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Intracellular leucine-rich alpha-2-glycoprotein-1 competes with Apaf-1 for binding cytochrome c in protecting MCF-7 breast cancer cells from apoptosis

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Abstract

Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.

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Acknowledgements

The authors thank Tim Leonard for preparation of the figures, Celia Choh for help with the viability assays, and Samantha Van Hove for assisting with the lentivirus infections. This work was supported by grants from the Minnesota Medical Foundation and University of Minnesota Graduate School (to R.J.). The authors recognize the Center for Mass Spectrometry and Proteomics at the University of Minnesota and various supporting agencies, including the National Science Foundation for Major Research Instrumentation Grants 9871237 and NSF-DBI-0215759 used to purchase the instruments employed in this study and National Institutes of Health Grant R01 CA157971 (to A.K.).

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Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, MN, 55455, USA

    Ronald Jemmerson & Katherine Staskus

  2. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN, 55455, USA

    LeeAnn Higgins

  3. Department of Genetics, Cell Biology, and Biophysics, Masonic Cancer Center, Minneapolis, MN, 55455, USA

    Kathleen Conklin

  4. Department of Laboratory Medicine and Pathology, Masonic Cancer Center, Minneapolis, MN, 55455, USA

    Ameeta Kelekar

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  1. Ronald Jemmerson
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  3. LeeAnn Higgins
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  5. Ameeta Kelekar
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Contributions

RJ conceived, planned, and supervised the project; carried out transfections, sub-cellular fractionation experiments, and Western blotting; performed viability assays, SDS-PAGE, and immunostaining for confocal microscopy; and wrote the manuscript. KS assisted with lrg transfection and captured images by confocal microscopy. LH analyzed the MS data. K.C. prepared recombinant LRG shRNA lentivirus. AK performed nitrogen cavitation; provided advice on sub-cellular fractionation, transfections, and Western blotting; and edited the manuscript.

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Correspondence to Ronald Jemmerson.

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The University of Minnesota and/or R.J. have commercially licensed two of the monoclonal antibodies employed in this study. There are no other potentially conflicting interests.

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Jemmerson, R., Staskus, K., Higgins, L. et al. Intracellular leucine-rich alpha-2-glycoprotein-1 competes with Apaf-1 for binding cytochrome c in protecting MCF-7 breast cancer cells from apoptosis. Apoptosis 26, 71–82 (2021). https://doi.org/10.1007/s10495-020-01647-9

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