Identification of lysosomal Npc1-binding proteins: Cathepsin D activity is regulated by NPC1.
Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1-binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032-1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC-MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.
Secció d'Errors Congènits del Metabolisme (IBC), Servei de Bioquímica i Genètica Molecular, Hospital Clínic, Barcelona, Spain.|CIBER de Enfermedades Raras (CIBERER), Madrid, Spain.|Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.
Judit
J
Macías-Vidal
Department of Cell Biology, Immunology and Neurosciences, University of Barcelona, Barcelona, Spain.
Martina
M
Guerrero-Hernández
Proteomic Unit, Centres Científics i Tecnològics, University of Barcelona, Barcelona, Spain.
Josep Maria
JM
Estanyol
CIBER de Enfermedades Raras (CIBERER), Madrid, Spain.|Laboratory of Cellular Biology, Centro de Investigación Príncipe Felipe, Valencia, Spain.
Carmen
C
Aguado
CIBER de Enfermedades Raras (CIBERER), Madrid, Spain.|Laboratory of Cellular Biology, Centro de Investigación Príncipe Felipe, Valencia, Spain.
Erwin
E
Knecht
Secció d'Errors Congènits del Metabolisme (IBC), Servei de Bioquímica i Genètica Molecular, Hospital Clínic, Barcelona, Spain.|CIBER de Enfermedades Raras (CIBERER), Madrid, Spain.|Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.
Maria Josep
MJ
Coll
Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.|Department of Cell Biology, Immunology and Neurosciences, University of Barcelona, Barcelona, Spain.
Oriol
O
Bachs
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002402:Cathepsin D / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D002853:Chromatography, Liquid; D004792:Enzyme Precursors / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000378:metabolism*; D060066:Protein Interaction Maps; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D002352:Carrier Proteins; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D011506:Proteins; C018589:lysosomal proteins; C058345:procathepsin D; D002402:Cathepsin D
Affinity chromatography; Biomedicine; Cathepsin D; LC-MS/MS; NPC1; Npc1-binding proteins