HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate endoplasmic reticulum stress-induced apoptosis

BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants

NGF/p75NTR or HSPB1-mediated regulation of cell survival under stress

Triple negative breast cancers (TNBC) lack estrogen, progesterone and HER-2 receptors rendering them insensitive to current hormone targeted therapies. However, TNBC have been reported to express and secrete an additional growth factor, Nerve Growth Factor (NGF). They also express the two cognate receptors, the 75 kDa neurotrophin receptor (p75NTR) and the tropomyosin related kinase A receptor (TrkA) to which NGF can bind. Identifying NGF -mediated p75NTRsignaling is a potential therapeutic target for TNBC. Using MDA-MB-231 cells, a representative of TNBC I confirmed a role for NGF-mediated pro-survival signaling through p75NTR. It is well known that NGF signaling through TrkA mediates cancer cell proliferation, while its signaling through p75NTR protects breast cancer cells from apoptosis. Therefore, targeting NGF/p75NTR signaling could potentially sensitize cells to chemotherapeutics. In this study, shRNA targeted to p75NTR to knock down the receptor or inhibitors designed to disrupt the NGF/p75NTR signaling were used and it was found that disrupting NGF/p75NTR signaling sensitized TNBC cells to apoptosis. The findings also illustrate that NGF regulates the levels of p75NTR through two distinct mechanisms, post-translational and transcriptional. Firstly, using Western blotting it was found that exogenous NGF inhibits the endogenous processing of p75NTR. Secondly, it was shown that exogenous NGF increased p75NTR mRNA transcription as determined by RT-PCR. A differential sensitivity of the cells to apoptosis induction was observed with and without induction of p75NTR by NGF. This regulation of p75NTR by NGF is linked to increased resistance of these cells to chemotherapeutic drugs. Alteration in NGF signaling is not only implicated in resistance of breast cancer to chemotherapeutic reagents but it is also associated with a number of other disease states. Previous work in our group involved the design and generation of NGF variants with a view to using them to selectively target Trk or p75NTR receptors. Here these variants were produced in HEK293T cells and their biological signaling was examined. Eight NGF variants were chosen and expressed into the media of HEK293T cells. The concentration of the variants was determined using an NGF ELISA followed by the analysis of TrkA-induced pro-survival signaling. The I31R and R69D NGF variants displayed no TrkA pro-survival activity as they were unable to protect PC12 cells from TG induced cell death. Interestingly I31R displayed no TrkA activity but retained binding affinity to p75NTR. I31 residue on NGF was identified as a target to disrupt TrkA signaling in disease conditions. The second part of the thesis focused on the pro-survival signaling mediated by the intracellular ER chaperone protein HSPB1 in response to ER stress. HSPB1 that belongs to the family of small heat shock proteins is a potent regulator of apoptosis. However, the role of HSPB1 in ER-stress induced apoptosis has not been delineated. Previous work in the lab has shown that thermal pre-conditioning can protect cells from ER stress induced cell death through its ability to regulate BH3 only protein, BIM. Here it is shown that overexpression of HSPB1 protected PC12 cells from ER stress induced apoptosis through ERK-mediated phosphorylation of BH3 only protein, BIM leading to proteasomal degradation of BIM. It is also shown that HSPB1 interacts with phospho-ERK 1/2 and BIM to mediate a cytoprotective effect against thapsigargin-induced ER stress. I also characterized the effect of mutations in HSPB1 in response to ER stress in PC12 cells. PC12 cells that stably expressed mutations in HSPB1 showed an inability to confer protection in response to ER stress and were unable to mediate degradation of BIM as they did in their wild type counterpart.2020-02-0

Nerve growth factor (NGF)-mediated regulation of p75(NTR) expression contributes to chemotherapeutic resistance in triple negative breast cancer cells

Triple negative breast cancer [TNBC] cells are reported to secrete the neurotrophin nerve growth factor [NGF] and express its receptors, p75 neurotrophin receptor [p75(NTR)] and TrkA, leading to NGF-activated pro-survival autocrine signaling. This provides a rationale for NGF as a potential therapeutic target for TNBC. Here we show that exposure of TNBC cells to NGF leads to increased levels of p75(NTR), which was diminished by NGF-neutralizing antibody or NGF inhibitors [Ro 08-2750 and Y1086]. NGF-mediated increase in p75(NTR) levels were partly due to increased transcription and partly due to inhibition of proteolytic processing of p75(NTR). In contrast, proNGF caused a decrease in p75(NTR) levels. Functionally, NGF-induced increase in p75(NTR) caused a decrease in the sensitivity of TNBC cells to apoptosis induction. In contrast, knock-down of p75(NTR) using shRNA or small molecule inhibition of NGF-p75(NTR) interaction [using Ro 08-2750] sensitized TNBC cells to drug-induced apoptosis. In patient samples, the expression of NGF and NGFR [the p75(NTR) gene] mRNA are positively correlated in several subtypes of breast cancer, including basal-like breast cancer. Together these data suggest a positive feedback loop through which NGF-mediated upregulation of p75(NTR) can contribute to the chemo-resistance of TNBC cells.Higher Education Authority [PRTLI 5] and the Beckman Fund, School of Natural Sciences, NUI Galway, Ireland. K.M. is funded by an Irish Research Council Fellowship (GOIPD/2014/53).2017-08-2

Phosphorylation of BRCA1 by ATM upon double-strand breaks impacts ATM function in end-resection: A potential feedback loop

BRCA1 maintains genome stability by promoting homologous recombination (HR)-mediated DNA double-strand break (DSB) repair. Mutation of mouse BRCA1-S1152, corresponding to an ATM phosphorylation site in its human counterpart, resulted in increased genomic instability and tumor incidence. In this study, we report that BRCA1-S1152 is part of a feedback loop that sustains ATM activity. BRCA1-S1152A mutation impairs recruitment of the E3 ubiquitin ligase SKP2. This in turn attenuates NBS1-K63 ubiquitination by SKP2 at DSB, impairs sustained ATM activation, and ultimately leads to deficient end resection, the commitment step in the HR repair pathway. Auto-phosphorylation of human ATM at S1981 is known to be important for its kinase activation; we mutated the corresponding amino acid residue in mouse ATM (S1987A) to characterize potential roles of mouse ATM-S1987 in the BRCA1-SKP2-NBS1-ATM feedback loop. Unexpectedly, MEFs carrying the ATM-S1987A knockin mutation maintain damage-induced ATM kinase activation, suggesting a species-specific function of human ATM auto-phosphorylation

Generation of rationally-designed nerve growth factor (NGF) variants with receptor specificity

Nerve growth factor (NGF) is the prototypic member of the neurotrophin family and binds two receptors, TrkA and the 75 kDa neurotrophin receptor (p75NTR), through which diverse and sometimes opposing effects are mediated. Using the FoldX protein design algorithm, we generated eight NGF variants with different point mutations predicted to have altered binding to TrkA or p75NTR. Of these, the I31R NGF variant exhibited specific binding to p75NTR. The generation of this NGF variant with selective affinity for p75NTR can be used to enhance understanding of neurotrophin receptor imbalance in diseases and identifies a key targetable residue for the development of small molecules to disrupt binding of NGF to TrkA with potential uses in chronic pain.This material is based upon works supported by the Science Foundation Ireland under Grant No. 09/RFP/BMT2153, Enterprise Ireland (IP 2016 0480), the Higher Education Authority [PRTLI 5] and the Beckman Fund, School of Natural Sciences, NUI Galway, Ireland.2018-11-0

Hspb1 facilitates erk-mediated phosphorylation and degradation of bim to attenuate endoplasmic reticulum stress-induced apoptosis

BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants

Hspb1 facilitates erk-mediated phosphorylation and degradation of bim to attenuate endoplasmic reticulum stress-induced apoptosis

BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants

Staphylococcal phosphatidylglycerol antigens activate human T cells via CD1a

Expressed on epidermal Langerhans cells, CD1a presents a range of self-lipid antigens found within the skin; however, the extent to which CD1a presents microbial ligands from bacteria colonizing the skin is unclear. Here we identified CD1a-dependent T cell responses to phosphatidylglycerol (PG), a ubiquitous bacterial membrane phospholipid, as well as to lysylPG, a modified PG, present in several Gram-positive bacteria and highly abundant in Staphylococcus aureus. The crystal structure of the CD1a–PG complex showed that the acyl chains were buried within the A′- and F′-pockets of CD1a, while the phosphoglycerol headgroup remained solvent exposed in the F′-portal and was available for T cell receptor contact. Using lysylPG and PG-loaded CD1a tetramers, we identified T cells in peripheral blood and in skin that respond to these lipids in a dose-dependent manner. Tetramer+CD4+ T cell lines secreted type 2 helper T cell cytokines in response to phosphatidylglycerols as well as to co-cultures of CD1a+ dendritic cells and Staphylococcus bacteria. The expansion in patients with atopic dermatitis of CD4+ CD1a–(lysyl)PG tetramer+ T cells suggests a response to lipids made by bacteria associated with atopic dermatitis and provides a link supporting involvement of PG-based lipid-activated T cells in atopic dermatitis pathogenesis