Dihydrotestosterone Receptors

*Significantly different from the H2O2-treated control cells ( 0

*Significantly different from the H2O2-treated control cells ( 0.01). Cell viability and nuclear morphology analyses revealed that depletion of endogenous PINK1 by PINK1 siRNA-1 or PINK1 siRNA-2 enhanced the susceptibility of PC12 cells to oxidative-stress-induced apoptosis (Physique 5DC5F). Presence of PINK1 and Oxidative Stress (A) Lysates from untransfected (UT) or transfected PC12 cells expressing wild-type (WT) PINK1 or the indicated mutant PINK1 were immunoprecipitated with anti-TRAP1 antibody, followed by immunoblotting using antibodies against TRAP1 and cytochrome c (Cyt. c). Input lane shows the levels of endogenous TRAP1 and cytochrome c in the lysate of untransfected PC12 cells.(B) PC12 cells expressing wild-type PINK1 or vector-transfected controls were incubated in the presence or absence of 400 M H2O2 for 16 h. Cell lysates (Input) were subjected to immunoprecipitation with anti-TRAP1, followed by immunoblotting using antibodies against TRAP1 and cytochrome c (Cyt. c). (1.2 MB TIF) pbio.0050172.sg003.tif (1.1M) GUID:?AE53EFEC-8780-4E65-85C8-615AADA16506 Abstract Mutations in the gene cause an autosomal recessive form of Parkinson disease (PD). So far, no substrates of PINK1 have been reported, and the mechanism by which PINK1 mutations lead to neurodegeneration is unknown. Here we report the identification of TNF receptor-associated protein 1 (TRAP1), a mitochondrial molecular chaperone also known as heat shock protein 75 (Hsp75), as a cellular substrate for PINK1 kinase. PINK1 binds and colocalizes with TRAP1 in the mitochondria and phosphorylates TRAP1 both in vitro and in vivo. We show that PINK1 protects against oxidative-stress-induced cell death by suppressing cytochrome c release from mitochondria, and this protective action of PINK1 depends on its kinase activity to phosphorylate TRAP1. Moreover, we find that the ability of PINK1 to promote TRAP1 phosphorylation and cell survival is usually impaired by PD-linked PINK1 G309D, L347P, and W437X mutations. Our findings suggest a novel pathway by which PINK1 phosphorylates downstream effector TRAP1 to prevent oxidative-stress-induced apoptosis and implicate the dysregulation of this mitochondrial pathway in PD pathogenesis. Author Summary Parkinson disease (PD) is usually characterized by the selective loss of midbrain dopaminergic neurons. Although the cause of PD is unknown, pathological analyses have suggested the involvement of oxidative stress and mitochondrial dysfunction. Recently, an inherited form of early-onset PD has been linked to mutations in both copies of the gene encoding the mitochondrial protein PINK1. Furthermore, increasing evidence indicates that single-copy mutations in PINK1 are a significant risk factor in the development of later-onset PD. Here we show that PINK1 is usually a protein kinase that phosphorylates the mitochondrial molecular chaperone TRAP1 to promote cell survival. We find that PINK1 normally protects against oxidative-stress-induced cell death by suppressing cytochrome c release from mitochondria. The PINK1 mutations linked to PD impair the ability of PINK1 to phosphorylate TRAP1 and SDZ 220-581 promote cell survival. Our findings reveal a novel anti-apoptotic signaling pathway that is disrupted by mutations in PINK1. We suggest that this pathway has a role in PD pathogenesis and may be a target for therapeutic intervention. Introduction Parkinson disease (PD) is the second most common neurodegenerative disease, characterized by the selective loss of dopaminergic neurons in the substantia nigra [1]. The cause of PD, particularly the sporadic disease, is unclear, but it likely involves both genetic and environmental factors. Genetic studies have identified a number of genes associated with familial PD [2]. Postmortem analyses reveal a deficiency in the mitochondrial complex I function in patients with sporadic PD [3]. Furthermore, exposure to environmental toxins that inhibit the mitochondrial complex I can lead to PD-like phenotypes in animal models [4], suggesting the involvement of mitochondrial dysfunction in PD pathogenesis. Mutations in the gene were originally discovered in three pedigrees with recessively inherited PD. Two homozygous mutations were initially identified: a truncating nonsense mutation SDZ 220-581 (W437X) and a G309D missense mutation [5]. Subsequently, multiple additional types ATP1B3 of PD-linked mutations or truncations in SDZ 220-581 have been reported, making the second most common causative gene of recessive PD [6,7]. Interestingly, despite autosomal recessive transmission of allele have been associated with late-onset PD [6C10]. The pathogenic mechanisms by which mutations lead to neurodegeneration are unknown. encodes a 581-amino-acid protein with a predicted N-terminal mitochondrial targeting sequence and a conserved serine/threonine kinase domain name [5]. PINK1 protein has been shown to localize in the mitochondria [5,11C13] and exhibit autophosphorylation activity in vitro [11,12,14]. The in vivo substrate(s) and biochemical function of PINK1 remain unknown. In cultured mammalian cells, overexpression of wild-type PINK1 protects cells against apoptotic stimuli [5,15], whereas small interfering RNA (siRNA)Cmediated depletion of PINK1 increases the susceptibility to apoptotic cell death [16]. In loss of PINK1 leads to mitochondrial defects and degeneration of muscle and dopaminergic neurons [17C20]. Despite ample evidence indicating an essential role of PINK1 in cytoprotection, the mechanism by which PINK1 protects against apoptosis is not understood. Here, we describe the characterization of mitochondrial serine/threonine kinase PINK1 and report the identification of TNF.