Project description:One of the downstream effects of mitophagy is the transient activation of mitochondrial biogenesis and, in turn, enhanced mitochondrial respiration. To gain insights into the transcriptional changes produced in ECs by incorporating exogenous mitochondria through either co-culture with MSCs (mitoT-ECs) or artificial transplantation (mitoAT-ECs), we performed RNA-seq analysis.
Project description:During early post-natal stage, cardiomyocytes undergo dramatic structural, metabolic, electrophysiological and cell cycle alterations towards maturation. Among them, the metabolic shift from carbohydrates to fatty acids metabolism is achieved along with mitochondrial biogenesis, dynamic switch and mitophagy. However, the regulatory mechanisms responsible for coordinated mitochondrial dynamics and mitophagy in mitochondrial maturation remain unclear. Here, we found that ablation of PDK1 in post-natal cardiomyocytes impairs mitochondrial maturation and metabolism, characterizing by arrest of mitochondria in neonatal stage, low levels of a subset of fatty acids and acylcarnitines. In addition, loss of PDK1 results in dysregulated mitochondrial dynamics and mitophagy. An imbalance of the AMPK-mTOR pathway and reduced phosphorylation of PDK1 substrates, including PKA and PKC family members, are observed. These results demonstrated that PDK1 ensures mitochondrial maturation and metabolic shift through kinase-dependent substrate phosphorylation and maintenance of the AMPK-mTOR axis to coordinate mitochondrial dynamics and mitophagy.
Project description:Mechanisms by which Porphyromonas gingivalis (P. gingivalis) infection enhances oral tumor growth or resistance to cell death remain elusive. Here, we determined that P. gingivalis infection mediates therapeutic resistance via inhibiting lethal mitophagy in cancer cells and tumors. Mechanistically, P. gingivalis targets the LC3B-ceramide complex by associating with LC3B via bacterial major fimbriae (FimA) protein, preventing ceramide-dependent mitophagy in response to various therapeutic agents. Moreover, ceramide-mediated mitophagy is induced by Annexin A2 (ANXA2)-ceramide association, involving the E142 residue of ANXA2. Inhibition of ANXA2-ceramide-LC3B complex formation by wild-type P. gingivalis prevented ceramide-dependent mitophagy. Moreover, a FimA-deletion mutant P. gingivalis variant had no inhibitory effects on ceramide-dependent mitophagy. Further, 16S rRNA sequencing of oral tumors indicated that P. gingivalis infection altered the microbiome of the tumor macroenvironment in response to ceramide analog treatment in mice. Thus, these data provide a mechanism describing the pro-survival roles of P. gingivalis in oral tumors.
Project description:Mitophagy is essential to maintain mitochondrial function and prevent diseases. It activates upon mitochondria depolarization, which causes PINK1 stabilization on the mitochondrial outer membrane. Strikingly, a number of conditions, including mitochondrial protein misfolding, can induce mitophagy without a loss in membrane potential. The underlying molecular details remain unclear. Here, we report that a loss of mitochondrial protein import, mediated by the pre-sequence translocase-associated motor complex PAM, is sufficient to induce mitophagy in polarized mitochondria. A genome-wide CRISPR/Cas9 screen for mitophagy inducers identifies components of the PAM complex. Protein import defects are able to induce mitophagy without a need for depolarization. Upon mitochondrial protein misfolding, PAM dissociates from the import machinery resulting in decreased protein import and mitophagy induction. Our findings extend the current mitophagy model to explain mitophagy induction upon conditions that do not affect membrane polarization, such as mitochondrial protein misfolding.
Project description:Down syndrome (DS), a complex genetic disorder caused by chromosome 21 trisomy, is associated with mitochondrial dysfunction leading to the accumulation of damaged mitochondria. Here we report that mitophagy, a form of selective autophagy activated to clear damaged mitochondria is deficient in primary human fibroblasts derived from individuals with DS leading to accumulation of damaged mitochondria with consequent increases in oxidative stress. We identified two molecular bases for this mitophagy deficiency: PINK1/PARKIN impairment and abnormal suppression of macroautophagy. First, strongly downregulated PARKIN and the mitophagic adaptor protein SQSTM1/p62 delays PINK1 activation to impair mitophagy induction after mitochondrial depolarization by CCCP or antimycin A plus oligomycin. Secondly, mTOR is strongly hyper-activated, which globally suppresses macroautophagy induction and the transcriptional expression of proteins critical for autophagosome formation such as ATG7, ATG3 and FOXO1. Notably, inhibition of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) using AZD8055 (AZD) restores autophagy flux, PARKIN/PINK initiation of mitophagy, and the clearance of damaged mitochondria by mitophagy. These results recommend mTORC1-mTORC2 inhibition as a promising candidate therapeutic strategy for Down Syndrome.
Project description:Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.
Project description:Parkin, an E3 ubiquitin ligase, plays an essential role in mitochondrial quality control. However, the mechanisms by which Parkin connects mitochondrial homeostasis to cellular metabolism in adipose tissue remain unclear. Here, we demonstrate that Park2 gene (encodes Parkin) deletion specifically from adipose tissue protects mice against high-fat diet and aging-induced obesity. Despite a mild reduction in mitophagy, mitochondrial DNA (mtDNA) content and mitochondrial function are significantly increased in Park2 deficient white adipocytes. Moreover, Park2 gene deletion robustly elevates mitochondrial biogenesis by increasing Pgc1α protein stability through mitochondrial superoxide-activated Nqo1. Both in vitro and in vivo studies show that Nqo1 overexpression elevates Pgc1α protein level and mtDNA content and enhances mitochondrial activity in mouse and human adipocytes. Taken together, our findings indicate that Parkin regulates mitochondrial homeostasis by balancing mitophagy and Pgc1α-mediated mitochondrial biogenesis in white adipocytes, suggesting a potential therapeutic target in adipocytes to combat obesity and obesity-associated disorders.
Project description:Bladder cancer is one of the most common malignancy in the urinary tract with high recurrence and drug resistance in clinics. Alternative treatments from existing drugs might be a promising strategy. Nitazoxanide (NTZ), an FDA-approved antiprotozoal drug, has got increasingly noticed because of its favorable safety profile and antitumor potential, yet the effects in bladder cancer and underlying mechanisms remain poorly understood. Herein, we find that NTZ induces mitochondrial damage and mitophagy initiation through PINK1-generated phospho-ubiquitin(pS65-Ub) and autophagy receptor-mediated pathway even in the absence of Atg5/Beclin1. Meanwhile, NTZ inhibits lysosomal degradation activity, leading to mitophagy flux impairment at late stage. Mitochondrial reactive oxygen species (ROS) production is critical in this process, as eliminating ROS with N-acetylcysteine (NAC) efficiently inhibits PINK1 signaling-mediated mitophagy initiation and alleviates lysosomal dysfunction. Co-treatment with NTZ and autophagy inhibitor Chloroquine (CQ) to aggravate mitophagy flux impairment promotes NTZ-induced apoptosis, while alleviation of mitophagy flux impairment with ROS scavenger reduced cell death. Moreover, we also discover a similar signaling response in the 3D bladder tumor spheroid after NTZ exposure. In vivo study reveals a significant inhibition of orthotopic bladder tumors with no obvious systemic toxicity. Together, our results uncover the anti-tumor activities of NTZ with the involvement of ROS-mediated mitophagy modulation at different stages and demonstrate it as a potential drug candidate for fighting against bladder tumors.
Project description:The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and thereby disease maintenance and progression in acute myeloid leukemia. The process of mitophagy is orchestrated by a variety of mitophagy receptors whose interplay in AML is not well understood. Here, we established a dual multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and individual contributions of mitophagy receptors and gain a deeper understanding of the functional interactome governing mitophagy in AML.