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Cited by 158Cholesterol and Lipid MetabolismSteroid Chemistry and BiochemistryLipid metabolism and biosynthesis

Regulation and immunolocalization of acyl-coenzyme A: cholesterol acyltransferase in mammalian cells as studied with specific antibodies

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Catherine C. Y., Chang, Chen, Jun, Thomas, Matthew A., Cheng, Dong, Veronica A., Del Priore, Newton, Roger S., Pape, Michael E., Chang, Ta-Yuan·1995·Journal of Biological Chemistry
DOI 10.1074/jbc.270.49.29532
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Abstract

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.

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  • Inflammatory signals such as LPS, pro-inflammatory cytokines, reactive oxygen species, advanced glycation end products, and oxidized LDL cause IKKβ phosphorylation; this phosphorylates IκBα, leading to its ubiquitination and degradation, which releases NF-κB to enter the nucleus and induce pro-inflammatory genes.

    “Extracellular inflammatory signals including lipopolysaccharide (LPS), pro-inflammatory cytokines, reactive oxygen species, advanced glycation end products, and oxidized low-density lipoprotein trigger the phosphorylation of IKKβ, which in turn phosphorylates IκBα for ubiquitination and degradation by proteasome [13–18].”

    From Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes (2013) · cites this paper as [20]

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  1. Daumerie, C. M., Woollett, L. A., and Dietschy, J. M. (1992) Proc. Natl. Acad. Sci.
  2. Goldstein, J. L., Dana, S. E., and Brown, M. S. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4288 -4292
  3. Sheng, Z., Otani, H., Brown, M. S., and Goldstein, J. L. (1995) Proc. Natl. Acad. Sci.
  4. Uelmen, P. J., Oka, K., Sullivan, M., Chang, C. C. Y., Chang, T. Y., and Chan, L. (1995) J. Biol. Chem., in press

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Authors
Catherine C. Y., C., Chen, J., Thomas, M. A., Cheng, D., Veronica A., D. P., Newton, R. S., Pape, M. E., & Chang, T.-Y.
Affiliations
Dartmouth College (US)
Corresponding author
Chang, Ta-Yuan
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1995-12
Volume
270
Issue
49
Pages
29532-29540
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.270.49.29532
OpenAlex
W1996953344
Cited by
158
References
45
Citations by year
2026: 1 · 2025: 2 · 2024: 1 · 2023: 4 · 2022: 3 · 2021: 3 · 2020: 4 · 2019: 3 · 2016: 1 · 2015: 7 · 2014: 6 · 2013: 1 · 2012: 3
FWCI
10.33
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (HL 36709)
Topics
  • Health Sciences › Medicine › Surgery › Cholesterol and Lipid Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Lipid metabolism and biosynthesis
Keywords
acyl-CoA:cholesterol acyltransferase, ACAT, polyclonal antibodies, immunolocalization, endoplasmic reticulum, cholesterol esterification, immunodepletion, human cell lines, CHO cells, HepG2 cells, fibroblasts
MeSH terms
Animals, Antibodies, Cell Line, Sterol O-Acyltransferase, Endoplasmic Reticulum, Cricetinae, Humans, Molecular Weight, Rabbits, Immunoblotting, CHO Cells

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