I seem incapable of getting this grant funded, so I am putting it out here in case someone else wants to pick up all or part of the project. There are no figures here (despite references in the text), but most of the figures/data has now been published here. If anyone is interested, email me and I will send you the full proposal with figures.
Note added in proof: to clarify, I have moved on from this project. My intent here is that someone else might be interested in doing some or all of this work.
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are universal regulators of intracellular calcium signaling. IP3Rs are calcium channels present primarily on endoplasmic reticulum (ER) membranes, and are gated by the second messenger inositol 1,4,5-trisphosphate (IP3). Generation of IP3 by the enzyme phospholipase C is stimulated by activation of many cell surface receptors, including G-protein coupled receptors, tyrosine kinase receptors, and death receptors . Our laboratory is interested in the regulation of IP3R channels during apoptosis, and in particular the role of protein binding partners in regulating IP3R-mediated apoptotic calcium release. In preliminary experiments, we have found that the breast cancer type 1 susceptibility protein (BRCA1) binds and regulates IP3R activity. Loss of function mutations in BRCA1 are causative in a majority of familial breast and ovarian cancers, and sporadic breast and ovarian tumors often are associated with BRCA1 downregulation. BRCA1 is thought to function primarily in the nucleus as a component of the DNA recombination/repair machinery to maintain genomic integrity. However, it is also well established that BRCA1 is also present in non-nuclear compartments , and an increasing amount of evidence suggests that BRCA1 present in non-nuclear compartments mediates apoptotic cell death under conditions of cytotoxic/genotoxic stress. Thus, an attractive hypothesis has been put forward that BRCA1 integrates both genome repair and cell death signaling pathways to protect against cancer development . Remarkably, the mechanisms by which BRCA1 stimulates apoptotic cell death are almost entirely unknown. In preliminary studies we have found that BRCA1 localizes to the ER, binds to IP3R calcium channels, and regulates apoptotic calcium release . These results suggest that the BRCA1 protein directly modulates intracellular calcium homeostasis and apoptosis, possibly by enhancing pro-apoptotic IP3R signaling. We hypothesize that BRCA1 translocates to the ER and binds IP3R channels during apoptosis, increasing apoptotic calcium release and cell death. We will test this hypothesis in two Specific Aims.
Specific Aim 1: Determine if BRCA1 directly modulates apoptotic calcium release from IP3R calcium channels.
Many pro-apoptotic proteins bind to and modulate IP3R activity during apoptosis . We hypothesize that BRCA1 binds to and activates IP3R channels during apoptosis. We will examine in detail the functional consequences of BRCA1 binding to IP3R channels in vitro and in intact cells undergoing cell death. The structural requirements for binding will be mapped by mutagenesis, and the functional effects of BRCA1 on IP3R activity will be investigated in intact cells and with single channel recording. Finally, cell permeant peptides based upon the binding interface between BRCA1 and IP3R will be developed to examine if they can activate the IP3R in vitro and in intact cells to activate cell death pathways.
Specific Aim 2: Determine the mechanism by which BRCA1 is recruited to ER membranes.
Many studies have suggested that BRCA1 interacts with subcellular organelles outside of the nucleus such as mitochondria (reviewed in [7, 8, 10]), however, the mechanism(s) are largely unknown. We hypothesize that BRCA1 translocates to the ER during apoptosis via a lipid binding motif, and this translocation is critical for its pro-apoptotic function. We will systematically analyze the dynamic subcellular distribution of BRCA1 under resting conditions and during apoptosis using biochemical and optical techniques. Our preliminary data indicates that the BRCA1 BRCT domain has lipid binding activity. We have found that BRCA1 binds to phosphatidic acid (PA), and furthermore that PA is produced by PKR-like ER kinase (PERK) at ER membranes during cell death. We will test whether cell death stimuli activate the ER stress response, PERK activation, and subsequent recruitment of BRCA1 to ER membranes using therapeutically relevant stimuli such as cisplatin and paclitaxel.
If we are able to establish that BRCA1 mediates its pro-apoptotic effects by enhancing IP3R-mediated calcium release, this would identify a novel pathway by which BRCA1 exerts its tumor suppressor activities. Furthermore, we have identified a novel lipid binding motif in BRCA1 that specifically targets BRCA1 to the ER membranes during cell death. Thus, we have identified two previously uncharacterized functional activities for BRCA1 with relevance to apoptotic signaling in cancer. Our model would strongly support the hypothesis that BRCA1, like p53, is central to integrating the DNA damage response to cell death. These studies, if successful, will have significant implications for understanding cancer progression in both hereditary and sporadic breast and ovarian cancers. Furthermore, these studies may reveal the IP3R as a novel therapeutic target.
Calcium, IP3Rs, and apoptosis
There are many regulatory proteins and pathways which participate in programmed (apoptotic) cell death. Alterations in calcium homeostasis is a prominent feature of many models of cell death, and has been appreciated for more than a quarter century [11-14]. In particular, decreases in ER calcium and increases in cytosolic and mitochondrial calcium have profound effects on the apoptotic program, including stimulation of release of apoptotic factors from mitochondria such as cytochrome c and Smac/Diablo. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular calcium channels which reside on ER membranes in close apposition to mitochondria, and calcium release from IP3R can directly influence mitochondrial calcium levels [15, 16]. In normal physiology, calcium released from IP3Rs results in a direct stimulation of mitochondrial metabolism . Therefore, it is logical to suspect that IP3R channels can directly influence mitochondrial calcium levels during apoptosis. In support of this, anti-sense or genetic knockout of IP3R gene(s) results in cytoprotection in multiple apoptotic modalities, including g-irradiation, growth factor deprivation, B-cell antigen and dexamethasone stimulation [18, 19], and furthermore apoptotic stimuli result in IP3R-dependent increases in mitochondrial calcium and subsequent permeability (reviewed in ). It is also known that both pro- and anti-apoptotic Bcl-2 proteins exert some of their effects by regulating ER calcium release via the IP3R [21-24]. In addition to regulating mitochondrial release of pro-apoptotic factors, IP3R-dependent increases in calcium also orchestrate many other aspects of apoptotic signaling, including activation of kinases, phosphatases, endonucleases, flippases, and proteases [6, 9, 25, 26].
We have shown that IP3R activity is regulated during apoptosis by canonical regulators such as calcium and IP3, and by novel interacting proteins such as cytochrome c [3-5, 27-32]. Other groups have shown that IP3R activity is regulated during apoptosis by caspases [33-35], Bcl-2 family members [21, 36, 37], and tumor suppressors such as promyelocytic leukemia (PML)  and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) . We and others have proposed that the multitude of binding partners of the IP3R is central to its role a calcium signal integrator [6, 40], orchestrating all calcium-dependent cellular responses. As such, it can be predicted that dysregulation of IP3R-mediated apoptosis would be associated with disease states such as cancer (defective cell death) or neurodegeneration (excessive cell death). However, a direct role for IP3Rs in cancer development is still unclear. Hereditary breast cancer is associated with mutations in breast cancer type 1 susceptibility protein (BRCA1), and sporadic cancers often downregulate BRCA1 expression. A multitude of studies have found that BRCA1, in addition to its role in maintaining genome integrity, has pro-apoptotic activity, but the mechanisms are essentially unknown (reviewed in [7, 8, 10]). We have found in preliminary studies that BRCA1 directly interacts with IP3Rs and modulates apoptotic calcium release.
BRCA1 in breast and ovarian cancer
Hereditary breast and ovarian cancer is caused primarily by mutations in the BRCA1 and/or BRCA2 genes, resulting in an increased lifetime risk for developing breast and/or ovarian cancer . The genetic basis of hereditary breast and ovarian cancer is an inherited germline mutation in one allele of either the BRCA1 or BRCA2 genes and subsequent loss of heterozygosity in somatic tissues. In the presence of BRCA1 mutations, patients have a 65–74% risk of developing breast cancer and a 39–46% risk of developing ovarian cancer . Both genes belong to the tumor suppressor gene family and it is thought that their primary function is to repair damaged DNA through double-stranded DNA repair . Therefore, an inherited mutation in either of these genes combined with loss of heterozygosity predisposes cells to chromosomal instability and greatly increased probability of malignant transformation and cancer development. Studies have reported that BRCA1-mutated breast and ovarian tumors have a better outcomes [43, 44]. This is likely due to increased sensitivity of BRCA mutated cells to chemotherapeutics targeting DNA such as anti-metabolites, alkylating agents, and topoisomerase inhibitors . After loss of BRCA expression, the main backup mechanism for the cell to repair damaged DNA is single strand repair/base excision repair. The rate limiting enzyme in this pathway is poly (ADP-ribose) polymerase (PARP). PARP inhibitors as monotherapy or in combination with other agents such as carboplatin are in late stage clinical development and show great promise for BRCA mutated cancers .
Perhaps not surprising, sporadic breast cancer is also associated with downregulation of BRCA1 protein levels. It has been known for almost 20 years that sporadic breast cancers downregulate of BRCA1 function [47-49]. The “BRCAness” of a tumor refers to the loss of BRCA function, and has significant implications for clinical management of the cancer . The mechanisms by which tumors downregulate BRCA1 expression are beginning to be elucidated. For example, upregulation of miR-182 by breast and ovarian cancers directly downregulates BRCA1 protein levels [51, 52]. Regardless of the mechanisms, it is clear that loss of BRCA1 expression is also driver of sporadic breast and ovarian cancers. Thus, understanding the biological roles of BRCA1 is essential for understanding the pathogenesis of all breast and ovarian cancers. In addition, identifying novel roles of BRCA1 in tumor biology could potentially be exploited therapeutically. For example, therapeutic activation of a pathway which is inactivated following BRCA1 loss could potentially inhibit tumor growth. We hypothesize that BRCA1 binds to and activates apoptotic calcium release from the IP3R calcium channel. Small molecules which mimic the actions of BRCA1 on the IP3R could potentially drive apoptosis in cancer cells which have lost BRCA1 expression. This will be tested in Aim 1.
BRCA1 structure, and function
The canonical function of BRCA1 is to maintain genome integrity. BRCA1 generally functions as part of macromolecular complexes in the nucleus in diverse cellular processes including G2-M checkpoint control, DNA replication, DNA end resection, homologous recombination-mediated DNA repair, non-homologous end joining, and the Fanconi anemia pathway (reviewed in ). BRCA1 is also found in non-nuclear compartments, where it has been proposed to regulate centrosome assembly, cell death and other processes (see below; [7, 54]).
Structurally, BRCA1 is a large protein (1863 amino acids) composed of a N-terminal RING domain and two C-terminal BRCA1 C-terminal (BRCT) repeats. The RING domain is associated with E3 ubiquitin ligase activity, while the BRCT domains are phospho-serine binding modules. It also contains two nuclear localization (NLS) sequences, and one nuclear export sequence (NES). BRCA1 is imported into the nucleus via two pathways, the canonical importin/karyopherin pathway and a “piggyback” pathway together with BRCA1-associated RING domain protein 1 (BARD1). Importin alpha binds to one of the basic NLS sequences in BRCA1 (located at amino acids 503-508 and 606-615), which brings BRCA1 to nuclear pores and translocation into the nucleoplasm in a GTP-dependent manner [55, 56]. A second method for nuclear import for BRCA1 is to “piggyback” into the nucleus with BARD1. BRCA1 and BARD1 form stable heterodimers via the RING domains present on each protein. Heterodimer formation leads to masking of the nuclear export sequences on both proteins, stabilizing them in the nuclear compartment . Heterodimer formation greatly increases the E3 ubiquitin ligase activity of BRCA1, and it is thought this is critical for BRCA1-dependent ubiquitination of substrates present at DNA damage-induced foci . Multiple studies have shown that there is a stable/constitutive pool of BRCA1 in non-nuclear compartments [8, 58-64], including our own work .
BRCA1 localization to non-nuclear compartments
BRCA1 has been purported to have multiple roles in non-nuclear compartments. The most well established role is by binding gamma-tubulin and regulating centrosome assembly and function . BRCA1 accomplishes this task by ubiquitinating centrosomal proteins . Relevant to its function in regulating gamma-tubulin, BRCA1 also regulates mitotic spindle assembly and the mitotic spindle checkpoint . In addition to the centrosome, BRCA1 has also been implicated as a mitochondrial protein with relevance to cell death. Some reports have shown localization to the mitochondrial matrix, where it may function to repair damaged mitochondrial DNA [59, 67]. Other reports have suggested that the BRCA1 binding partner BARD1 is selectively targeted to mitochondria, whereas BRCA1 is present at much lower amounts . The function of BRCA1 at mitochondria has been said to be anti-apoptotic , pro-apoptotic [63, 67, 68], or both . The reason for the lack of consensus is unknown, but like all BRCA1 literature a significant question is always the specificity of the chosen antibodies . Furthermore, most studies rely on biochemical techniques such as subcellular fractionation. As there is a direct physical coupling between ER and mitochondrial membranes (so called mitochondrial associated membranes, or MAMs), it is nearly impossible to separate these two organelles. We have recently shown that BRCA1 is recruited to ER membranes during apoptosis, and this requires expression of the ER-resident IP3R channel . We overcame some of the above difficulties by using YFP-tagged BRCA1 and showing recruitment to IP3R channels in living cells during cell death using FRET imaging. Furthermore, we have validated the specificity of our BRCA1 antibody using siRNA mediated knockdown of endogenous BRCA1 (Figure 6; . Thus, using improved tools we have solid evidence that a significant fraction of BRCA1 is ER localized, and this localization increases during cell death.
BRCA1 as a pro-apoptotic protein
Shortly after the discovery of the BRCA1 gene, it was found that ectopic expression of BRCA1 sensitized cells to serum deprivation and calcium ionophore-induced apoptosis . Since this initial observation, multiple studies have shown that BRCA1 likely functions as a pro-apoptotic protein, particularly when localized to non-nuclear compartments. BRCA1 stimulates apoptosis of ovarian and breast cancer cell lines treated with multiple stressors, including anticancer agents, whereas dominant-negative BRCA1 constructs reverses these effects . Of note, one study suggested that BRCA1 selectively increased sensitivity of BRCA1 null cell lines to spindle poisons such as paclitaxel, whereas it inhibited DNA damage-induced apoptosis (presumably by stimulating DNA repair functions) . Expression of BARD1 inhibits the pro-apoptotic activity of BRCA1, indirectly suggesting that sequestration of BRCA1 in the nucleus inhibits its pro-apoptotic functions . Several studies have indicated that caspase-3 cleavage of BRCA1 stimulates its nuclear export and pro-apoptotic functions, amplifying the apoptotic signal [74-76]. In one of the few mechanistic studies examining the pro-apoptotic role of BRCA1, it was found that BRCA1 sequesters X-linked inhibitor of apoptosis protein (XIAP), and this interaction is disrupted by phosphorylation of BRCA1 by the ATM and Rad3- related kinase (ATR), thereby de-repressing caspase-3 . However, it is unclear how cytosolic BRCA1/XIAP complexes could be regulated by the nuclear kinase ATR. As BRCA1 is phosphorylated by at least seven kinases , hyperphosphorylation and redistribution of BRCA1 to non-nuclear compartments may represent a generalized mechanism for BRCA1 pro-apoptotic signaling. Finally, as mentioned above BRCA1 pro-apoptotic function may be related to mitochondrial localization [59, 63, 69], however these studies are mostly correlative.
Summary of the proposal
Our preliminary and published data indicate that BRCA1 binds to IP3R channels via the RING domain . This leads to increased IP3R activity and apoptotic calcium release. A major question is the mechanism by which BRCA1 is recruited to IP3R channels during apoptosis. We have found that the first BRCT domain of BRCA1 is a phosphatidic acid (PA) binding module. During ER stress PA is produced at the ER by the PERK, and our preliminary data indicates this is a signal for recruitment of BRCA1 to the ER. Once at the ER, BRCA1 can bind to the IP3R channel via the RING domain. Thus, the lipid binding activity of the BRCT domain is a potential mechanism for the stimulus-dependent recruitment of BRCA1 to the ER, where it then can bind IP3R channels during cell death (Figure 1).
As described above, it is clear that non-nuclear BRCA1 is a critical mediator of apoptotic cell death. The mechanism(s) mediating the pro-apoptotic effects are largely unexplored. The innovation in this proposal is the conceptual notion that BRCA1 localizes to ER membranes to exert its pro-apoptotic effects. Specifically, we hypothesize that BRCA1 translocates to the ER and binds IP3R channels during apoptosis, increasing apoptotic calcium release and cell death. In the course of these studies, we have identified two major previously undescribed functions for BRCA1. First, BRCA1 binds and activates the IP3R channel at ER membranes during cell death. Second, we have found that the first BRCT repeat, classically associated with binding phosphoproteins, is also a phospholipid binding motif. Our hypothesis is that coordination of both of these functions is essential for the pro-apoptotic function of BRCA1 with significant implications for tumor biology. Finally, we will try to exploit this functional relationship to generate new tools to stimulate cell death of cancer cells. In particular, we will generate cell permeant peptides based upon the BRCA1 binding interface to see if they can directly stimulate cell death. If successful, this would be the starting point for the generation of pro-apoptotic drugs targeting IP3R channels. This is relevant not only for hereditary and sporadic breast and ovarian cancers, but potentially all cancers as IP3R channels are generally considered universal regulators of cell death .
General overview of approach
In this proposal, we will be using cell biological approaches to investigate the potential involvement of BRCA1 in modulating IP3R activity during apoptosis. Our approach will closely follow our previous work investigating the translocation of cytochrome c from mitochondria to IP3Rs residing on ER membranes, and functional effects of this interaction [3-5]. In particular, we will take advantage of biochemical techniques such as subcellular fractionation and immunoprecipitation. We will also utilize extensively optical techniques such as calcium imaging and fusions of green fluorescent protein variants to examine trafficking and binding via fluorescence resonance energy transfer (FRET). Of course, a variety of apoptosis assays will also be employed to examine the effects of BRCA1/IP3R binding on calcium-dependent cell death. Of significant importance is the choice of cell lines and apoptotic stimulus in these assays. In part, our preliminary studies described below utilize a BRCA1 null cell line derived from a metastatic ovarian tumor (UWB1.289 cells) and the same line with stable re-introduction of recombinant BRCA1. We will also independently confirm key findings in another cell line derived from a tumor with loss of BRCA1 expression, HCC1937 cells, derived from a primary mammary ductal carcinoma. This cell line is homozygous for a BRCA1 nonsense 5382C mutation. As models of ovarian and breast cancer which express wild-type BRCA1, we will utilize SKOV-3 cells (ovarian adenocarcinoma) [80, 81] and MCF7 cells (breast adenocarcinoma) . Although some of our (partly published) preliminary data was acquired in HeLa and DT40 cells, these will not be used in future experiments with the exception of single channel recording of IP3R channels on DT40 cell nuclei. For these experiments, the cellular context is irrelevant.
Specific Aim 1: Determine if BRCA1 directly modulates apoptotic calcium release from IP3R calcium channels.
In this Aim, we will:
- Determine the functional consequences of BRCA1 binding to IP3R
- Develop pro-apoptotic cell permeant peptides based on the BRCA1 binding interface
Many pro-apoptotic proteins bind to and modulate IP3R activity during apoptosis . The main rationale driving this Aim is that BRCA1 binds to and directly activates IP3R channels during cell death. We will determine the kinetics of binding of BRCA1 to IP3R channels and physiologic consequences using well established techniques which were developed in our laboratory and in collaboration with Dr. David Yule at the University of Rochester. Additionally, we hypothesize that small peptides derived from the BRCA1 binding domain may directly activate apoptotic calcium release from the IP3R in vitro and in intact cells. As proof of principle that this is a feasible approach, peptides derived from another IP3R binding protein, Bcl-XL, have direct effects on channel gating and function .
Preliminary data to support the rationale, hypothesis, and approach
BRCA1 binds to the IP3R
In a yeast-two-hybrid experiment using the C-terminal tail domain of IP3R (a.a. 2589-2749) as bait, the RING domain of BRCA1 was isolated as an interacting clone (a.a. 1-112). To confirm this direct interaction, we used purified recombinant protein in an in vitro binding experiment. Purified RING domain was covalently conjugated to cyanogen bromide activated agarose beads and the ability of purified GST-IP3R tail domain and modulatory domain (a.a. 923-1582) to bind to the conjugated beads was tested. The RING domain of BRCA1 was able to specifically pull down IP3R tail domain, suggesting a direct protein-protein interaction between BRCA1 and the IP3R tail (Figure 2A). In order to test for a physical interaction between full length BRCA1 and IP3R in cells, we performed a co-immunoprecipitation experiment. Using an IP3R antibody we were able to co-immunoprecipitate endogenous IP3R-1 with endogenous BRCA1 in HeLa cells (Figure 2B). Thus, yeast-2-hybrid, co-immunoprecipitation, and direct binding of purified components strongly suggest a direct physical interaction between BRCA1 and IP3R mediated by the BRCA1 RING domain and the IP3R tail domain. Subcellular fraction experiments confirm a significant amount of cellular BRCA1 is localized to ER membranes where it would have the potential to bind IP3R channels (Figure 2C).
BRCA1 increases IP3R open probability
To determine the direct effects of BRCA1 on IP3R activity, we recorded single channel currents on isolated nuclei which express recombinant rat IP3R-1 . As shown in Figure 3A-B, when the BRCA1 GST-RING domain was included in the patch pipette the open probability of the IP3R-1 channel in the presence of a subsaturating concentration of IP3 (1 µM) was dramatically increased (control: 0.21 +/- 0.02, GST-RING: 0.57 +/- 0.06, GST only: 0.21 +/- 0.02). This was due to a destabilization of the closed state of the channel (Figure 3C). Thus, BRCA1 directly and potently increases IP3R activity by modulating channel gating.
BRCA1 modulates IP3R function in intact cells
We next chose to examine the effect of BRCA1 on IP3R function by expressing full length BRCA1 fused to the C-terminus of yellow fluorescent protein (YFP) and examining the response to escalating doses of histamine. The YFP-BRCA1 fusion protein has been previously characterized and is functionally comparable to the wild type protein [60, 73]. We measured calcium release in response to 100nM, 1µM, and 10µM histamine in cells expressing YFP-BRCA1 and adjacent cells not expressing the fusion protein. As shown in Figure 4A-C, BRCA1 expression significantly sensitized HeLa cells to 100nM histamine challenge, increasing both peak release (Figure 4A-B) and the number of responding cells (Figure 4C). Expression of BRCA1 did not appear to affect peak release of calcium in response to 1µM histamine or a saturating dose of 10µM histamine (Figure 4D). However, at both of these doses there is a significant increase in the oscillation frequency, which would be expected to have profound implications for downstream signaling events . Thus, expression of BRCA1 has significant stimulatory effects on calcium signaling through IP3R-coupled pathways. Conversely, knockdown of the endogenous BRCA1 expression had precisely the opposite effect, leading to decreased calcium release from the IP3R (data not shown; ). It has been shown that BRCA1 the BRCA1 binding protein and homologue BARD1 are targeted to mitochondria [59, 63, 64, 67-69], and our subcellular fractionation results indicate a significant amount of BRCA1 is present in a fraction which also contains mitochondria (Figure 2C). We hypothesized that BRCA1 may facilitate calcium transfer into mitochondria. To test this hypothesis, we stimulated HeLa cells with 10µM histamine and measured calcium uptake into mitochondria using Rhod-2. As shown in Figure 4G, BRCA1 expression has no effect on calcium uptake into mitochondria.
BRCA1 promotes apoptotic calcium release and cell death
We next examined the effect of BRCA1 expression on paclitaxel-induced apoptosis. In these experiments we used the patient derived, BRCA1-mutated cell line UWB 1.289 (UWB) isolated from an ovarian carcinoma and this same cell line stably rescued with wild type BRCA1 (UWB-BRCA1). The BRCA1 mutations in the parental UWB line eliminate expression of the BRCA1 protein. Treatment of UWB cells with paclitaxel for 24 hours did not cause an elevation of cytosolic calcium, whereas UWB cells with rescued BRCA1 expression had significantly elevated calcium consistent with apoptotic calcium release (Figure 5A). This indicates that BRCA1 expression is required for paclitaxel-induced apoptotic calcium release via the IP3R. Measurement of apoptosis by both caspase-3 enzymatic activity and propidium iodide staining both indicated that BRCA1 expression was required for efficient paclitaxel-induced cell death of ovarian carcinoma cells (Figure 5B-C). This suggests that BRCA1 expression restores paclitaxel-sensitivity to the BRCA1-null cells. This is consistent with similar findings by other groups (40).
Aim 1, Subaim 1: Determine the functional consequences of BRCA1 binding to IP3R
In preliminary studies, we have found that BRCA1 stimulates apoptotic calcium release induced by paclitaxel treatment (Figure 5). We have also found that BRCA1 increases IP3R activity in vitro and in intact cells (Figures 3,4). We will expand upon these results by examining in detail the functional impact that BRCA1 has on IP3R Ca2+ channels. To accomplish this task we will use two approaches: 1) single channel recording of IP3R activity and 2) long-term optical imaging of apoptotic calcium release induced by paclitaxel, cisplatin, and staurosporine. Apoptotic calcium release will be measured in UWB cells or UWB with rescue of BRCA1 expression and in SKOV-3 cells with or without knockdown of the endogenous BRCA1 protein.
Single channel recording
The most definitive method of determining direct modulation of IP3R activity by BRCA1 is by single channel recording of IP3R activity. As shown in Figure 3, BRCA1 has dramatic effects on the single channel activity of the IP3R by altering channel gating. This experiment was done at optimal Ca2+ and ATP concentrations and a subsaturating dose of IP3. In this subaim we will characterize in detail the effects of BRCA1 on IP3R activity. The experiment in Figure 3 was done with 30nM BRCA1 RING domain. We will perform a full dose curve to determine the binding affinity of BRCA1 for the IP3R. We will then examine in the regulation of the channel by IP3, Ca2+, and ATP at various concentrations in the presence and absence of BRCA1. A more detailed examination of the effects of BRCA1 on IP3R gating will be accomplished by examining burst kinetics as described . All of these experiments will be performed by the nuclear patch clamp technique on DT40 cell nuclei which are expressing recombinant type 1 IP3R . This subaim will be accomplished by collaborators David Yule and Larry Wagner at the University of Rochester.
Apoptotic calcium release
Our data clearly show that BRCA1 has signficant effects on agonist (histamine)-induced calcium release (Figure 4; ). Furthermore, we demonstrated that paclitaxel treatment for 24 hours resulted in significantly increased cytosolic calcium in cells expressing BRCA1 (Figure 5; ). This prelimnary result is supportive of apoptotic calcium release, but detailed kinetics of calcium release in the presence and absence of BRCA1 is required to conclude BRCA1 contributes to apoptotic calcium release.. Using the genetically encoded calcium indicator protein GCaMP6s, we now have the ability to measure cytosolic calcium for many hours during cell death . We will perform long-term (8-12 hours) continuous calcium imaging of UWB cells or UWB cells stably expressing BRCA1 during cell death induced by paclitaxel, cisplatin, and staurosporine. The rationale for using paclitaxel and cisplatin is that they are both clinically relevant therapeutics with completely different mechanisms of action. Staurosporine will be used as a positive control, as the role of calcium ions in cell death induced by this stimulus is very well established (including our own work; [3, 4, 27, 32]). The same experiments will be performed in SKOV-3 cells with or without siRNA-mediated knockdown of BRCA1. As shown in Figure 6, we can achieve essentially complete knockdown of BRCA1 using two commercially available siRNA oligos (Ambion/Life Technologies). One oligo also increased IP3R expression (siRNA-s458; Figure 6) which we interpreted as compensatory upregulation . Thus, for these experiments we will use siRNA-459. Of note, Figure 6 also proves the specificity of the antibody used in our studies (Cell Signaling Technologies Cat#9010). As part of our model (Figure 1) we have included cytochrome c binding to IP3R as an early and contributory event to apoptotic calcium release and recruitment of BRCA1. To determine the relative contribution of cytochrome c binding to apoptotic calcium release, we will repeat the above experiments in the presence of well-characterized dominant-negative cell peptides which inhibit cytochrome binding to IP3R [4, 5].
Aim 1, Subaim 2: Develop pro-apoptotic cell permeant peptides based on the BRCA1 binding interface
We will first systematically investigate the binding determinants of IP3R/BRCA1 interactions. We will use a mammalian expression system which we have used successfully in the past to determine the binding interface of the IP3R with cytochrome c . We will first utilize a series of deletion constructs of the BRCA1 RING domain that can be expressed in mammalian cells and contains an HA epitope for immunoprecipitation. The ability of these constructs to bind to endogenous full-length IP3R or recombinant IP3R C-terminal tail will be tested by co-immunoprecipitation as in Figure 2. Our goal is to refine to the binding interface to ~20 amino acids or less, and determine if synthetic BRCA1 peptides can mimic the effects of the full length RING domain on IP3R gating as in Figure 3. Similar experiments demonstrated that a peptide derived from the Bcl-XL BH4 domain had direct effects on IP3R gating . As Bcl-XL also binds to the IP3R C-terminus, we feel this is proof-of-principle that this endeavor is feasible. If we are successful in generating peptide(s) which alter IP3R activity, we will conjugate them to the lipophilic fluorophore BODIPY 577/618, which we have shown renders hydrophilic peptides cell permeant . We will then see if this peptide, by itself, can induce apoptotic calcium release and cell death of UWB and SKOV-3 cells. These effects should be independent of BRCA1 expression but absolutely dependent on IP3R expression.
Alternative Approaches Aim 1
We feel we now have strong preliminary data supporting the feasibility of our experiments examining the effects of BRCA1 on IP3R function, and we do not expect any technical difficulties. The development of peptides which modulate IP3R activity may be considerably more difficult. Of particular concern is whether fragments of the RING domain are soluble when exogenously expressed in cells. An alternative approach is to simply skip the cell-based approach and synthesize overlapping peptides covering the length of the RING domain. Since this domain is only ~100 amino acids, this is not necessarily an overly expensive or difficult task. The structure of the BRCA1 RING domain is known , and there are three major helices, one of which is embedded in a globular domain. The N- and C-terminal helices are known to participate in protein-protein interactions and thus are likely candidates for further investigation.
Specific Aim 2: Determine the mechanism by which BRCA1 is recruited to ER membranes.
In this Aim, we will:
- Determine the subcellular redistribution of endogenous and recombinant BRCA1 to ER membranes in response to apoptotic stimuli.
- Characterize the lipid binding activity of the BRCA1 BRCT domain.
- Determine if PERK activation following ER stress is the mechanism by which BRCA1 is recruited to ER membranes during cell death.
Many studies have suggested that BRCA1 directly activates apoptotic cell death (reviewed in [7, 8, 10]), but the mechanisms remain obscure. Our preliminary data indicates that BRCA1 localizes to ER membranes where it stimulates apoptotic calcium release through IP3R channels on the ER. The primary rationale driving this Aim is that during cell death BRCA1 is recruited to ER membranes by directly binding phosphatidic acid produced during cell death. We hypothesize that this recruitment is mediated by a novel lipid binding motif present in the first BRCT domain of BRCA1.
Preliminary data to support the rationale, hypothesis, and approach
BRCA1 is recruited to the ER and binds IP3R channels during apoptosis
We hypothesize that BRCA1 binding to IP3R on ER membranes increases during cell death, and this is an essential component of its tumor suppressor activity. In order to measure changes in the BRCA1/IP3R interaction in living cells during apoptosis, we used the FRET pair CFP-IP3R and YFP-BRCA1. We used paclitaxel to induce apoptosis, and measured dynamic changes in the FRET ratio (and thus binding) in cells transfected with CFP-IP3R-1 and YFP-BRCA1 or YFP alone (Figure 7). We found a significant increase in cytosolic FRET after treatment with paclitaxel (1µM) in cells expressing both CFP-IP3R-1 and YFP-BRCA1 (Figure 7A). The kinetics of association are consistent with the time course of activation of cell death proteins such as JNK1 in response to paclitaxel treatment . We saw no increase in FRET after treatment with paclitaxel in the nucleus of cells transfected with CFP-IP3R-1 and YFP-BRCA1 or in cells transfected with CFP-IP3R-1 and YFP only (Figure 7A-B). These results indicate that BRCA1 is recruited to IP3R channels on the ER during apoptosis, and support the hypothesis that under resting (i.e., non-apoptotic) conditions only a subpopulation of IP3Rs are bound to BRCA1.
BRCA1 has a novel lipid binding domain
We hypothesized that recruitment of BRCA1 to the ER may be mediated by an intrinsic lipid binding activity in the BRCA1 protein. We identified a potential lipid binding domain within the C-terminal tandem BRCT domain of BRCA1 using Adaptive-BLAST [87, 88]. Specifically, we found that amino acid residues 1664-1696 within the first BRCT repeat have a strong potential for lipid binding based upon homology to fatty acid binding proteins (Figure 8A). Examination of high scoring residues within this sequence identified a basic region with several threonine residues common to lipid binding pockets, including phosphatidic acid binding proteins (Figure 8B; ). When mapped onto the structure of the tandem BRCT domains, these residues were solvent accessible and thus potentially able to participate in lipid binding (Figure 8C). To experimentally test this hypothesis, we purified recombinant GST-BRCT and performed a lipid strip overlay experiment. GST-RING domain was used as a negative control. GST-BRCT strongly bound phosphatidic acid (PA), whereas GST-RING was unable to bind to any lipids (Figure 8D). Thus, the BRCT domain of BRCA1 has a previously uncharacterized lipid binding activity which may mediate localization at ER membranes.
Phosphatidic acid is produced at the ER during paclitaxel-induced cell death
In Figure 8 we show that the BRCA1 BRCT domain binds to phosphatidic acid (PA). Phosphatidic acid can be produced from multiple enzymes. The two best characterized are phospholipase D and diacylglycerol kinase. Recently, it has been shown that the ER-resident protein PKR-like ER kinase (PERK) can produce PA by phosphorylating diacylglycerol at the ER . PERK is activated by the ER stress response. We and others have shown that activation of the IP3R during apoptosis leads to calcium store depletion and PERK activation [30, 90, 91]. Thus, we hypothesize that during apoptosis IP3R calcium release leads to PERK activation and PA production. This would in turn recruit BRCA1 to ER membranes leading to more apoptotic calcium release in a feed-forward mechanism. To determine if PA is produced at the ER during paclitaxel-induced apoptosis, we used the PA indicator protein phosphatidic acid biosensor with superior sensitivity (GFP-PASS; ). We co-expressed ER-localized dsRed and measured the FRET signal as an indicator of GFP-PASS recruitment to ER membranes (and thus PA production). As shown in Figure 9A, paclitaxel resulted in increased FRET at ER membranes. This could be completely abrogated by pre-incubating the cells with a PERK inhibitor (Figure 9B). Thus, PA production by PERK at ER membranes is an early event in paclitaxel-mediated cell death and may be a mechanism for BRCA1 recruitment to ER membranes.
Aim 2, Subaim 1: Determine the subcellular redistribution of endogenous and recombinant BRCA1 to ER membranes in response to apoptotic stimuli.
In this subaim, we will investigate endogenous wild-type BRCA1 localization in SKOV-3 cells and recombinant untagged and YFP tagged BRCA1 localization in BRCA1 null UWB cells using biochemical approaches. We will use established subcellular fractionation techniques used extensively by our group [3-5, 30] to enrich for nuclear, mitochondrial, cytosolic, and ER fractions. Careful evaluation of purity will be monitored as in Figure 2. Our focus will be on the apoptosis-dependent translocation of BRCA1 between these different fractions. Our choice of apoptotic stimuli will be empirically determined. Our preliminary data indicates that therapeutically relevant doses of paclitaxel result in apoptotic calcium release, caspase-3 activation, and cell death (Figure 5), and thus will be the starting point for these studies. We will also use two other stimuli as described and justified in Aim 1: cisplatin and staurosporine. Apoptosis induction in all experimental paradigms will be confirmed by quantifying caspase-3 enzymatic rates in cytosolic fractions (100,000 xg supernatants) isolated in the fractionation procedure as described previously by our group [3, 5, 28]. In cells expressing endogenous BRCA1 (SKOV-3, MCF-7), it would be expected that a proportion of BRCA1 would remain nuclear localized to support nuclear functions of BRCA1. We will test this by examining the relative levels of nuclear to ER localized BRCA1 during apoptosis induced by paclitaxel and cisplatin. Furthermore, we will monitor the nuclear function of BRCA1 by analyzing DNA foci formation using immunohistochemical techniques as described .
We will monitor in real time in live cells translocation of YFP-BRCA1 in response to the apoptotic stimuli described above in stably expressing UWB cells. Co-localization to subcellular fractions will be determined by FRET using dsRed fusion proteins targeted to ER, mitochondrial, and nuclear fractions as we have shown in Figure 8 using ER targeting dsRed. To complement these studies, traditional immunofluorescence and co-localization analysis of endogenous BRCA1 (in SKOV-3 cells) and recombinant untagged BRCA1 (in UWB cells) will be performed. A BRCA1 construct lacking the BRCT repeats will be used as a control. In subaim 2 below we will map in detail the lipid binding determinants of the first BRCT domain of BRCA1. Once generated, BRCA1 mutants deficient in lipid binding will be tested for recruitment to ER and other organelles as described above.
Aim 2, Subaim 2: Characterize the lipid binding activity of the BRCA1 BRCT domain
We have found that the purified BRCT domain binds most strongly in vitro to phosphatidic acid (PA) using PIP strips (Fig. 7D). In this subaim we will further investigate the lipid binding activity and specificity of the BRCT domain of BRCA1. We will measure purified BRCT binding to large unilamellar vesicles composed of phosphatidylcholine (PC) and the test lipids phosphatidic acid (PA) or phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3), phosphatidylinositol 3,5-bisphosphate (PI3,5P2), or phosphatidylinositol 3,5-bisphosphate (PI4,5P2). These lipids all have at least partial BRCT binding activity as determined by PIP strips (Figure 8). As a control we will use PC/phosphatidylethanolamine (PE) vesicles. Binding will be performed using a standard sucrose-loaded liposome sedimentation assay , which we are now performing in the lab after the initial assistance of our neighbor Dr. Guangwei Du, who also provided the GFP-PASS shown in Figure 9. We will next determine the sequence-specific requirements of lipid binding using site-directed mutagenesis. As noted in Figure 8, we have identified using bioinformatics approaches conserved resides which likely mediate lipid binding. These will be mutated individually and in combination to alanine and tested for the ability to bind both PIP strips and liposomes. If amino acids within this conserved domain do not mediate binding, we will make a series of deletion constructs and examine binding as above. Once we have identified the PA/lipid binding residues, mutation of these residues on full length BRCA1 and YFP-BRCA1 will be made and tested as above for stimulus-dependent localization to ER membranes. They will also be tested for their ability to rescue apoptotic calcium release and cell death in UWB cells as in Figure 5.
Aim 2, Subaim 3: Determine if PERK activation following ER stress is the mechanism by which BRCA1 is recruited to ER membranes during cell death.
The IP3R is now recognized as a central mediator of the ER stress response. Our group and others have shown that under cellular stress conditions when apoptotic calcium release from the IP3R is prominent, ER stress is activated downstream of ER calcium store depletion [30, 94-96]. As such, most apoptotic stimuli which stimulate apoptotic calcium release from the IP3R also activate ER stress pathways. Relevant to this proposal, ER stress is known to be activated downstream of cell death induced by paclitaxel , cisplatin , and staurosporine . We determine if paclitaxel, cisplatin, and staurosporine induced ER stress in our model systems of BRCA1 mutated (UWB, HCC1937) and BRCA1 wild type (SKOV-3, MCF-7) cell lines. ER stress will be measured as previously described by us using western blotting for phosphorylated PERK, phosphorylate IRE-1 and upregulated BiP . We have also shown a compensatory upregulation of calnexin and calreticulin during IP3R-mediated ER stress , and as such will also measure the levels of these proteins. Cell death will be monitored by caspase-3 enzymatic activity.
PERK activation and PA production during ER stress
The main hypothesis driving this subaim is that PERK activation leads to PA production during ER stress, and this leads to recruitment of BRCA1 to ER membranes. We will quantitatively measure production of PA at ER membranes using FRET imaging as in our preliminary data in Figure 9. We will measure increased FRET between the PA sensor GFP-PASS and ER-dsRed during ER stress induction in living cells treated with paclitaxel, cisplatin, and staurosporine. As a negative control we will use a GFP-PASS sensor with point mutations which eliminate PA binding (described in ). As another control we will test whether inhibition of PERK activity with GSK2606414 (as in Figure 9B) or siRNA-mediated knockdown of PERK prevent GFP-PASS accumulation at ER membranes.
PERK activation and BRCA1 recruitment to ER membranes
Our previous work and preliminary data indicate that BRCA1 binds PA (Figure 8;). We will test whether PERK activation and subsequent PA production is responsible for BRCA1 recruitment to ER membranes during cell death. Using the biochemical and optical approaches outlined in Subaim 1 above, we will determine if PERK inhibition and PERK knockdown inhibits BRCA1 recruitment to ER membranes after stimulation with paclitaxel, cisplatin, and staurosporine. As a control we will use BRCA1 mutants deficient in lipid binding generated in subaim 2.
IP3R activation and BRCA1 recruitment to ER membranes
Our model outlined in Figure 1 is that IP3R activation is the most proximal event leading to Ca2+ store depletion, ER stress, and subsequent PA production and BRCA1 recruitment. BRCA1 would then bind IP3R channels to amplify the apoptotic signal. This model is analogous to our previous work on cytochrome c binding to the IP3R [3-5, 29]. We will test whether chelation of cytosolic calcium with BAPTA-AM (which is well known to block apoptotic calcium release; e.g. ) or inhibition of IP3R channels with the “IP3 sponge”  inhibits BRCA1 translocation to ER membranes as in Subaim 1. Furthermore, we will also test whether peptides which block cytochrome c binding to IP3R [4, 5] also have an effect on BRCA1 localization to determine if this is an obligate step in BRCA1 recruitment to the ER. We will also monitor induction of the ER stress response under these conditions by Western blotting as described above. Finally, to test the role of ER PA production in mediating BRCA1-dependent activation of IP3R channels, we will image apoptotic calcium release as in Aim 1, subaim 1 using UWB cells stably expressing either wild type or BRCA1 with point mutations which eliminate PA binding and recruitment to ER membranes.
Alternative Approaches Aim 2
From a technical standpoint, we have extensive experience with all of the assays described in the Aim. The biggest question is whether BRCA1 specifically binds PA or whether it has more promiscuous lipid binding activities. That will certainly become evident early in this Aim, and we can modify the plan accordingly. We anticipate that it is unlikely that BRCA1 binds to the phosphoinositol lipids shown to bind BRCA1 by the PIP strip assay in Figure 8. These lipids are relatively rare and are enriched primarily at the plasma membrane. We find no evidence for BRCA1 enrichment at the PM under basal or stimulated conditions, however we do find recruitment to ER membranes during cell death . Thus, we feel our model is the most logical based upon our current published and preliminary data.
- Hedgepeth, S.C., et al., The BRCA1 Tumor Suppressor Binds to Inositol 1,4,5-Trisphosphate Receptors to Stimulate Apoptotic Calcium Release. J Biol Chem, 2015.
- Bobrovnikova-Marjon, E., et al., PERK utilizes intrinsic lipid kinase activity to generate phosphatidic acid, mediate Akt activation, and promote adipocyte differentiation. Mol Cell Biol, 2012. 32(12): p. 2268-78.
- Boehning, D., et al., Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nat Cell Biol, 2003. 5(12): p. 1051-61.
- Boehning, D., et al., A peptide inhibitor of cytochrome c/inositol 1,4,5-trisphosphate receptor binding blocks intrinsic and extrinsic cell death pathways. Proc Natl Acad Sci U S A, 2005. 102(5): p. 1466-71.
- Wozniak, A.L., et al., Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis. J Cell Biol, 2006. 175(5): p. 709-14.
- Patterson, R.L., D. Boehning, and S.H. Snyder, Inositol 1,4,5-trisphosphate receptors as signal integrators. Annu Rev Biochem, 2004. 73: p. 437-65.
- Henderson, B.R., Regulation of BRCA1, BRCA2 and BARD1 intracellular trafficking. Bioessays, 2005. 27(9): p. 884-93.
- Yang, E.S. and F. Xia, BRCA1 16 years later: DNA damage-induced BRCA1 shuttling. Febs J, 2010. 277(15): p. 3079-85.
- Joseph, S.K. and G. Hajnoczky, IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond. Apoptosis, 2007. 12(5): p. 951-68.
- Thompson, M.E., BRCA1 16 years later: nuclear import and export processes. Febs J, 2010. 277(15): p. 3072-8.
- Schanne, F.A., et al., Calcium dependence of toxic cell death: a final common pathway. Science, 1979. 206(4419): p. 700-2.
- Fleckenstein, A., et al., Myocardial fiber necrosis due to intracellular Ca overload-a new principle in cardiac pathophysiology. Recent Adv Stud Cardiac Struct Metab, 1974. 4: p. 563-80.
- Leonard, J.P. and M.M. Salpeter, Agonist-induced myopathy at the neuromuscular junction is mediated by calcium. J Cell Biol, 1979. 82(3): p. 811-9.
- Orrenius, S., B. Zhivotovsky, and P. Nicotera, Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol, 2003. 4(7): p. 552-65.
- Rizzuto, R., et al., Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science, 1998. 280(5370): p. 1763-6.
- Csordas, G., A.P. Thomas, and G. Hajnoczky, Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria. Embo J, 1999. 18(1): p. 96-108.
- Hajnoczky, G., et al., Decoding of cytosolic calcium oscillations in the mitochondria. Cell, 1995. 82(3): p. 415-24.
- Jayaraman, T. and A.R. Marks, T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis. Mol Cell Biol, 1997. 17(6): p. 3005-12.
- Khan, A.A., et al., Lymphocyte apoptosis: mediation by increased type 3 inositol 1,4,5-trisphosphate receptor. Science, 1996. 273(5274): p. 503-7.
- Hajnoczky, G., et al., Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals. Cell Calcium, 2000. 28(5-6): p. 349-63.
- Chen, R., et al., Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate. J Cell Biol, 2004. 166(2): p. 193-203.
- Scorrano, L., et al., BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science, 2003. 300(5616): p. 135-9.
- Oakes, S.A., et al., Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum. Proc Natl Acad Sci U S A, 2005. 102(1): p. 105-10.
- Oakes, S.A., et al., Regulation of endoplasmic reticulum Ca2+ dynamics by proapoptotic BCL-2 family members. Biochem Pharmacol, 2003. 66(8): p. 1335-40.
- Hajnoczky, G., E. Davies, and M. Madesh, Calcium signaling and apoptosis. Biochem Biophys Res Commun, 2003. 304(3): p. 445-54.
- Szalai, G., R. Krishnamurthy, and G. Hajnoczky, Apoptosis driven by IP(3)-linked mitochondrial calcium signals. Embo J, 1999. 18(22): p. 6349-61.
- Akimzhanov, A.M. and D. Boehning, Monitoring dynamic changes in mitochondrial calcium levels during apoptosis using a genetically encoded calcium sensor. J Vis Exp, 2011(50).
- Akimzhanov, A.M., et al., T-cell receptor complex is essential for Fas signal transduction. Proc Natl Acad Sci U S A, 2010. 107(34): p. 15105-10.
- Boehning, D., R.L. Patterson, and S.H. Snyder, Apoptosis and calcium: new roles for cytochrome c and inositol 1,4,5-trisphosphate. Cell Cycle, 2004. 3(3): p. 252-4.
- Jeschke, M.G., et al., Calcium and ER stress mediate hepatic apoptosis after burn injury. J Cell Mol Med, 2009. 13(8B): p. 1857-65.
- Steinmann, C., et al., Requirement of inositol 1,4,5-trisphosphate receptors for tumor-mediated lymphocyte apoptosis. J Biol Chem, 2008. 283(20): p. 13506-9.
- Akimzhanov, A.M., J.M. Barral, and D. Boehning, Caspase 3 cleavage of the inositol 1,4,5-trisphosphate receptor does not contribute to apoptotic calcium release. Cell Calcium, 2013. 53(2): p. 152-8.
- Assefa, Z., et al., Caspase-3-induced truncation of type 1 inositol trisphosphate receptor accelerates apoptotic cell death and induces inositol trisphosphate-independent calcium release during apoptosis. J Biol Chem, 2004. 279(41): p. 43227-36.
- Haug, L.S., S.I. Walaas, and A.C. Ostvold, Degradation of the type I inositol 1,4,5-trisphosphate receptor by caspase-3 in SH-SY5Y neuroblastoma cells undergoing apoptosis. J Neurochem, 2000. 75(5): p. 1852-61.
- Hirota, J., T. Furuichi, and K. Mikoshiba, Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner. J Biol Chem, 1999. 274(48): p. 34433-7.
- Li, C., et al., Bcl-X(L) affects Ca(2+) homeostasis by altering expression of inositol 1,4,5-trisphosphate receptors. Proc Natl Acad Sci U S A, 2002. 99(15): p. 9830-5.
- Li, C., et al., Apoptosis regulation by Bcl-x(L) modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating. Proc Natl Acad Sci U S A, 2007. 104(30): p. 12565-70.
- Giorgi, C., et al., PML regulates apoptosis at endoplasmic reticulum by modulating calcium release. Science, 2010. 330(6008): p. 1247-51.
- Bononi, A., et al., Identification of PTEN at the ER and MAMs and its regulation of Ca(2+) signaling and apoptosis in a protein phosphatase-dependent manner. Cell Death Differ, 2013. 20(12): p. 1631-43.
- Berridge, M.J., P. Lipp, and M.D. Bootman, The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol, 2000. 1(1): p. 11-21.
- Hereditary breast and ovarian cancer syndrome. Gynecol Oncol, 2009. 113(1): p. 6-11.
- Scully, R., et al., Genetic analysis of BRCA1 function in a defined tumor cell line. Mol Cell, 1999. 4(6): p. 1093-9.
- Johannsson, O.T., et al., Survival of BRCA1 breast and ovarian cancer patients: a population-based study from southern Sweden. J Clin Oncol, 1998. 16(2): p. 397-404.
- Yang, D., et al., Association of BRCA1 and BRCA2 mutations with survival, chemotherapy sensitivity, and gene mutator phenotype in patients with ovarian cancer. JAMA, 2011. 306(14): p. 1557-65.
- Vencken, P.M., et al., Chemosensitivity and outcome of BRCA1- and BRCA2-associated ovarian cancer patients after first-line chemotherapy compared with sporadic ovarian cancer patients. Ann Oncol, 2011. 22(6): p. 1346-52.
- Lee, J.M., J.A. Ledermann, and E.C. Kohn, PARP Inhibitors for BRCA1/2 mutation-associated and BRCA-like malignancies. Ann Oncol, 2014. 25(1): p. 32-40.
- Turner, N.C., et al., BRCA1 dysfunction in sporadic basal-like breast cancer. Oncogene, 2007. 26(14): p. 2126-32.
- Mueller, C.R. and C.D. Roskelley, Regulation of BRCA1 expression and its relationship to sporadic breast cancer. Breast Cancer Res, 2003. 5(1): p. 45-52.
- Thompson, M.E., et al., Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression. Nat Genet, 1995. 9(4): p. 444-50.
- Turner, N., A. Tutt, and A. Ashworth, Hallmarks of ‘BRCAness’ in sporadic cancers. Nat Rev Cancer, 2004. 4(10): p. 814-9.
- Moskwa, P., et al., miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors. Mol Cell, 2011. 41(2): p. 210-20.
- Krishnan, K., et al., MicroRNA-182-5p targets a network of genes involved in DNA repair. RNA, 2013. 19(2): p. 230-42.
- Huen, M.S., S.M. Sy, and J. Chen, BRCA1 and its toolbox for the maintenance of genome integrity. Nat Rev Mol Cell Biol, 2010. 11(2): p. 138-48.
- Irminger-Finger, I. and C.E. Jefford, Is there more to BARD1 than BRCA1? Nat Rev Cancer, 2006. 6(5): p. 382-91.
- Chen, C.F., et al., The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor. J Biol Chem, 1996. 271(51): p. 32863-8.
- Li, S., et al., Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs. J Biol Chem, 1998. 273(11): p. 6183-9.
- Brzovic, P.S., et al., Structure of a BRCA1-BARD1 heterodimeric RING-RING complex. Nat Struct Biol, 2001. 8(10): p. 833-7.
- Chen, Y., et al., Aberrant subcellular localization of BRCA1 in breast cancer. Science, 1995. 270(5237): p. 789-91.
- Coene, E.D., et al., Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria. Mol Biol Cell, 2005. 16(2): p. 997-1010.
- Fabbro, M., et al., BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export. J Biol Chem, 2002. 277(24): p. 21315-24.
- Thangaraju, M., S.H. Kaufmann, and F.J. Couch, BRCA1 facilitates stress-induced apoptosis in breast and ovarian cancer cell lines. J Biol Chem, 2000. 275(43): p. 33487-96.
- Rodriguez, J.A., W.W. Au, and B.R. Henderson, Cytoplasmic mislocalization of BRCA1 caused by cancer-associated mutations in the BRCT domain. Exp Cell Res, 2004. 293(1): p. 14-21.
- Tembe, V. and B.R. Henderson, BARD1 translocation to mitochondria correlates with Bax oligomerization, loss of mitochondrial membrane potential, and apoptosis. J Biol Chem, 2007. 282(28): p. 20513-22.
- Henderson, B.R., The BRCA1 Breast Cancer Suppressor: Regulation of Transport, Dynamics, and Function at Multiple Subcellular Locations. Scientifica (Cairo), 2012. 2012: p. 796808.
- Parvin, J.D., The BRCA1-dependent ubiquitin ligase, gamma-tubulin, and centrosomes. Environ Mol Mutagen, 2009. 50(8): p. 649-53.
- Starita, L.M., et al., BRCA1-dependent ubiquitination of gamma-tubulin regulates centrosome number. Mol Cell Biol, 2004. 24(19): p. 8457-66.
- Brodie, K.M. and B.R. Henderson, Differential modulation of BRCA1 and BARD1 nuclear localisation and foci assembly by DNA damage. Cell Signal, 2010. 22(2): p. 291-302.
- Laulier, C., et al., Bcl-2 inhibits nuclear homologous recombination by localizing BRCA1 to the endomembranes. Cancer Res, 2011. 71(10): p. 3590-602.
- Maniccia, A.W., et al., Mitochondrial localization, ELK-1 transcriptional regulation and growth inhibitory functions of BRCA1, BRCA1a, and BRCA1b proteins. J Cell Physiol, 2009. 219(3): p. 634-41.
- Wilson, C.A., et al., BRCA1 protein products: antibody specificity. Nat Genet, 1996. 13(3): p. 264-5.
- Shao, N., et al., Induction of apoptosis by the tumor suppressor protein BRCA1. Oncogene, 1996. 13(1): p. 1-7.
- Quinn, J.E., et al., BRCA1 functions as a differential modulator of chemotherapy-induced apoptosis. Cancer Res, 2003. 63(19): p. 6221-8.
- Fabbro, M., et al., BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention. Exp Cell Res, 2004. 298(2): p. 661-73.
- Zhan, Q., et al., Caspase-3 mediated cleavage of BRCA1 during UV-induced apoptosis. Oncogene, 2002. 21(34): p. 5335-45.
- Dizin, E., et al., Caspase-dependent BRCA1 cleavage facilitates chemotherapy-induced apoptosis. Apoptosis, 2008. 13(2): p. 237-46.
- O’Donnell, J.D., et al., BRCA1 185delAG truncation protein, BRAt, amplifies caspase-mediated apoptosis in ovarian cells. In Vitro Cell Dev Biol Anim, 2008. 44(8-9): p. 357-67.
- Martin, S.A. and T. Ouchi, BRCA1 phosphorylation regulates caspase-3 activation in UV-induced apoptosis. Cancer Res, 2005. 65(23): p. 10657-62.
- Rosen, E.M., et al., BRCA1 gene in breast cancer. J Cell Physiol, 2003. 196(1): p. 19-41.
- Harr, M.W. and C.W. Distelhorst, Apoptosis and autophagy: decoding calcium signals that mediate life or death. Cold Spring Harb Perspect Biol, 2010. 2(10): p. a005579.
- Zhou, C., P. Huang, and J. Liu, The carboxyl-terminal of BRCA1 is required for subnuclear assembly of RAD51 after treatment with cisplatin but not ionizing radiation in human breast and ovarian cancer cells. Biochem Biophys Res Commun, 2005. 336(3): p. 952-60.
- Wiltshire, T., et al., BRCA1 contributes to cell cycle arrest and chemoresistance in response to the anticancer agent irofulven. Mol Pharmacol, 2007. 71(4): p. 1051-60.
- Sevcik, J., et al., The BRCA1 alternative splicing variant Delta14-15 with an in-frame deletion of part of the regulatory serine-containing domain (SCD) impairs the DNA repair capacity in MCF-7 cells. Cell Signal, 2012. 24(5): p. 1023-30.
- Monaco, G., et al., Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor. PLoS One, 2013. 8(8): p. e73386.
- Wagner, L.E., 2nd and D.I. Yule, Differential regulation of the InsP(3) receptor type-1 and -2 single channel properties by InsP(3), Ca(2)(+) and ATP. J Physiol, 2012. 590(Pt 14): p. 3245-59.
- Borahay, M.A., et al., Simvastatin Potently Induces Calcium-Dependent Apoptosis of Human Leiomyoma Cells. J Biol Chem, 2014.
- Lee, L.F., et al., Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). J Biol Chem, 1998. 273(43): p. 28253-60.
- Hong, Y., et al., Phylogenetic Profiles Reveal Structural and Functional Determinants of Lipid-binding. J Proteomics Bioinform, 2009. 2: p. 139-149.
- Ko, K.D., et al., Phylogenetic profiles reveal structural/functional determinants of TRPC3 signal-sensing antennae. Commun Integr Biol, 2009. 2(2): p. 133-7.
- Stace, C.L. and N.T. Ktistakis, Phosphatidic acid- and phosphatidylserine-binding proteins. Biochim Biophys Acta, 2006. 1761(8): p. 913-26.
- Jeschke, M.G. and D. Boehning, Endoplasmic reticulum stress and insulin resistance post-trauma: similarities to type 2 diabetes. J Cell Mol Med, 2011.
- Song, J., et al., Severe burn-induced endoplasmic reticulum stress and hepatic damage in mice. Mol Med, 2009. 15(9-10): p. 316-20.
- Zhang, F., et al., Temporal production of the signaling lipid phosphatidic acid by phospholipase D2 determines the output of extracellular signal-regulated kinase signaling in cancer cells. Mol Cell Biol, 2014. 34(1): p. 84-95.
- Peng, M., et al., BACH1 is a DNA repair protein supporting BRCA1 damage response. Oncogene, 2006. 25(15): p. 2245-53.
- Kiviluoto, S., et al., Regulation of inositol 1,4,5-trisphosphate receptors during endoplasmic reticulum stress. Biochim Biophys Acta, 2013. 1833(7): p. 1612-24.
- Li, G., et al., Role of ERO1-alpha-mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress-induced apoptosis. J Cell Biol, 2009. 186(6): p. 783-92.
- Deniaud, A., et al., Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene, 2008. 27(3): p. 285-99.
- Liao, P.C., et al., Involvement of endoplasmic reticulum in paclitaxel-induced apoptosis. J Cell Biochem, 2008. 104(4): p. 1509-23.
- Mandic, A., et al., Cisplatin induces endoplasmic reticulum stress and nucleus-independent apoptotic signaling. J Biol Chem, 2003. 278(11): p. 9100-6.
- Short, D.M., et al., Apoptosis induced by staurosporine alters chaperone and endoplasmic reticulum proteins: Identification by quantitative proteomics. Proteomics, 2007. 7(17): p. 3085-96.Approach: 3