The National Institute of Diabetes & Digestive & Kidney Diseases

Our area of research is the cell biology of lipid metabolism. We study lipids, lipases, and specific proteins uniquely involved in lipid metabolic processes in cells and tissues. Our objective is to learn how these molecules are translocated in normal cells and to identify defects in cells from humans and animals with genetic lesions in lipid metabolism. To this end we use confocal light microscopy and electron microscopy in combination with immunocytochemistry and cytochemistry to locate unesterified cholesterol, other lipids, hepatic lipase, lipoprotein lipase, hormone sensitive lipase, and a unique adipocyte phosphoprotein, perilipin, in tissues and cultured cells.

A mainstay technique of our research is cryo-immunogold electron microscopy. With this technique we identified perilipin as the first protein to be located at the monolayered surface of intracellular stores of neutral lipid. Subsequently other proteins of lipid metabolism (including hormone sensitive lipase) have been identified at this site. Thus intracellular lipid stores appear to have a similar intimate structural association with their regulatory proteins as that between neutral lipids and apoproteins in plasma lipoproteins (chylomicrons, very low density and low density lipoproteins).

Our studies on a human genetic defect in cholesterol metabolism (Niemann Pick Type C disease) led to the discovery that intracellular trafficking of LDL-derived cholesterol involves post-lysosomal transport to the Golgi apparatus. Using freeze-fracture electron microscopy transport of intracellular cholesterol through the Golgi was found to be defective in cultured fibroblasts from NPC patients, resulting in cellular cholesterol lipidosis. Recent discovery of the Niemann Pick Type C gene (Science, July 1997) has allowed us to investigate the cellular processing of the NPC protein and the relationship of this protein to sites of cholesterol accumulation in normal and mutant cells. These studies on the role of the NPC protein should increase our knowledge of how cells deal with cholesterol, in particular how free cholesterol moves out of lysosomes and to other parts of the cell to exert feedback homeostatic responses.

1. Kishida T, Kostetskii I, Zhang Z, Martinez F, Liu P, Walkley SU, Dwyer NK, Blanchette-Mackie EJ, Radice GL, Strauss JF 3rd  Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism.  J Biol Chem (279): 19276-85, 2004. [Full Text/Abstract]

2. Neufeld EB, Stonik JA, Demosky SJ Jr, Knapper CL, Combs CA, Cooney A, Comly M, Dwyer N, Blanchette-Mackie J, Remaley AT, Santamarina-Fojo S, Brewer HB Jr  The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease.  J Biol Chem (279): 15571-8, 2004. [Full Text/Abstract]

3. Zhang M, Sun M, Dwyer NK, Comly ME, Patel SC, Sundaram R, Hanover JA, Blanchette-Mackie EJ  Differential trafficking of the Niemann-Pick C1 and 2 proteins highlights distinct roles in late endocytic lipid trafficking.  Acta Paediatr Suppl (92): 63-73; discussion 45, 2003. [Full Text/Abstract]

4. Zhang M, Liu P, Dwyer NK, Christenson LK, Fujimoto T, Martinez F, Comly M, Hanover JA, Blanchette-Mackie EJ, Strauss JF 3rd  MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.  J Biol Chem (277): 33300-10, 2002. [Full Text/Abstract]

5. Zhang M, Dwyer NK, Love DC, Cooney A, Comly M, Neufeld E, Pentchev PG, Blanchette-Mackie EJ, Hanover JA  Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease.  Proc Natl Acad Sci U S A (98): 4466-71, 2001. [Full Text/Abstract]

6. Zhang M, Dwyer NK, Neufeld EB, Love DC, Cooney A, Comly M, Patel S, Watari H, Strauss JF 3rd, Pentchev PG, Hanover JA, Blanchette-Mackie EJ  Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes.  J Biol Chem (276): 3417-25, 2001. [Full Text/Abstract]

7. Blanchette-Mackie EJ  Intracellular cholesterol trafficking: role of the NPC1 protein.  Biochim Biophys Acta (1486): 171-83, 2000. [Full Text/Abstract]

8. Shamburek RD Pentchev PG Zech LA Blanchette-Mackie J Carstea ED VandenBroek JM Cooper PS Neufeld EB Phair RD Brewer HB Jr Brady RO Schwartz CC  Intracellular trafficking of the free cholesterol derived from LDL cholesteryl ester is defective in vivo in Niemann-Pick C disease: insights on normal metabolism of HDL and LDL gained from the NP-C mutation.  J Lipid Res (38): 2422-35, 1997. [Full Text/Abstract]

9. Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings C, Gu J, Rosenfeld MA, Pavan WJ, Krizman DB, Nagle J, Polymeropoulos MH, Sturley SL, Ioannou YA, Higgins ME, Comly M, Cooney A, Brown A, Kaneski CR, Blanchette-Mackie EJ, Dwyer NK, Neufeld EB, Chang TY, Liscum L, Tagle DA. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277(5323): 228-231, 1997. [Full Text/Abstract]

10. Blanchette-Mackie EJ Dwyer NK Barber T Coxey RA Takeda T Rondinone CM Theodorakis JL Greenberg AS Londos C  Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes.  J Lipid Res (36): 1211-26, 1995. [Full Text/Abstract]