BARD1 translocation to mitochondria correlates with Bax oligomerization, loss of mitochondrial membrane potential, and apoptosis

The breast cancer regulatory protein-1 (BRCA1)-associated RING domain 1 (BARD1) gene is mutated in a subset of breast/ovarian cancers. BARD1 functions as a heterodimer with BRCA1 in nuclear DNA repair. BARD1 also has a BRCA1-independent apoptotic activity. Here we investigated the link between cytoplasmic localization and apoptotic function of BARD1. We used immunofluorescence microscopy and deconvolution analysis to resolve BARD1 cytoplasmic staining patterns and detected endogenous BARD1 at mitochondria. BARD1 was also detected in mitochondrial cell fractions by immunoblotting. The targeting of BARD1 to mitochondria was modestly stimulated by DNA damage and did not require BRCA1 as indicated by RNA interference and peptide-competition experiments. Transiently expressed yellow fluorescence protein-BARD1 localized to mitochondria, and the targeting sequences were mapped to both the N and C terminus of BARD1. Ectopic yellow fluorescence protein-BARD1 induced apoptosis and loss of mitochondrial membrane potential in MCF-7 breast tumor cells. BARD1 apoptotic function was associated with stimulation of Bax oligomerization at mitochondria. This distinguishes it from BRCA1, which is pro-apoptotic but did not induce Bax oligomerization. The cancer-associated BARD1 splice-variant DeltaRIN (lacks the BRCA1 binding domain and ankyrin repeats) was recruited to mitochondria but did not stimulate apoptosis or alter membrane permeability. We propose that BARD1 has two main sites of action in its cellular response to DNA damage, the nucleus, where it promotes cell survival through DNA repair, and the mitochondria, where BARD1 regulates apoptosis.     

Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.     

RecQL4 cytoplasmic localization: Implications in mitochondrial DNA oxidative damage repair

RecQL4, one of the five human RecQ helicases, is crucial for genomic stability and RecQL4 when mutated leads to premature aging phenotypes in humans. Unlike other human RecQ helicases, RecQL4 is found both in the nucleus and the cytoplasm. While the nuclear localization signal (NLS) and the retention domain at the N-terminus are responsible for the nuclear localization of RecQL4, the signal for its cytoplasmic localization is essentially unknown. In this study, two functional nuclear exporting signals (NESs; pNES2 and pNES3) were identified at the C-terminus of RecQL4. Deletion of pNES2 drastically diminished the cytoplasmic localization of RecQL4. Strikingly, addition of ubiquitination tail at the C-terminus of RecQL4 substantially enriched the cytoplasmic fraction of RecQL4 only in the presence of functional pNES2. Immunofluorescence studies revealed that the cytoplasmic RecQL4 was localized in mitochondria. Consistent with its mitochondrial localization, a regulatory role for RecQL4 in the maintenance of mitochondrial DNA (mtDNA) copy number was demonstrated. Elevation of ectopic expression of RecQL4 increased the mtDNA copy number in HEK293 cells while RecQL4 knock down markedly decreased the mtDNA copy number in U2OS cells. Additionally, a substantially increased level of mitochondrial superoxide production, and a markedly decreased repair capacity for oxidative DNA damage were observed in the mitochondria of both RecQL4 deficient human fibroblasts and RecQL4-suppressed cancer cells. These data strongly suggest a regulatory role for RecQL4 in mitochondrial stability and function. Collectively, our study demonstrates that NES-mediated RecQL4 export to the cytoplasm is essential for the maintenance of mitochondrial genome stability.     

TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells

The protein transduction domain (PTD) from the HIV-1 TAT protein has been widely utilized to deliver biologically active macromolecules, including full-length proteins, into a variety of cell types in vitro and in vivo. Without additional targeting signals, the intracellular localization of the proteins delivered in this fashion appears to be cytoplasmic, nuclear or, as recently reported, endosomal. In this study, we show that the presence of the mitochondrial targeting signal (MTS) from hMnSOD on the N-terminus of TAT-fusion proteins directs them into mitochondria of breast cancer cells. We generated and purified fusion proteins containing GFP (MTS-GFP-TAT) or Exonuclease III (MTS-ExoIII-TAT) from Escherichia coli. The results of Western blots of subcellular fractions and fluorescent microscopic analyses revealed efficient protein transduction and mitochondrial localization of the fusion proteins. Specific exonuclease activity was found in the mitochondrial extracts isolated from MTS-ExoIII-TAT transduced cells. This increased exonuclease activity reduced the repair of mtDNA damage following oxidative stress. This diminished mtDNA repair led to a decrease in survival of breast cancer cells. Thus, the present study demonstrates the applicability of this new approach for intramitochondrial targeting of TAT-fusion proteins capable of modulating mitochondrial function and cell survival.     

The T cell receptor gamma chain alternate reading frame protein (TARP), a prostate-specific protein localized in mitochondria

We previously showed that mRNA encoding TARP (T cell receptor gamma chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.     

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.     

The 90-kDa heat-shock protein (Hsp90)-binding immunophilin FKBP51 is a mitochondrial protein that translocates to the nucleus to protect cells against oxidative stress

Confocal microscopy images revealed that the tetratricopeptide repeat motif (TPR) domain immunophilin FKBP51 shows colocalization with the specific mitochondrial marker MitoTracker. Signal specificity was tested with different antibodies and by FKBP51 knockdown. This unexpected subcellular localization of FKBP51 was confirmed by colocalization studies with other mitochondrial proteins, biochemical fractionation, and electron microscopy imaging. Interestingly, FKBP51 forms complexes in mitochondria with the glucocorticoid receptor and the Hsp90/Hsp70-based chaperone heterocomplex. Although Hsp90 inhibitors favor FKBP51 translocation from mitochondria to the nucleus in a reversible manner, TPR domain-deficient mutants of FKBP51 are constitutively nuclear and fully excluded from mitochondria, suggesting that a functional TPR domain is required for its mitochondrial localization. FKBP51 overexpression protects cells against oxidative stress, whereas FKBP51 knockdown makes them more sensitive to injury. In summary, this is the first demonstration that FKBP51 is a major mitochondrial factor that undergoes nuclear-mitochondrial shuttling, an observation that may be related to antiapoptotic mechanisms triggered during the stress response.     

BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export

BRCA1 is a tumor suppressor with several important nuclear functions. BRCA1 has no known cytoplasmic functions. We show here that the two previously identified nuclear localization signals (NLSs) are insufficient for nuclear localization of BRCA1 due to the opposing action of an NH2-terminal nuclear export signal. In transfected breast cancer cells, BRCA1 nuclear localization requires both the NLSs and NH2-terminal RING domain region; mutating either of these sequences shifts BRCA1 to the cytoplasm. The BRCA1 RING element mediates nuclear import via association with BARD1, and this is not affected by cancer-associated RING mutations. Moreover, BARD1 directly masks the BRCA1 nuclear export signal, and the resulting block to nuclear export is requisite for efficient import and nuclear localization of ectopic and endogenous BRCA1. Our results explain why BRCA1 exon 11 splice variants, which lack the NLSs but retain the RING domain, are frequently detected in the nucleus and in nuclear foci in vivo. In fact, co-expression of BARD1 promoted formation of DNA damage-induced nuclear foci comprising ectopic wild-type or NLS-deficient BRCA1, implicating BARD1 in nuclear targeting of BRCA1 for DNA repair. Our identification of BARD1 as a BRCA1 nuclear chaperone has regulatory implications for its reported effects on BRCA1 protein stability, ubiquitin ligase activity, and DNA repair.     

Macromolecular crowding effect is critical for maintaining SIRT1's nuclear localization in cancer cells

SIRT1 is a principle class III histone deacetylase which exhibits versatile functions in stress response, development, and pathological processes including cancer. Although SIRT1 deacetylates a wide range of nuclear and cytoplasmic proteins, its subcellular localization in cancer cells has been controversial. In this study, we uncovered the inconsistent reports about SIRT1 subcellular localization is partially due to different analysis approaches. While immunofluorescence and live cell imaging reveal a predominant nuclear localization of SIRT1, conventional cell fractionation often results in a severe leaking of SIRT1 into the cytoplasm. Such a leakage is mainly caused by loss of cytoplasmic macromolecular crowding effect as well as hypotonic dwelling during the isolation of the nuclei. We also developed an improved cell fractionation procedure which maintains SIRT1 in its original subcellular localization. Analyzing a variety of human cancer cell lines using this approach and other methods demonstrate that SIRT1 predominantly localizes to the nucleus in cancer cells.     

FANCL supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and Oligomycin and Antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner.     

A rapid method for the fractionation of isolated hepatocytes

The fractionation of rat liver hepatocytes using a mechanical disruption technique followed by centrifugation is reported; the whole procedure requires approximately 10 min. Marker enzyme distribution data are in good agreement with distribution data from standard techniques connected with the production of three subcellular fractions—cytoplasmic, mitochondrial, and microsomal. Electrophoretic analysis of the mitochondrial and microsomal fractions show total band correspondence between the fractions produced by the method and traditional techniques. Examination of the fractions by electron microscopy supports the view that the mitochondrial fraction is comprised of both intact mitochondria and mitochondria from which the outer membrane has been removed. The microsomal fraction contains discrete vesicles derived from both rough and smooth endoplasmic reticulum.     

Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis

Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. However, the molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells.     

A comprehensive approach to determining BER capacities and their change with aging in Drosophila melanogaster mitochondria by oligonucleotide microarray

DNA repair mechanisms are key components for the maintenance of the essential mitochondrial genome. Among them, base excision repair (BER) processes, dedicated in part to oxidative DNA damage, are individually well known in mitochondria. However, no large view of these systems in differential physiological conditions is available yet. Combining the use of pure mitochondrial fractions and a multiplexed oligonucleotide cleavage assay on a microarray, we demonstrated that a large range of glycosylase activities were present in Drosophila mitochondria. Most of them were quantitatively different from their nuclear counterpart. Moreover, these activities were modified during aging.