Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis

Abstract

Nitric oxide and other reactive nitrogen species target multiple sites in the mitochondria to impact cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose dependent progressive increase in NO-dependent DAF fluorescence that corresponded to mitochondrial membrane potential loss, and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially-derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.

cIAP1 and cIAP2 limit macrophage necroptosis by inhibiting Rip1 and Rip3 activation

“…In addition, we saw that phosphorylation of Rip1 rapidly Upon SM treatment, Rip3 and to a lesser extent Rip1 showed increased expression. Similarly, increased Rip3 expression in necroptotic cells has recently been reported, 38 and analysis of different cell lines has shown that sensitivity to TNF-a/zVAD-FMK-induced necrosis is determined by Rip3 expression. 11 Quantitative RT-PCR revealed that upregulation of Rips following SM treatment is likely post-transcriptional.…”

NF-κB Protects Cells from Gamma Interferon-Induced RIP1-Dependent Necroptosis

“…Images were acquired using a Carl Zeiss 510 Meta confocal system with excitation at 488 nm and 561 nm for ROS and ⌬m, respectively. Five images were collected randomly for each sample, and fluorescence change was quantified using Image J software (13,39 and rela Ϫ/Ϫ MEFs with IFN-␥ and monitored cell survival over a period of 72 h. As a positive control, we used TNF-␣, which is known to trigger apoptosis in the absence of RelA (5). As expected, TNF-␣ induced robust cell death of rela Ϫ/Ϫ MEFs within 12 to 24 h of challenge ( Fig.…”

Neuronal complex I deficiency occurs throughout the Parkinson’s disease brain, but is not associated with neurodegeneration or mitochondrial DNA damage

“…The NDUFB8 subunit is essential for the assembly, structure stability and function of the complex [39,46] and its knockdown decreases complex I activity in animals [10]. We, therefore, reasoned that NDUFB8 deficiency should reflect the entire complex.…”

“…Serial 3 μm thick sections were stained with primary antibodies (all from Abcam) against complex I (NDUFB8, ab110242, dilution 1:300), complex II (anti-SDHA, ab14715, dilution 1:4000), complex III (anti-UQCRC2, ab14745, dilution 1:10,000), complex IV (COX-I, ab14705, dilution 1:10,000), complex V (ATP-synthase, ab14748, dilution 1:10,000), and the mitochondrial membrane marker porin (VDAC1, ab14734, dilution 1:10,000). We chose the NDUFB8 subunit because it is central to complex I assembly and function [10,39,46] and has been widely used as a marker of the integrity of the complex [16,40,41]. Sections were deparaffinised in Histoclear and rehydrated in graded ethanol.…”