GAPDHcysNO in Bacterial Virulence: Induction of Macrophage Apoptosis
Nowhere perhaps is the significance of GAPDH as a moonlighting protein illustrated as well as with its role in apoptosis, including its “activation” by nitric oxide forming GAPDHcys-NO (Hara et al. 2006). Most notably, this is highlighted by the role of GAPDHcys-NO in the complex intracellular pathways through which cells program their own destruction. These biochemical pathways exhibit defined protein: protein and protein: nucleic acid interactions, affinity-mediated changes in protein binding, changes in intracellular localization, uncovering of dormant protein activities, sequential post-translational modifications, induction of gene expression, and, lastly, the role of GAPDHcys-NO as a transnitrosylase. All of the above are initiated subsequent to a single event, that is, NO modification of GAPDH at its active site cysteine. To initiate apoptosis, cytoplasmic GAPDHcys-NO binds to Siah1, an E3 ubiquitin ligase which catalyzes protein degradation
(Hara et al. 2005; Hara and Snyder 2006). This protein complex translocates to the nucleus where not only does Siah1 catalyze the degradation of nuclear proteins, but also, through the formation of a second nuclear protein complex GAPDHcys-NO: Siahl: P300/CBP, induces a cell death program of gene involving the regulation of p53, PUMA, Bax, and p21 (Sen et al. 2008). Further analysis indicated that GAPDHcys-NO may act a as a transnitrosylase, shifting enzymatically its NO moiety to an acceptor protein which now may assume new functions (Kornberg et al. 2010; Kohr et al. 2014; Rodriguez-Ortigosa et al. 2014).
As if these were not, by themselves, a series of complex cellular events, other studies indicate the presence of a competing mechanism which may be intended to protect cells from the apoptotic effects of GAPDHcys-NO. These investigations identified a novel protein, GOSPEL (GADPH's competitor of Siah Protein Enhances Life), which binds GAPDHcys-NO thereby preventing the formation of the GAPDHcys-NO: Siahl complex (Sen et al. 2009). This protein: protein interaction vitiates Siahl nuclear translocation and all the subsequent nuclear apopotic effects. The kinetic relationship between the two protein: protein complexes (GAPDHcys-NO: Siahl and GAPDHcys-NO: GOSPEL) is unknown. However, it was suggested that a ternary intermediate complex may be formed by interaction of Siahl with GAPDHcys-NO: GOSPEL, resulting in the formation of GAPDHcys-NO: Siahl and the dissociation of GOSPEL (Sirover 20ll).
Bacterial virulence may be maintained by inducing apoptosis in host macrophages. In this manner, the respective pathogen may evade immunopro- tective defenses by destroying those cells responsible for that protection. Three separate studies identified that both nitric oxide and GAPDH were required for bacteria to induce macrophage-programmed cell death. First, Marriott et al. (2004) demonstrated using iNOS inhibitors that S. pneumonia-induced human macrophage apoptosis was dependent on its ability to produce nitric oxide. In those studies, infection resulted in stimulation of iNOS activity and rates of apoptosis. The addition of now-classical iNOS inhibitors diminished both the former and the latter in a coordinated manner.
Second, Ulett and Adderson (2005) reported that S. agalactiae infection of murine macrophages induced apoptosis. Using inhibitor analysis, they observed that iNOS expression was required. Third, in both S. agalactiae and in S. aureus infection of murine macrophages, Oliveira et al. (20l2) demonstrated that extracellular bacterial GAPDH was required for the induction of apoptosis by each pathogen. In particular, depletion of bacterial GAPDH from culture supernatants diminished programmed cell death. Cumulatively, these results suggest, but do not prove that, each pathogen forms bacterial GAPDHcys-NO as the active molecule which induces macrophage apoptosis, thereby providing an immuno- evasive pathway. This may be tested experimentally.