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Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

ABSTRACT: The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.

SUBMITTER: Eraso P

PROVIDER: S-EPMC3692421 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

Eraso Pilar P Portillo Francisco F Mazón María J MJ

PloS one 20130625 6

The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterize ...[more]

PMID: 23825623

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Project description:BackgroundWhile tumor suppressor p53 functions primarily as a transcription factor in the nucleus, cellular stress can cause p53 to translocate to the mitochondria and directly trigger a rapid apoptotic response. We have previously shown that fusing p53 (or its DNA binding domain, DBD, alone) to the mitochondrial targeting signal (MTS) from Bak or Bax can target p53 to the mitochondria and induce apoptosis in gynecological cancer cell lines including cervical cancer cells (HeLa; wt p53), ovarian cancer cells (SKOV-3; p53 267del non-expressing), and breast cancer cells (T47D; L194F p53 mutation). However, p53 with Bak or Bax MTSs have not been previously tested in cancers with strong dominant negative (DN) mutant p53 which are capable of inactivating wt p53 by homo-oligomerization. Since p53-Bak or Bax MTS constructs act as monomers, they are not subject to DN inhibition. For this study, the utility of p53-Bak or p53-Bax MTS constructs was tested for ovarian cancers which are known to have varying p53 statuses, including a strong DN contact mutant p53 (Ovcar-3 cells), a p53 DN structural mutant (Kuramochi cells), and a p53 wild type, low expressing cells (ID8).ResultsOur mitochondrial p53 constructs were tested for their ability to localize to the mitochondria in both mutant non-expressing p53 (Skov-3) and p53 structural mutant (Kuramochi) cell lines using fluorescence microscopy and a nuclear transcriptional activity assay. The apoptotic activity of these mitochondrial constructs was determined using a mitochondrial outer membrane depolarization assay (TMRE), caspase assay, and a late stage cell death assay (7-AAD). We also tested the possibility of using our constructs with paclitaxel, the current standard of care in ovarian cancer treatment. Our data indicates that our mitochondrial p53 constructs are able to effectively localize to the mitochondria in cancer cells with structural mutant p53 and induce apoptosis in many ovarian cancer cell lines with different p53 statuses. These constructs can also be used in combination with paclitaxel for an increased apoptotic effect.ConclusionsThe results suggest that targeting p53 to mitochondria can be a new strategy for ovarian cancer treatment.

Dataset Information

Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

Publications

Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

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