1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

Intracellular cholesterol trafficking is dependent upon NPC2 interaction with Lysobisphosphatidic Acid

Research Article
  • Cited 16
  • Views 2,282
  • Annotations
Cite this article as: eLife 2019;8:e50832 doi: 10.7554/eLife.50832

Abstract

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files

The following previously published data sets were used

Article and author information

Author details

  1. Leslie A McCauliff

    Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Annette Langan

    Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Ran Li

    Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Olga Ilnytska

    Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Debosreeta Bose

    Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Miriam Waghalter

    Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Kimberly Lai

    Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Peter C Kahn

    Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Judith Storch

    Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
    For correspondence
    storch@sebs.rutgers.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5482-1777

Funding

Ara Parseghian Medical Research Foundation

  • Olga Ilnytska
  • Judith Storch

American Heart Association (11PRE7330012)

  • Leslie A McCauliff

American Heart Association (18CDA34110230)

  • Olga Ilnytska

American Heart Association (14GRNT19990014)

  • Judith Storch

National Institutes of Health (GM 1125866)

  • Judith Storch

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Publication history

  1. Received: August 4, 2019
  2. Accepted: October 2, 2019
  3. Accepted Manuscript published: October 3, 2019 (version 1)
  4. Version of Record published: November 14, 2019 (version 2)

Copyright

© 2019, McCauliff et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,282
    Page views
  • 407
    Downloads
  • 16
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Fatima Alghoul et al.
    Research Article

    During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The ‘start-stop’ sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.

    1. Biochemistry and Chemical Biology
    M'Lynn E Fisher et al.
    Research Article

    The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.