Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage.
Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids (SLs) caused by defects in either NPC1 or NPC2 proteins. We previously demonstrated that NPC1 human skin fibroblasts overexpressing endosomal Rab proteins (Rab7 or Rab9) showed a correction in the storage disease phenotype. In the current study, we used protein transduction to further investigate Rab9-mediated reduction of stored lipids in NPC cells. Recombinant human Rab9 fused with the herpes simplex virus VP22 protein fragment was overexpressed, purified, and added to culture medium to induce protein transduction. When VP22-Rab9 was transduced into NPC1 fibroblasts, nearly all cells showed significant reduction in cellular free cholesterol levels, with no cytotoxicity up to 5 microM. A fraction of the VP22-Rab9 that was transduced into the cells was shown to bind to rab GDP dissociation inhibitor, suggesting that this pool of VP22-Rab9 had become prenylated. The reduction in cellular free cholesterol was associated with correction of abnormal intracellular trafficking of BODIPY-lactosylceramide and an increase of sterols in the culture media. The clearance of lysosomal free cholesterol was also associated with a decrease in LDL-receptor levels. In addition, we demonstrated reduction of intracellular cholesterol by VP22-Rab9 transduction in NPC2 fibroblasts and in cultured mouse NPC1 neurons. These observations provide important new information about the correction of membrane traffic in NPC cells by Rab9 overexpression and may lead to new therapeutic approaches for treatment of this disease.
Department of Biochemistry and Molecular Biology Mayo Clinic and Foundation Rochester, Minnesota 55905, USA.
Keishi
K
Narita
Amit
A
Choudhury
Kostantin
K
Dobrenis
Deepak K
DK
Sharma
Eileen L
EL
Holicky
David L
DL
Marks
Steven U
SU
Walkley
Richard E
RE
Pagano
D001692:Biological Transport; D002352:Carrier Proteins; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006023:Glycoproteins / Q000172:deficiency; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000172:deficiency; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000188:drug therapy* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D033921:Vesicular Transport Proteins; D015678:Viral Structural Proteins; D020691:rab GTP-Binding Proteins / Q000494:pharmacology*
D002352:Carrier Proteins; D006023:Glycoproteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; C104585:NPC2 protein, human; D000077296:Niemann-Pick C1 Protein; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D002784:Cholesterol; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins