| PROJECT DESCRIPTION |
FINAL STATUS REPORT Dated 8/31/2001 |
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| Recently, the primary gene causing NPC disease was isolated. However, the function(s) of this novel gene are still elusive. We have developed a novel system for looking at the function of NPC1 and have determined that this protein functions as a permease, a protein that assists certain molecules in crossing membranes. These observations have significant implications in our understanding of NPC disease pathogenesis. Better understanding of the function of NPC1 will lead to rational design of treatment regiments. Following identification of NPC1, we identified a second gene that shares extensive homology with NPC1, NPC1L1. Preliminary characterization reveals that this gene is expressed in all tissues with liver and muscles showing the highest levels of NPC1L1 mRNA. In addition, the subcellular location of NPC1L1 protein is different from NPC1 in that the former is located in the Golgi apparatus whereas the latter resides in late endosomes. Based on the fact that these two genes share extensive sequence (52% amino acid homology) and structural (predicted to have the same number of transmembrane domains as NPC1) homologies, we hypothesize that NPC1L1 belongs in the same family as NPC1. If that is the case, then the two proteins may facilitate the movement of similar molecules at different locations in the cell. We will use the same novel system, described for NPC1, to characterize the molecules that NPC1L1 transports and compare these results to the type of molecules transported by NPC1. If these two proteins are, in fact, members of the same family (all preliminary evidence suggest that they are) then we could envision modulating the activity of NPC1L1 in NPC1 negative cells in order to complement for the deficiency in NPC1. In addition, to better characterize NPC1L1, we are going to generate mice that are going to be defective for NPC1L1. Characterization of these mice and comparison to NPC1-defective mice should allow for a direct assessment of the phenotype created by a deficiency of NPC1L1. These data should further our understanding of NPC1 and NPC1L1 and expand our knowledge of this new family of proteins. Understanding how these proteins work will also increase our understanding of how they cause disease when deficient. | We are happy to report that during this one year of funding by NNPDF we were able to accomplish our specific aims with respect to the preliminary characterization of NPC1L1 and the generation of a mouse knockout model of NPC1L1. Thus, we have determined that NPC1L1 belongs in the RND family of permeases, together with NPC1 and, as recently described, with Patched. As proposed in our application we set out to generate a mouse knockout model of NPC1L1 since no human disease or animal models due to defects in this gene are currently known. This will allow us to investigate both the intracellular and physiological effects of absent NPC1L1 expression. We have already successfully generated chimeric mice and a colony of NPC1L1 homozygous knockout mice is currently being produced for our studies. Mice that were heterozygous and homozygous for the knockout allele on a homogeneous C57BL/6J background have now been successfully obtained. The heterozygotes are clearly able to breed and homozygotes survive at least until the second week after birth. These mice will be characterized to reveal any phenotype caused by the lack of NPC1L1 expression, such as alterations in their life span, growth rate and weight, as well as any anatomical abnormalities. In addition, we will investigate the effect of various lipid-rich or poor diets on their growth and survival. | |
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PUBLICATIONS: http://www.jbc.org/cgi/reprint/280/13/12710.pdf |
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