Nucleophosmin, a Critical Bax Cofactor in Ischemia-Induced Cell Death

Abstract

We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediated cell death. To test this hypothesis, Bax activation was induced by metabolic stress. During stress, nucleolar NPM translocated into the cytosol, NPMBax complexes formed, and both NPM and Bax accumulated in mitochondria. Expression of a cytosol-restricted NPM mutant (NPM-⌬NLS), but not a nucleus-restricted NPM mutant, increased NPM-Bax complex formation, mitochondrial NPM and Bax accumulation, mitochondrial membrane injury, caspase 3 activation, and ischemia-induced cell death. Coexpression of NPM-⌬NLS with constitutively active Bax mutants caused nearly universal cell death in the absence of metabolic stress, whereas expression of active Bax or NPM-⌬NLS alone did not. A Bax peptide that disrupts NPM-Bax interaction significantly reduced cell death caused by exposure to metabolic inhibitors in vitro and preserved kidney function after ischemia in vivo. Thus, NPM-Bax interaction enhances mitochondrial Bax accumulation, organelle injury, and cell death. NPM-Bax complex formation is a novel target for preventing ischemic tissue injury. Although Bax toxicity is well characterized, the events that signal Bax translocation to mitochondria, a major target of Baxmediated apoptosis, are unknown (1). Interestingly, more than 75% of Bax colocalizes with mitochondria during apoptogenic insults, whereas the remaining Bax translocates to nuclei and endoplasmic reticulum or exists unbound in the cytosol (2). Bax lacks a mitochondrial localizing sequence, and a mitochondrial Bax receptor has yet to be identified (3, 4), complicating efforts to dissect the molecular signals that drive intracellular Bax movement and toxicity. The possibility that a molecular chaperone facilitates mitochondrial Bax accumulation has been entertained recently (5, 6).Several Bax chaperones have been proposed (7), including humanin, Ku-70, Bax interacting factor 1 (bif-1), clusterin, specific 14-3-3 isoforms, and nucleophosmin (NPM; also called B23). NPM is an abundant, ubiquitously expressed, 35-kDa phosphoprotein that localizes to the nucleolar region of resting cells (8). In healthy cells, NPM promotes RNA metabolism, as well as the packaging and transport of cell proteins, by reversibly shuttling between the cytosol and the nucleolar region (9, 10). NPM also migrates into the cytosol during cell division to promote ribosomal protein synthesis and cell proliferation before returning to the nucleolar region (5,11,12). These NPM functions are essential, since its elimination causes embryonic death in NPM knockout mice (11).In cell-free systems, NPM interacts with Bax (6, 13). However, NPM only binds Bax that has undergone conformational change characterized by exposure of the amino-terminal 6A7 epitope (6, 14). The site responsible for binding NPM has been identified and localizes to the Bax carboxy-terminal domain (14). Furthermore, a synthetic, 21-amino-acid Bax peptide that includes this carboxyterminal NPM binding site disrupts NPM-Bax interaction in...

Regulated Cell Death in AKI

“…Bax activation may involve the interaction with specific proteins, such as Bid, p53, humanin, 14-3-3 protein, ku70, and Bif-1. [66][67][68][69][70][71][72] In renal tubular cells, Borkan and colleagues 73 have recently shown the interaction of nucleophosmin with Bax, which seems to be critical to Bax activation during metabolic stress in vitro and ischemic AKI in vivo. As discussed below, the activation of Bax and Bak also involves a striking change of mitochondrial morphology and consequent alterations of the membrane property.…”

The Role of BCL-2 Family Members in Acute Kidney Injury

“…42,43 Remarkably, neither conformational bax activation (6A7 epitope exposure) nor cytosolic NPM accumulation are sufficient to cause death in renal epithelial cells. 44 In contrast, coexpression of conformationally active bax and a cytosol-restricted NPM mutant cause marked renal cell death even in the absence of a pro-apoptotic insult. 44 NPM immunoprecipitates with active bax and both accumulate in mitochondria subjected to a proapoptotic insult.…”

“…44 In contrast, coexpression of conformationally active bax and a cytosol-restricted NPM mutant cause marked renal cell death even in the absence of a pro-apoptotic insult. 44 NPM immunoprecipitates with active bax and both accumulate in mitochondria subjected to a proapoptotic insult. 44 Interestingly, conformationally active bax has been detected in nuclei and, in at least some circumstances, actually releases nuclear NPM into the cytosol, a novel bax effect that is not antagonized by bcl-xl.…”

“…44 NPM immunoprecipitates with active bax and both accumulate in mitochondria subjected to a proapoptotic insult. 44 Interestingly, conformationally active bax has been detected in nuclei and, in at least some circumstances, actually releases nuclear NPM into the cytosol, a novel bax effect that is not antagonized by bcl-xl. 45 Taken together, these observations show a collaborative but fail-safe system in which bax and NPM are mutually dependent coconspirators in precipitating cell death.…”

The role of the nucleolus in neurodegeneration.

“…Surprisingly, NPM has been proposed to play contradictory roles depending on the context. In a rodent model of stroke, NPM has been implicated as a chaperone for the pro-apoptotic BCL2 Associated X Protein (BAX) [111,249]. Conversely, in cultured rodent neurons, anti-excitotoxic effects were also demonstrated as NPM inhibited the Seven In Absentia Homolog 1 (SIAH1)-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) death cascade [136,159].…”