weight intermediate forms of Prosap and intermediate forms of Cath D accumulated inside the cells, while the formation of saposins and mature Cath D was impaired.

Saposin B binds and transfers phospholipids.

Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known.

The N370S (Asn370-->Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C.

The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370-->Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity.

Glucosylceramidase mass and subcellular localization are modulated by cholesterol in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values.

Interaction of saposin D with membranes: effect of anionic phospholipids and sphingolipids.

Saposin (Sap) D is an endolysosomal protein that, together with three other similar proteins, Sap A, Sap B and Sap C, is involved in the degradation of sphingolipids and, possibly, in the solubilization and transport of gangliosides. We found that Sap D is able to destabilize and disrupt membranes containing each of the three anionic phospholipids most abundant in the acidic endolysosomal compartment, namely lysobisphosphatidic acid (LBPA), phosphatidylinositol (PI) and phosphatidylserine (PS). The breakdown of the membranes, which occurs when the Sap D concentration on the lipid surface reaches a critical value, is a slow process that gives rise to small particles.