Characterization of a Bcl-X<sub>L</sub>-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells

Chen, Yan; Sternberg, Paul; Cai, Jiyang

doi:10.1167/iovs.07-1121

Cited by 13 publications

(11 citation statements)

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“…The PRL-1 binding proteins identified by Lee et al included TPD54, NDUFB8 (NADH:ubiquinone oxidoreductase subunit B8), and FKBP38. NDUFB8 and FKBP38 are known to have mitochondrial localizations [43]. Now, we have shown that TPD54 also has a mitochondrial distribution and interacts with PDH E1α in breast cancer cells, indicating its potentially important role in the metabolic regulation in cancer.…”

Section: Discussionmentioning

confidence: 86%

The novel function of tumor protein D54 in regulating pyruvate dehydrogenase and metformin cytotoxicity in breast cancer

Zhuang

Frazier

et al. 2019

Cancer Metab

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BackgroundThe role of tumor protein D54 in breast cancer has not been studied and its function in breast cancer remains unclear. In our previous pharmacogenomic studies using lymphoblastoid cell line (LCL), this protein has been identified to affect metformin response. Although metformin has been widely studied as a prophylactic and chemotherapeutic drug, there is still a lack of biomarkers predicting the response to metformin in breast cancer. In this study, we revealed the novel function of TPD54 in breast cancer through understanding how TPD54 altered the cancer cell sensitivity to metformin.MethodsThe role of TPD54 in altering cellular sensitivity to metformin treatment was carried out by either knockdown or overexpression of TPD54, followed by measuring cell viability and reactive oxygen species (ROS) production in MCF7 breast cancer cell line and breast cancer patient-derived xenografts. Functional analysis of TPD54 in breast cancer cells was demonstrated by studying TPD54 protein localization and identification of potential binding partners of TPD54 through immunoprecipitation followed by mass spectrometry. The effect of TPD54 on pyruvate dehydrogenase (PDH) protein regulation was demonstrated by western blot, immunoprecipitation, and site-directed mutagenesis.ResultsTPD54 inhibited colony formation and enhanced cellular sensitivity to metformin treatment in MCF7 cells and breast cancer patient-derived xenografts. Mechanistic study indicated that TPD54 had mitochondrial localization, bound to and stabilized pyruvate dehydrogenase E1α by blocking pyruvate dehydrogenase kinase 1 (PDK1)-mediated serine 232 phosphorylation. TPD54 knockdown increased PDH E1α protein degradation and led to decreased PDH enzyme activity, which reduced mitochondrial oxygen consumption and reactive oxygen species (ROS) production, thus contributing to the resistance of breast cancer cells to metformin treatment.ConclusionWe have discovered a novel mechanism by which TPD54 regulates pyruvate dehydrogenase and affects the sensitivity of breast cancer to metformin treatment. Our findings highlight the important post-translational regulation of PDK1 on PDH E1α and the potential application of TPD54 as a biomarker for selecting tumors that may be sensitive to metformin therapy. These provide new insights into understanding the regulation of PDH complexes and the resistance mechanisms of cancer cells to metformin treatment.Electronic supplementary materialThe online version of this article (10.1186/s40170-018-0193-4) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 86%

The novel function of tumor protein D54 in regulating pyruvate dehydrogenase and metformin cytotoxicity in breast cancer

Zhuang

Frazier

et al. 2019

Cancer Metab

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“…One milligram of the cell lysate was incubated overnight with 20 μ L anti–Hsc70 antibody (200 μ g/mL; Santa Cruz Biotechnology, Santa Cruz, CA) or 15 μ L anti–HNE antibody (200 μ g/mL; R&D Systems, Minneapolis, MN). Protein G-agarose beads (Upstate, Lake Placid, NY) were used to pull down the antibody and the bound proteins 26. After stringent washes, the immunoprecipitates were boiled in 2× Laemmli sample buffer (Bio-Rad), separated by 12% SDS-polyacrylamide gel, and subjected to Western blot analyses.…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylinositol 3 Kinase Pathway and 4-Hydroxy-2-Nonenal-Induced Oxidative Injury in the RPE

Chen

Sternberg

et al. 2009

Invest. Ophthalmol. Vis. Sci.

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Purpose 4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation product in the retina and the retinal pigment epithelium. The purpose of the present study was to investigate how NF-E2–related factor-2 (Nrf2) and phosphatidylinositol 3 (PI3K) pathways affect the responses of cultured human retinal pigment epithelial (RPE) cells to 4-HNE. Methods Cultured ARPE-19 cells were treated with different concentrations of 4-HNE and a PI3K inhibitor, LY294002. Intracellular glutathione (GSH) was measured by high-performance liquid chromatography (HPLC). The transcriptional activity of Nrf2 was measured by dual luciferase assay after transient transfection with reporter plasmids. The mRNA level of glutamate cysteine ligase (GCL) was quantified by real-time RT-PCR. Formation of HNE adduct on heat shock cognate protein 70 (Hsc70) was measured by immunoprecipitation and Western blot analyses. Results Treatment with 4-HNE increased Nrf2 activity and GSH synthesis in a dose-dependent manner in cultured RPE cells. The modulatory subunit of GCL was upregulated by 4-HNE. Antioxidant responses were largely abolished by pretreatment with LY294002. The modification of Hsc70 by 4-HNE was increased when PI3K was inhibited. Conclusions The Nrf2-dependent antioxidant response protects against 4-HNE toxicity, and this protective mechanism is dependent on the functions of the PI3K pathway.

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“…24 Detection of senescence-associate heterochromatin foci (SAHF) was performed as described in the literature. 25,26 Briefly cells were fixed with 4% formaldehyde and then permeabilized with 0.2% Triton X-100.…”

Section: Indirect Immunofluorescence Staining and Confocal Microscopymentioning

confidence: 99%

Subcellular Distribution and Activity of Mechanistic Target of Rapamycin in Aged Retinal Pigment Epithelium

Zhao

et al. 2014

Investigative Ophthalmology & Visual Science

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Characterization of a Bcl-X_L-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells

Abstract: FKBP8 and its novel splice variant are Bcl-XL-interacting proteins and regulate the apoptotic signaling pathways in the RPE.

Cited by 13 publications

References 47 publications

The novel function of tumor protein D54 in regulating pyruvate dehydrogenase and metformin cytotoxicity in breast cancer

The novel function of tumor protein D54 in regulating pyruvate dehydrogenase and metformin cytotoxicity in breast cancer

Phosphatidylinositol 3 Kinase Pathway and 4-Hydroxy-2-Nonenal-Induced Oxidative Injury in the RPE

Subcellular Distribution and Activity of Mechanistic Target of Rapamycin in Aged Retinal Pigment Epithelium

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Characterization of a Bcl-XL-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells

Abstract: FKBP8 and its novel splice variant are Bcl-XL-interacting proteins and regulate the apoptotic signaling pathways in the RPE.

Cited by 13 publications

References 47 publications

The novel function of tumor protein D54 in regulating pyruvate dehydrogenase and metformin cytotoxicity in breast cancer

The novel function of tumor protein D54 in regulating pyruvate dehydrogenase and metformin cytotoxicity in breast cancer

Phosphatidylinositol 3 Kinase Pathway and 4-Hydroxy-2-Nonenal-Induced Oxidative Injury in the RPE

Subcellular Distribution and Activity of Mechanistic Target of Rapamycin in Aged Retinal Pigment Epithelium

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Characterization of a Bcl-X_L-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells