THESIS
2022
1 online resource (xiv, 161 pages) : illustrations (some color)
Abstract
BCL-2 proteins are tail-anchored (TA) proteins localized to the mitochondrial outer membrane
(MOM). Although their roles in regulating apoptosis are well understood, how they are inserted
into the mitochondria remains to be characterized. As many TA proteins are inserted by an
ASNA1-mediated pathway, here I examined if ASNA1 is involved in BCL-2 protein insertion.
Furthermore, despite the pivotal role of ASNA1 in regulating many cellular functions, the
essentiality of ASNA1 in cancer and normal cells is still to be fully addressed. Using a
conditional knockout system, I discovered that ASNA1 was essential for the survival of HeLa
and RPE-1 cells. MCL-1, an anti-apoptotic member of the BCL-2 family, was found to interact
with ASNA1 using co-immunoprecipitation assays. Moreover, a positiv...[
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BCL-2 proteins are tail-anchored (TA) proteins localized to the mitochondrial outer membrane
(MOM). Although their roles in regulating apoptosis are well understood, how they are inserted
into the mitochondria remains to be characterized. As many TA proteins are inserted by an
ASNA1-mediated pathway, here I examined if ASNA1 is involved in BCL-2 protein insertion.
Furthermore, despite the pivotal role of ASNA1 in regulating many cellular functions, the
essentiality of ASNA1 in cancer and normal cells is still to be fully addressed. Using a
conditional knockout system, I discovered that ASNA1 was essential for the survival of HeLa
and RPE-1 cells. MCL-1, an anti-apoptotic member of the BCL-2 family, was found to interact
with ASNA1 using co-immunoprecipitation assays. Moreover, a positive correlation in protein
expression was identified between MCL-1 and ASNA1. Depletion of ASNA1 downregulated
the expression of MCL-1. Conversely, overexpression of ASNA1 stabilized MCL-1 protein.
Moreover, knockout of ASNA1 sensitized cells to a MCL-1 inhibitor, which suggests a
potential synergism between ASNA1 and MCL-1 in inducing cell death. In addition to ASNA1,
the essentiality of BCL-2 and BCL-XL in cancer cells was also addressed using the condition
knockout system. BCL-XL, but not BCL-2, was essential for cell survival in unperturbed and
mitotic-arrested HeLa cells. Knockout of BCL-XL promoted cell death in a BAX-dependent
manner. Surprisingly, co-depletion of BCL-2 and BCL-XL synergistically induced cell death,
implicating their functional redundancy in cell survival. Altogether, these results extend our
understanding of the regulation of BCL-2 proteins and provide insights into therapeutics
development for targeting BCL-2 proteins.
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