Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin

  1. Rima S Chakrabarti
  2. Sally A Ingham
  3. Julia Kozlitina
  4. Austin Gay
  5. Jonathan C Cohen
  6. Arun Radhakrishnan
  7. Helen H Hobbs  Is a corresponding author
  1. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
  2. University of Texas Southwestern Medical Center, United States

Abstract

Cholesterol partitions into accessible and sequestered pools in cell membranes. Here we describe a new assay using fluorescently-tagged anthrolysin O, a cholesterol-binding bacterial toxin, to measure accessible cholesterol in human red blood cells (RBCs). Accessible cholesterol levels were stable within individuals but varied >10-fold among individuals. Significant variation was observed among ethnic groups (Blacks>Hispanics>Whites). Variation in accessibility of RBC cholesterol was unrelated to the cholesterol content of RBCs or plasma, but was associated with the phospholipid composition of the RBC membranes and with plasma triglyceride levels. Pronase treatment of RBCs only modestly altered cholesterol accessibility. Individuals on hemodialysis, who have an unexplained increase in atherosclerotic risk, had significantly higher RBC cholesterol accessibility. Our data indicate that RBC accessible cholesterol is a stable phenotype with significant inter-individual variability. Factors both intrinsic and extrinsic to the RBC contribute to variation in its accessibility. This assay provides a new tool to assess cholesterol homeostasis among tissues in humans.

Article and author information

Author details

  1. Rima S Chakrabarti

    Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  2. Sally A Ingham

    Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  3. Julia Kozlitina

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  4. Austin Gay

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  5. Jonathan C Cohen

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  6. Arun Radhakrishnan

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7266-7336
  7. Helen H Hobbs

    Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    helen.hobbs@utsouthwestern.edu
    Competing interests
    Helen H Hobbs, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8700-9897

Funding

Howard Hughes Medical Institute

  • Rima S Chakrabarti
  • Sally A Ingham
  • Helen H Hobbs

National Institutes of Health (PO1 HL20948)

  • Austin Gay
  • Jonathan C Cohen
  • Arun Radhakrishnan
  • Helen H Hobbs

Welch Foundation (I-1793)

  • Arun Radhakrishnan

American Heart Association (12SDG12040267)

  • Arun Radhakrishnan

National Institutes of Health (5T32-GM008203)

  • Austin Gay

National Institutes of Health (UL1TR001105)

  • Julia Kozlitina
  • Helen H Hobbs

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

Ethics

Human subjects: All study protocols were approved by the Institutional Review Board (IRB) of the University of Texas Southwestern Medical Center, and all subjects provided written informed consent. Each participant completed a detailed staff-administered survey, including questions about socioeconomic status, medical history and medication use. Ancestry was self-reported.

Reviewing Editor

  1. Stephen G Young, University of California, Los Angeles, United States

Publication history

  1. Received: November 16, 2016
  2. Accepted: January 28, 2017
  3. Accepted Manuscript published: February 7, 2017 (version 1)
  4. Accepted Manuscript updated: February 8, 2017 (version 2)
  5. Version of Record published: February 23, 2017 (version 3)

Copyright

© 2017, Chakrabarti 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

  • 2,477
    Page views
  • 473
    Downloads
  • 29
    Citations

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

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)

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

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

  1. Rima S Chakrabarti
  2. Sally A Ingham
  3. Julia Kozlitina
  4. Austin Gay
  5. Jonathan C Cohen
  6. Arun Radhakrishnan
  7. Helen H Hobbs
(2017)
Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin
eLife 6:e23355.
https://doi.org/10.7554/eLife.23355
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Andrea Volante, Juan Carlos Alonso, Kiyoshi Mizuuchi
    Research Article Updated

    Three-component ParABS partition systems ensure stable inheritance of many bacterial chromosomes and low-copy-number plasmids. ParA localizes to the nucleoid through its ATP-dependent nonspecific DNA-binding activity, whereas centromere-like parS-DNA and ParB form partition complexes that activate ParA-ATPase to drive the system dynamics. The essential parS sequence arrangements vary among ParABS systems, reflecting the architectural diversity of their partition complexes. Here, we focus on the pSM19035 plasmid partition system that uses a ParBpSM of the ribbon-helix-helix (RHH) family. We show that parSpSM with four or more contiguous ParBpSM-binding sequence repeats is required to assemble a stable ParApSM-ParBpSM complex and efficiently activate the ParApSM-ATPase, stimulating complex disassembly. Disruption of the contiguity of the parSpSM sequence array destabilizes the ParApSM-ParBpSM complex and prevents efficient ATPase activation. Our findings reveal the unique architecture of the pSM19035 partition complex and how it interacts with nucleoid-bound ParApSM-ATP.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Chris Furlan, Nipa Chongdar ... James A Birrell
    Research Article Updated

    Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers connected via a [4Fe–4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron–sulfur cluster domain: a ‘closed bridge’ and an ‘open bridge’ conformation, where a Zn2+ site may act as a ‘hinge’ allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site.