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HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation.

ABSTRACT: Neuronal stress or injury results in the activation of proteins, which regulate the balance between survival and apoptosis. However, the complex mechanism of cell signaling involving cell death and survival, activated in response to cellular stress is not yet completely understood. To bring more clarity about these mechanisms, a Boolean network was constructed that represented the apoptotic pathway in neuronal cells. FasL and neurotrophic growth factor (NGF) were considered as inputs in the absence and presence of heat shock proteins known to shift the balance toward survival by rescuing pro-apoptotic cells. The probabilities of survival, DNA repair and apoptosis as cellular fates, in the presence of either the growth factor or FasL, revealed a survival bias encoded in the network. Boolean predictions tested by measuring the mRNA level of caspase-3, caspase-8, and BAX in neuronal Neuro2a (N2a) cell line with NGF and FasL as external input, showed positive correlation with the observed experimental results for survival and apoptotic states. It was observed that HSP70 contributed more toward rescuing cells from apoptosis in comparison to HSP27, HSP40, and HSP90. Overexpression of HSP70 in N2a transfected cells showed reversal of cellular fate from FasL-induced apoptosis to survival. Further, the pro-survival role of the proteins BCL2, IAP, cFLIP, and NF?B determined by vertex perturbation analysis was experimentally validated through protein inhibition experiments using EM20-25, Embelin and Wedelolactone, which resulted in 1.27-, 1.26-, and 1.46-fold increase in apoptosis of N2a cells. The existence of a one-to-one correspondence between cellular fates and attractor states shows that Boolean networks may be employed with confidence in qualitative analytical studies of biological networks.

SUBMITTER: Vasaikar SV

PROVIDER: S-EPMC4548197 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation.

Vasaikar Suhas V SV Ghosh Sourish S Narain Priyam P Basu Anirban A Gomes James J

Frontiers in cellular neuroscience 20150825

Neuronal stress or injury results in the activation of proteins, which regulate the balance between survival and apoptosis. However, the complex mechanism of cell signaling involving cell death and survival, activated in response to cellular stress is not yet completely understood. To bring more clarity about these mechanisms, a Boolean network was constructed that represented the apoptotic pathway in neuronal cells. FasL and neurotrophic growth factor (NGF) were considered as inputs in the abse ...[more]

PMID: 26379495

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Project description:BackgroundThe oncoprotein MYC has the dual capacity to drive cell cycle progression or induce apoptosis, depending on the cellular context. BAG1 was previously identified as a transcriptional target of MYC that functions as a critical determinant of this cell fate decision. The BAG1 protein is expressed as multiple isoforms, each having an array of distinct biochemical functions; however, the specific effector function of BAG1 that directs MYC-dependent cell survival has not been defined.MethodsIn our studies the human osteosarcoma line U2OS expressing a conditional MYC-ER allele was used to induce oncogenic levels of MYC. We interrogated MYC-driven survival processes by modifying BAG1 protein expression. The function of the separate BAG1 isoforms was investigated by depleting cells of endogenous BAG1 and reintroducing the distinct isoforms. Flow cytometry and immunoblot assays were performed to analyze the effect of specific BAG1 isoforms on MYC-dependent apoptosis. These experiments were repeated to determine the role of the HSP70 chaperone complex in BAG1 survival processes. Finally, a proteomic approach was used to identify a set of specific pro-survival proteins controlled by the HSP70/BAG1 complex.ResultsLoss of BAG1 resulted in robust MYC-induced apoptosis. Expression of the larger isoforms of BAG1, BAG1L and BAG1M, were insufficient to rescue survival in cells with oncogenic levels of MYC. Alternatively, reintroduction of BAG1S significantly reduced the level of apoptosis. Manipulation of the BAG1S interaction with HSP70 revealed that BAG1S provides its pro-survival function by serving as a cofactor for the HSP70 chaperone complex. Via a proteomic approach we identified and classified a set of pro-survival proteins controlled by this HSP70/BAG1 chaperone complex that contribute to the BAG1 anti-apoptotic phenotype.ConclusionsThe small isoform of BAG1, BAG1S, in cooperation with the HSP70 chaperone complex, selectively mediates cell survival in MYC overexpressing tumor cells. We identified a set of specific pro-survival clients controlled by the HSP70/BAG1S chaperone complex. These clients define new nodes that could be therapeutically targeted to disrupt the survival of tumor cells driven by MYC activation. With MYC overexpression occurring in most human cancers, this introduces new strategies for cancer treatment.

Dataset Information

HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation.

Publications

HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation.

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