Browsing by Subject "Caspase-2"
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Item Open Access Metabolic Control of CaMKII-mediated Caspase-2 Suppression by B55β/PP2A(2015) Huang, BofuApoptosis is a programmed form of cell death, essential for maintaining tissue homeostasis and eliminating dysfunctional cells. The process of apoptosis is executed by a family of cysteine proteases called caspases. High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway (PPP). This metabolic suppression of caspase-2 is exerted via the inhibitory phosphorylation of S135 on the caspase-2 prodomain by activated Ca2+/Calmodulin-dependent protein kinase II (CaMKII). However, it was unclear how CaMKII activity is regulated by nutrient flux.
After investigating how nutrient flux leads to activation of CaMKII, a recent study reported that coenzyme A (CoA) can directly bind to and activate CaMKII. However, by performing mass spectrometry (MS) analysis of CaMKII, and other biochemical assays, including gel filtration assays, immuno-precipitation assays, immuno-depletion assays, and in vitro kinase assays, in the Xenopus egg extract system, our studies show that the complete nutrient-driven CaMKII activation requires the additional release of a "brake" through the dephosphorylation of CaMKII at novel sites (T393/S395). Furthermore, this metabolically-stimulated dephosphorylation of CaMKII is mediated by the metabolic activation of protein phosphatase 2A (PP2A) in complex with the B55β targeting subunit. Importantly, our findings have been successfully replicated in human 293T cells, including the metabolic activation of CaMKII, and also the suppression of this activation by B55β knockdown.
Our discovery represents a novel locus of CaMKII regulation and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and PP2A-regulated physiological processes, as both enzymes appear to be responsive to alterations in glucose metabolized via the PPP. Finally, our study reveals B55β as a potential target for cancer therapy, because of its importance in suppressing metabolic suppression of caspase-2 activation and apoptosis.
Item Open Access Metabolic Regulation of Caspase-2(2011) Buchakjian, Marisa RaeApoptosis is a form of programmed cellular "suicide" which is activated in response to a variety of pro-death stimuli. Apoptotic cell death is orderly and energy-dependent, and cellular constituents are packaged into membrane-bound vesicles for consumption by phagocytes. Toxic intracellular signals are never exposed to neighboring cells or to the extracellular environment, and a host inflammatory response does not occur. Apoptosis is executed by the coordinated activation of caspase family proteins. Caspase-2 is an apical protease in this family, and promotes cell death after receipt of cues from intracellular stressor signals. Caspase-2 helps to initiate apoptosis by responding to cellular death stimuli and signaling for downstream cytochrome c release and executioner caspase activation.
Several years ago our lab determined that Xenopus laevis oocyte death is partly controlled by the activation of caspase-2. In the setting of oocyte or egg extract nutrient depletion, caspase-2 was observed to be activated upstream of mitochondrial cytochrome c. In fact, caspase-2 is suppressed in response to the nutrient status of the oocyte: nutrient-replete oocytes with healthy pentose phosphate pathway flux and abundant NADPH production are able to inhibit caspase-2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. Phosphorylation of caspase-2 at S135 is critical in preventing oocyte cell death, and a caspase-2 mutant unable to be phosphorylated loses its ability to respond to suppressive NADPH signals.
In this dissertation we examine the converse mechanism of metabolically-regulated caspase-2 activation in the Xenopus egg extract. We now show that caspase-2 phosphorylated at S135 binds the interactor 14-3-3 zeta, thus preventing caspase-2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1, which directly binds caspase-2. Although caspase-2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3 zeta from caspase-2 is the point of metabolic control and allows for caspase-2 dephosphorylation. Accordingly, a caspase-2 mutant unable to bind 14-3-3 zeta is highly susceptible to activation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine caspase-2 by phosphorylation and 14-3-3 binding in mouse eggs.
In the second part of this dissertation we examine the paradigm of caspase-2 metabolic regulation in a mammalian somatic cell context. We observed that mammalian caspase-2 is a metabolically-regulated phosphoprotein in somatic cells, and that the site of regulation is caspase-2 S164. Phosphorylation at S164 appears to inhibit mammalian caspase-2 by preventing its induced proximity oligomerization, thus also preventing procaspase-2 autocatalytic processing. We further identify some of the molecular machinery involved in S164 phosphorylation and demonstrate conservation with the validated Xenopus regulators. Interestingly, we extend the findings of caspase-2 phosphorylation to a study of ovarian cancer, and show that caspase-2 S164 phosphorylation might be involved in determining cancer cell chemosensitivity. We further provide evidence that chemosensitivity can be modulated by the cellular metabolic status in a caspase-2-dependent manner. Thus, we have identified a novel phosphorylation site on mammalian caspase-2 in somatic cells, and are working further to understand the implications of caspase-2 signaling in the context of cancer cell responsiveness to chemotherapeutic treatments.
Item Open Access TRAF Regulation of Caspase-2-Dependent Apoptosis in Response to DNA Damage(2016) Robeson, AlexanderThe DNA of a cell operates as its blueprint, providing coded information for the production of the RNA and proteins that allow the cell to function. Cells can face a myriad of insults to their genomic integrity during their lifetimes, from simple errors during growth and division to reactive oxygen species to chemotherapeutic reagents. To deal with these mutagenic insults and avoid passing them on to progeny, cells are equipped with multiple defenses. Checkpoints can sense problems and halt a cell’s progression through the cell cycle in order to allow repairs. More drastically, cells can also prevent passing on mutations to progeny by triggering apoptosis, or programmed cell death. This work will present two separate discoveries regarding the regulation of DNA damage-induced apoptosis and the regulation of the spindle checkpoint.
The protease caspase-2 has previously been shown to be an important regulator of DNA damage-induced apoptosis. In unstressed cells caspase-2 is present as an inactive monomer, but upon sensing a stress caspase-2 dimerizes and becomes catalytically active. The mechanisms that regulate this dimerization are poorly understood. The first research chapter details our development of a novel method to study dimerized caspase-2, which in turn identified TRAF2 as a direct activator of caspase-2. Specifically, we utilized the Bimolecular Fluorescence Complementation technique, wherein complementary halves of the Venus fluorophore are fused to caspase-2: when caspase-2 dimerizes, the non-fluorescent halves fold into a functional Venus fluorophore. We combined this technique with a Venus-specific immunoprecipitation that allowed the purification of caspase-2 dimers. Characterization of the caspase-2 dimer interactome by MS/MS identified several members of the TNF Receptor Associated Factor (TRAF) family, specifically TRAF1, 2, and 3. Knockdown studies revealed that TRAF2 plays a primary role in promoting caspase-2 dimerization and downstream apoptosis in response to DNA damage. Identification of a TRAF Interacting Motif (TIM) on caspase-2 indicates that TRAF2 directly acts on caspase-2 to induce its activation. TRAF2 is known to act as an E3 ubiquitin ligase as well as a scaffold for other E3 ubiquitin ligases. Indeed, we identified three lysine residues in the caspase-2 prodomain (K15, K152, and K153) important for its ubiquitination and complex formation. Together these results revealed a novel role for TRAF2 as a direct activator of caspase-2 apoptosis triggered by DNA damage.
During mitosis, when the cell prepares to divide, great care is taken to ensure that the chromosomes are properly segregated between the two daughter cells by the mitotic spindle. This is primarily accomplished through the spindle checkpoint, which becomes activated when the mitotic spindle is not properly attached to each chromosome’s kinetochore. When activated, the primary effector of the spindle checkpoint, the mitotic checkpoint complex (MCC), inhibits the anaphase-promoting complex (APC/C) by binding to the APC/C co-activator, CDC20. This prevents the APC/C from targeting critical pro-mitotic proteins, like cyclin B and securin, to promote mitotic exit. Although the function of the MCC is well understood, its regulation is not, especially in regard to protein phosphatases To investigate this, we activated the spindle checkpoint with microtubule inhibitors and then treated with a variety of phosphatase inhibitors, examining the effect on the MCC and APC/C. We found that two separate inhibitors, calyculin A and okadaic acid (1uM), were able to promote the dissociation of the MCC. This led to the activation of the APC/C, but the cells remained in mitosis as evidenced by high levels of Cdk1 activity and chromosome condensation. This is the first time that phosphatases have been shown to be essential to maintaining the MCC and an active spindle checkpoint.
Item Open Access Uncovering a Novel Role of the Apoptotic Initiator Caspase, Caspase-2(2014) Segear Johnson, Erika LeeWith the prevalence of obesity and metabolic syndrome rising sharply world-wide, it has become increasingly important to define the molecular mechanisms underlying the pathogenesis and progression of diseases associated with lipid-induced cytotoxicity. Cardiovascular disease, type-2 diabetes mellitus, and nonalchoholic fatty liver disease (NAFLD) have all recently gained recognition as diseases that are exacerbated by lipoapoptosis. In this dissertation, we demonstrate a novel role for caspase-2 as an initiator of lipoapoptosis. Using an unbiased metabolomics approach, we discovered that the activation of caspase-2, the initiator of apoptosis in Xenopus egg extracts, is associated with an accumulation of long-chain fatty acid (LCFA) metabolites. Metabolic treatments that block the buildup of LCFAs potently inhibit caspase-2, while add-back of a saturated LCFA restores caspase activation in the extract setting. Extending these findings to mammalian cells, we show that caspase-2 is engaged and activated in response to treatment with the saturated LCFA, palmitate. Down-regulation of caspase-2 significantly impairs cell death induced by saturated LCFAs, revealing a conserved, critical role for caspase-2 in mediating LCFA-induced lipoapoptosis.
Since lipoapoptosis has been implicated as a key driver of the progression of NAFLD, we aimed to determine the therapeutic significance of our findings by evaluating the importance of caspase-2 in an in vivo model of this disease. We subjected wild-type and caspase-2 knockout mice to a diet which induces severe liver steatosis and the development nonalcoholic steatohepatitis (NASH), the most advanced stage of NAFLD characterized by liver fibrosis. Interestingly, we observed an increase in caspase-2 protein levels in the livers of wild-type mice fed a NASH-inducing diet. These findings were of particular importance, since caspase-2 expression was also significantly elevated in patients diagnosed with NASH. Most importantly, we demonstrated that caspase-2 knockout mice are protected from apoptosis and fibrosis when fed a NASH-inducing diet, suggesting that caspase-2 is major regulator of hepatocyte lipoapoptosis. Together, these findings reveal a previously unknown role for caspase-2 as an initiator of lipoapoptosis and suggest that caspase-2 may be an attractive therapeutic target for inhibiting pathological lipid-induced apoptosis.