Here, we demonstrate a role for the mitochondrial NAD-dependent deacetylase Sirt3 in the maintenance of basal ATP levels and as a regulator of mitochondrial electron transport. We note that Sirt3 ؊/؊ mouse embryonic fibroblasts have a reduction in basal ATP levels. Reconstitution with wild-type but not a deacetylase-deficient form of Sirt3 restored ATP levels in these cells. Furthermore in wild-type mice, the resting level of ATP correlates with organ-specific Sirt3 protein expression. Remarkably, in mice lacking Sirt3, basal levels of ATP in the heart, kidney, and liver were reduced >50%. We further demonstrate that mitochondrial protein acetylation is markedly elevated in Sirt3 ؊/؊ tissues. In addition, in the absence of Sirt3, multiple components of Complex I of the electron transport chain demonstrate increased acetylation. Sirt3 can also physically interact with at least one of the known subunits of Complex I, the 39-kDa protein NDUFA9. Functional studies demonstrate that mitochondria from Sirt3 ؊/؊ animals display a selective inhibition of Complex I activity. Furthermore, incubation of exogenous Sirt3 with mitochondria can augment Complex I activity. These results implicate protein acetylation as an important regulator of Complex I activity and demonstrate that Sirt3 functions in vivo to regulate and maintain basal ATP levels.acetylation ͉ sirtuins ͉ complex I ͉ electron transport

Mitochondrial reactive oxygen species and reactive nitrogen species are proven to be major sources of oxidative stress in the cell; they play a prominent role in a wide range of human disorders resulting from nonapoptotic cell death. The aim of this study is to examine the cytoprotective effect of the NecroX series against harmful stresses, including pro-oxidant (tertiarybutylhydroperoxide), doxorubicin, CCl₄, and hypoxic injury. In this study, these novel chemical molecules inhibited caspase-independent cell death with necrotic morphology, which is distinctly different from apoptosis, autophagy, and necroptosis. In addition, they displayed strong mitochondrial reactive oxygen species and ONOO⁻ scavenging activity. Further, oral administration of these molecules in C57BL/6 mice attenuated streptozotocin-induced pancreatic islet β-cell destruction as well as CCl₄-induced hepatotoxicity in vivo. Taken together, these results demonstrate that the NecroX series are involved in the blockade of nonapoptotic cell death against mitochondrial oxidative stresses. Thus, these chemical molecules are potential therapeutic agents in mitochondria-related human diseases involving necrotic tissue injury.

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Aims: Acetaminophen (APAP)-induced liver injury is mainly due to the excessive formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) through the formation of a reactive intermediate, N-acetyl-pbenzoquinone imine (NAPQI), in both humans and rodents. Here, we show that the indole-derived synthetic compound has a protective effect against APAP-induced liver injury in C57Bl/6 mice model. Results: NecroX-7 decreased tert-butylhydroperoxide (t-BHP)-and APAP-induced cell death and ROS/RNS formation in HepG2 human hepatocarcinoma and primary mouse hepatocytes. In mice, NecroX-7 decreased APAP-induced phosphorylation of c-Jun N-terminal kinase ( JNK) and 3-nitrotyrosine (3-NT) formation, and also protected mice from APAP-induced liver injury and lethality by binding directly to NAPQI. The binding of NecroX-7 to NAPQI did not require any of cofactors or proteins. NecroX-7 could only scavenge NAPQI when hepatocellular GSH levels were very low. Innovation: NecroX-7 is an indole-derived potent antioxidant molecule, which can be bound to some types of radicals and especially NAPQI. It is well known that the NAPQI is a major intermediate of APAP, which causes necrosis of hepatocytes in rodents and humans. Thus, blocking NAPQI formation or eliminating NAPQI are novel strategies for the treatment or prevention of APAP-induced liver injury instead of GSH replenishment. Conclusion: Our data suggest that the indole-derivative, NecroX-7, directly binds to NAPQI when hepatic GSH levels are very low and the NAPQI-NecroX-7 complex is secreted to the blood from the liver. NecroX-7 shows more preventive and similar therapeutic effects against APAP-induced liver injury when compared to the effect of N-acetylcysteine in C57Bl/6 mice.

Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits. The CKIIbeta subunit is thought to mediate the tetramer formation and interact with other target proteins. However, its physiological function remains obscure. In this study, point mutants of CKIIbeta that are defective for the L41 binding were isolated by using the reverse two-hybrid system. A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of CKIIbeta are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for CKIIbeta homodimerization. Two point mutants, R75 and R83, of CKIIbeta interacted with L5, topoisomerase IIbeta, and CKBBP1/SAG, but not with the wild-type CKIIbeta. This indicates that CKIIbeta homodimerization is not a prerequisite for its binding to target proteins. These CKIIbeta point mutants may be useful in exploring the biochemical physiological functions of CKIIbeta.