To cap off our Niemann-Pick C disease blog series — here are parts 1, 2, and 3 — we’ll take a closer look at the two ends of the NPC1 protein, and the roles they play in shaping how NPC1 proteins are transported from sites of production to sites of action inside cells. NPC1 protein transport is a particularly interesting feature of NPC pathophysiology because it gives us insights into the different functional consequences of different mutations in the NPC1 gene. In fact, the most commonly observed mutation in NPC patients (I1061T) is a mutation that reduces NPC1 protein transport to lysosomes, specifically endolysosomal compartments where NPC1 regulates cholesterol and other lipids. Point mutations like I1061T are a recurring theme across Mendelian diseases.

Lysosomes are membrane-bound organelles filled with soluble hydrolytic enzymes. They are formed in a process of gradual additions of protein-filled vesicles from the Golgi to membrane-bound compartments known as endosomes. Eventually, through a series of changes to early and late endosomes, the membrane-bound compartment can be considered a lysosome. (Jeyakumar et al., 2005)

For more info on lysosomes, I highly suggest you to check out our earlier blog post on the topic.

fig 1

The NPC1 protein is a 13 transmembrane domain molecule that sits in the membrane of lysosomes, and like all proteins has two ends: a C-terminus and a N-terminus. There are seven domains that extend within the lumen and another seven that extend into the cytosol. Some of you may have already noticed, that in a protein with an odd number of transmembrane domains, the topology must be such that the C-terminus and N-terminus are on opposing sides of the cell membrane. (Davies, 2000)

fig 2

fig 3 In the image to the right are two adjacent cells (though only the one on the left is visible) stained in red with an antibody for NPC1 protein. The C terminal ‘dileucine’ motif (LLNF) has been implicated in NPC1 protein’s appropriate trafficking to lysosomes. C-terminal NPC 1 truncation mutant proteins are not able to localize to lysosomes; note the reticular pattern around the nucleus, characteristic of ER retention in the visible cell on the left. (Watari et al., 1999)

fig 4 As a result, cholesterol accumulates in lysosomes, as seen in this Filipin stain. Note that the cell on the right is now visible. It’s not expressing NPC1 protein at all — it’s a null mutant — and shows the same cholesterol-accumulating phenotype as a NPC1 mutant protein that is able to fold but cannot reach lysosomes (Watari et al., 1999).

Similarly, the N terminus plays an important role in NPC1 protein function. In contrast to C-terminal truncation mutants, N-terminal truncation mutants successfully reach lysosomes, but are unable to mobilize cholesterol from lysosomes, as seen below. Image (A) shows the standard Filipin staining, with the ‘canonical’ cholesterol accumulation typical of NPC mutant cells. (B) is an immunostain for NPC1 protein, showing its localized to some membrane-bound compartment. (C) is an immunostain for a known lyososomal-localized protein, lpg95. Note that overlap of (B) and (C) staining in D. This confirms the hypothesis that NPC1 N-terminal truncation proteins reach the lysosome, but do not carry out their normal cholesterol-mobilizing function.

fig 5

These insights help drive our quest for precision small-molecule therapeutics that are targeted to specific mutations.


Davies, J. P. “Topological Analysis of Niemann-Pick C1 Protein Reveals That the Membrane Orientation of the Putative Sterol-sensing Domain Is Identical to Those of 3-Hydroxy-3-methylglutaryl-CoA Reductase and Sterol Regulatory Element Binding Protein Cleavage-activating Protein.” Journal of Biological Chemistry (2000): 24367-4374.

Watari, H. “Niemann-Pick C1 Protein: Obligatory Roles for N-terminal Domains and Lysosomal Targeting in Cholesterol Mobilization.” Proceedings of the National Academy of Sciences (1999): 805-10.

Jeyakumar, Mylvaganam, Raymond A. Dwek, Terry D. Butters, and Frances M. Platt. “Storage Solutions: Treating Lysosomal Disorders of the Brain.” Nature Reviews Neuroscience (2005).

Alberts, Bruce. Molecular Biology of the Cell. 4th ed. New York: Garland Science, 2002.


Image: (c) Can Stock Photo / olivier26

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