scholarly journals Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Michael Nguyen Trinh ◽  
Michael S Brown ◽  
Joachim Seemann ◽  
Joseph L Goldstein ◽  
Feiran Lu
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayer ◽  
Full Length ◽  
Cholesterol Transport ◽  
Transmembrane Helices ◽  
Terminal Domain ◽  
Data Support ◽  
Covalently Linked ◽  
Niemann Pick ◽  
Polytopic Membrane Protein

Niemann-Pick C1 (NPC1) is a polytopic membrane protein with 13 transmembrane helices that exports LDL-derived cholesterol from lysosomes by carrying it through the 80 Å glycocalyx and the 40 Å lipid bilayer. Transport begins when cholesterol binds to the N-terminal domain (NTD) of NPC1, which projects to the surface of the glycocalyx. Here, we reconstitute cholesterol transport by expressing the NTD as a fragment separate from the remaining portion of NPC1. When co-expressed, the two NPC1 fragments reconstitute cholesterol transport, indicating that the NTD has the flexibility to interact with the remaining parts of NPC1 even when not covalently linked. We also show that cholesterol can be transferred from the NTD of one full-length NPC1 to another NPC1 molecule that lacks the NTD. These data support the hypothesis that cholesterol is transported through interactions between two or more NPC1 molecules.

scholarly journals Signature of N-terminal domain (NTD) structural re-orientation in NPC1 for proper alignment of cholesterol transport: Molecular dynamics study with mutation

2020 ◽  
Author(s):  
Hye-Jin Yoon ◽  
Hyunah Jeong ◽  
Hyung Ho Lee ◽  
Soonmin Jang
Keyword(s):  
Molecular Dynamics ◽  
Full Length ◽  
Cholesterol Transport ◽  
Cholesterol Trafficking ◽  
Terminal Domain ◽  
Loop Region ◽  
Niemann Pick ◽  
Dynamics Simulations ◽  
And Function ◽  
Lumenal Domain

AbstractThe lysosomal membrane protein NPC1 (Niemann-Pick type C1) and NPC2 (Niemann-Pick type C2) are main players of cholesterol control in lysosome and it is known that mutation on these proteins leads to cholesterol trafficking related disease, called Niemann-Pick disease type C (NPC) disease. The mutation R518W or R518Q on NPC1 is one of such disease-related mutations, causing reduced cholesterol transport by half, resulting in accumulation of cholesterol and lipids in late endosomal/lysosomal region of the cell. Even though there has been significant progress in understanding cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of full length NPC1 in 2016, many details such as interaction of full length NPC1 with NPC2, molecular motions responsible for cholesterol transport during and after this interaction, and structure and function relations of many mutations are still not well understood.We report the extensive molecular dynamics simulations to gain insight into the structure and motions of NPC1 lumenal domain for cholesterol transport and disease behind the mutation (R518W). It is found that the mutation induces structural shift of NTD (N-terminal domain), toward the loop region in MLD (middle lumenal domain), which is believed to play central role in interaction with NPC2 protein, such that the interaction with NPC2 protein might be less favorable compare to wild NPC1. Also, the simulation indicates the possible re-orientation of the NTD, aligning to form an internal tunnel, after receiving the cholesterol from NPC2 with wild NPC1 unlike the mutated one, a possible pose for further action in cholesterol trafficking. We believe the current study can provide better understanding on the cholesterol transport by NPC1, especially the role of NTD of NPC1, in combination with NPC2 interaction.Synopsismodeling study of cholesterol binding protein NPC1

scholarly journals Structure and reconstitution of a hydrolase complex that releases peptidoglycan from the membrane after polymerization

2020 ◽  
Author(s):  
Kaitlin Schaefer ◽  
Tristan W. Owens ◽  
Julia E. Page ◽  
Marina Santiago ◽  
Daniel Kahne ◽  
...  
Keyword(s):  
Cell Wall ◽  
Membrane Protein ◽  
Transmembrane Helices ◽  
Cell Wall Assembly ◽  
Lipid Ii ◽  
Peptidoglycan Cell Wall ◽  
The Matrix ◽  
Peptide Precursor ◽  
Polytopic Membrane Protein ◽  
Wall Assembly

Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix2–4. A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix.

scholarly journals Triazoles inhibit cholesterol export from lysosomes by binding to NPC1

2016 ◽  
Vol 114 (1) ◽  
pp. 89-94 ◽  
Author(s):  
Michael N. Trinh ◽  
Feiran Lu ◽  
Xiaochun Li ◽  
Akash Das ◽  
Qiren Liang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Binding Site ◽  
Point Mutation ◽  
Lipid Bilayer ◽  
Cross Linking ◽  
Intact Cells ◽  
In Vitro System ◽  
Niemann Pick ◽  
Initial Binding

Niemann–Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764–4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.

scholarly journals Lipid-bound ApoE3 self-assemble into elliptical disc-shaped particles

2020 ◽  
Author(s):  
Andreas Haahr Larsen ◽  
Nicolai Tidemand Johansen ◽  
Michael Gajhede ◽  
Lise Arleth ◽  
Søren Roi Midtgaard
Keyword(s):  
Lipid Bilayer ◽  
Full Length ◽  
Cholesterol Transport ◽  
X Ray ◽  
Negative Stain ◽  
X Ray Scattering ◽  
Repeat Domain ◽  
Structural Modulation ◽  
Negative Stain Electron Microscopy ◽  
Repeat Segment

AbstractApolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.

scholarly journals Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer

1997 ◽  
Vol 327 (1) ◽  
pp. 37-43 ◽  
Author(s):  
S. PARVATHY ◽  
Sylvester Y. OPPONG ◽  
Eric H. KARRAN ◽  
Derek R. BUCKLE ◽  
Anthony J. TURNER ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Angiotensin Converting Enzyme ◽  
Lipid Bilayer ◽  
Proteolytic Processing ◽  
Full Length ◽  
Converting Enzyme ◽  
Pig Kidney ◽  
Zinc Metalloprotease ◽  
Metalloprotease Inhibitors ◽  
Triton X

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 μM, whereas TAPI-2 was slightly less potent (IC50 18 μM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2′ substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is related to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton X-100 and CHAPS, but not with n-octylβ-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.

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