Reciprocal Roles of Tom7 and OMA1 during Mitochondrial Import and Activation of PINK1.

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.

KPNB1 modulates the Machado-Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP.

Machado-Joseph disease (MJD) is characterized by a pathological expansion of the polyglutamine (polyQ) tract within the ataxin-3 protein. Despite its primarily cytoplasmic localization, polyQ-expanded ataxin-3 accumulates in the nucleus and forms intranuclear aggregates in the affected neurons. Due to these histopathological hallmarks, the nucleocytoplasmic transport machinery has garnered attention as an important disease relevant mechanism. Here, we report on MJD cell model-based analysis of the nuclear transport receptor karyopherin subunit beta-1 (KPNB1) and its implications in the molecular pathogenesis of MJD. Although directly interacting with both wild-type and polyQ-expanded ataxin-3, modulating KPNB1 did not alter the intracellular localization of ataxin-3. Instead, overexpression of KPNB1 reduced ataxin-3 protein levels and the aggregate load, thereby improving cell viability. On the other hand, its knockdown and inhibition resulted in the accumulation of soluble and insoluble ataxin-3. Interestingly, the reduction of ataxin-3 was apparently based on protein fragmentation independent of the classical MJD-associated proteolytic pathways. Label-free quantitative proteomics and knockdown experiments identified mitochondrial protease CLPP as a potential mediator of the ataxin-3-degrading effect induced by KPNB1. We confirmed reduction of KPNB1 protein levels in MJD by analyzing two MJD transgenic mouse models and induced pluripotent stem cells (iPSCs) derived from MJD patients. Our results reveal a yet undescribed regulatory function of KPNB1 in controlling the turnover of ataxin-3, thereby highlighting a new potential target of therapeutic value for MJD.

Mitochondrial Dysfunction Associated with mtDNA in Metabolic Syndrome and Obesity.

Metabolic syndrome (MetS) is a precursor to the major health diseases associated with high mortality in industrialized countries: cardiovascular disease and diabetes. An important component of the pathogenesis of the metabolic syndrome is mitochondrial dysfunction, which is associated with tissue hypoxia, disruption of mitochondrial integrity, increased production of reactive oxygen species, and a decrease in ATP, leading to a chronic inflammatory state that affects tissues and organ systems. The mitochondrial AAA + protease Lon (Lonp1) has a broad spectrum of activities. In addition to its classical function (degradation of misfolded or damaged proteins), enzymatic activity (proteolysis, chaperone activity, mitochondrial DNA (mtDNA)binding) has been demonstrated. At the same time, the spectrum of Lonp1 activity extends to the regulation of cellular processes inside mitochondria, as well as outside mitochondria (nuclear localization). This mitochondrial protease with enzymatic activity may be a promising molecular target for the development of targeted therapy for MetS and its components. The aim of this review is to elucidate the role of mtDNA in the pathogenesis of metabolic syndrome and its components as a key component of mitochondrial dysfunction and to describe the promising and little-studied AAA + LonP1 protease as a potential target in metabolic disorders.

Mitochondrial Caseinolytic Protease P: A Possible Novel Prognostic Marker and Therapeutic Target in Cancer.

Caseinolytic protease P (ClpP) is a mitochondrial serine protease. In mammalian cells, the heterodimerization of ClpP and its AAA+ ClpX chaperone results in a complex called ClpXP, which has a relevant role in protein homeostasis and in maintaining mitochondrial functionality through the degradation of mitochondrial misfolded or damaged proteins. Recent studies demonstrate that ClpP is upregulated in primary and metastatic human tumors, supports tumor cell proliferation, and its overexpression desensitizes cells to cisplatin. Interestingly, small modulators of ClpP activity, both activators and inhibitors, are able to impair oxidative phosphorylation in cancer cells and to induce apoptosis. This review provides an overview of the role of ClpP in regulating mitochondrial functionality, in supporting tumor cell proliferation and cisplatin resistance; finally, we discuss whether this protease could represent a new prognostic marker and therapeutic target for the treatment of cancer.

Mutations in LONP1, a mitochondrial matrix protease, cause CODAS syndrome.

Cerebral, ocular, dental, auricular, skeletal anomalies (CODAS) syndrome (MIM 600373) was first described and named by Shehib et al, in 1991 in a single patient. The anomalies referred to in the acronym are as follows: cerebral-developmental delay, ocular-cataracts, dental-aberrant cusp morphology and delayed eruption, auricular-malformations of the external ear, and skeletal-spondyloepiphyseal dysplasia. This distinctive constellation of anatomical findings should allow easy recognition but despite this only four apparently sporadic patients have been reported in the last 20 years indicating that the full phenotype is indeed very rare with perhaps milder or a typical presentations that are allelic but without sufficient phenotypic resemblance to permit clinical diagnosis. We performed exome sequencing in three patients (an isolated case and a brother and sister sib pair) with classical features of CODAS. Sanger sequencing was used to confirm results as well as for mutation discovery in a further four unrelated patients ascertained via their skeletal features. Compound heterozygous or homozygous mutations in LONP1 were found in all (8 separate mutations; 6 missense, 1 nonsense, 1 small in-frame deletion) thus establishing the genetic basis of CODAS and the pattern of inheritance (autosomal recessive). LONP1 encodes an enzyme of bacterial ancestry that participates in protein turnover within the mitochondrial matrix. The mutations cluster at the ATP-binding and proteolytic domains of the enzyme. Biallelic inheritance and clustering of mutations confirm dysfunction of LONP1 activity as the molecular basis of CODAS but the pathogenesis remains to be explored.

Activation of SIRT1 by silibinin improved mitochondrial health and alleviated the oxidative damage in experimental diabetic neuropathy and high glucose-mediated neurotoxicity.

BACKGROUND: Silibinin (SBN), a sirtuin 1 (SIRT1) activator, has been evaluated for its anti-inflammatory activity in many inflammatory diseases. However, its role in diabetes-induced peripheral neuropathy (DPN) remains unknown. The SIRT1 activation convalesces nerve functions by improving mitochondrial biogenesis and mitophagy. METHODS: DPN was induced by streptozotocin (STZ) at a dose of 55 mg/kg, i.p. in the male SD rats whereas neurotoxicity was induced in Neuro2A cells by 30 mM (high glucose) glucose. Neurobehavioural (nerve conduction velocity and nerve blood flow) western blot, immunohistochemistry, and immunocytochemistry were performed to evaluate the protein expression and their cellular localisation. RESULTS: Two-week SBN treatment improved neurobehavioural symptoms, SIRT1, PGC-1α, and TFAM expression in the sciatic nerve and HG insulted N2A cells. It has also maintained the mitophagy by up-regulating PARL, PINK1, PGAM5, LC3 level and provided antioxidant defence by upregulating Nrf2. CONCLUSION: SBN has shown neuroprotective potential in DPN through SIRT1 activation and antioxidant mechanism.

Insight into the mitochondrial unfolded protein response and cancer: opportunities and challenges.

The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved protective transcriptional response that maintains mitochondrial proteostasis by inducing the expression of mitochondrial chaperones and proteases in response to various stresses. The UPRmt-mediated transcriptional program requires the participation of various upstream signaling pathways and molecules. The factors regulating the UPRmt in Caenorhabditis elegans (C. elegans) and mammals are both similar and different. Cancer cells, as malignant cells with uncontrolled proliferation, are exposed to various challenges from endogenous and exogenous stresses. Therefore, in cancer cells, the UPRmt is hijacked and exploited for the repair of mitochondria and the promotion of tumor growth, invasion and metastasis. In this review, we systematically introduce the inducers of UPRmt, the biological processes in which UPRmt participates, the mechanisms regulating the UPRmt in C. elegans and mammals, cross-tissue signal transduction of the UPRmt and the roles of the UPRmt in promoting cancer initiation and progression. Disrupting proteostasis in cancer cells by targeting UPRmt constitutes a novel anticancer therapeutic strategy.

Mitochondrial Chaperones and Proteases in Cardiomyocytes and Heart Failure.

Heart failure is one of the leading causes of morbidity and mortality worldwide. In cardiomyocytes, mitochondria are not only essential organelles providing more than 90% of the ATP necessary for contraction, but they also play critical roles in regulating intracellular Ca2+ signaling, lipid metabolism, production of reactive oxygen species (ROS), and apoptosis. Because mitochondrial DNA only encodes 13 proteins, most mitochondrial proteins are nuclear DNA-encoded, synthesized, and transported from the cytoplasm, refolded in the matrix to function alone or as a part of a complex, and degraded if damaged or incorrectly folded. Mitochondria possess a set of endogenous chaperones and proteases to maintain mitochondrial protein homeostasis. Perturbation of mitochondrial protein homeostasis usually precedes disruption of the whole mitochondrial quality control system and is recognized as one of the hallmarks of cardiomyocyte dysfunction and death. In this review, we focus on mitochondrial chaperones and proteases and summarize recent advances in understanding how these proteins are involved in the initiation and progression of heart failure.

PINK1 import regulation at a crossroad of mitochondrial fate: the molecular mechanisms of PINK1 import.

PTEN-induced kinase 1 (PINK1) is a mitochondrial kinase whose activity is tightly regulated by the mitochondrial health status. In response to mitochondrial damage, activated PINK1 can promote mitophagy, an autophagic elimination of damaged mitochondria, by cooperating with Parkin ubiquitin ligase. Loss-of-function of PINK1/Parkin-mediated mitophagy results in the accumulation of dysfunctional mitochondria, which could be one aetiology of Parkinson's disease (PD). Within step-by-step signalling cascades of PINK1/Parkin-mediated mitophagy, mitochondrial damage-dependent PINK1 kinase activation is a critical step to trigger the mitophagy signal. Recent investigation of this process reveals that this stress-dependent PINK1 kinase activation is achieved by its regulated import into different mitochondrial compartments. Thus, PINK1 import regulation stands at an important crossroad to determine the mitochondrial fate-'keep' or 'remove'? In this review, we will summarize how the PINK1 import is regulated in a mitochondrial health status-dependent manner and how this process could be pharmacologically modulated to activate the PINK1/Parkin pathway.

AAA+ ATPases in Protein Degradation: Structures, Functions and Mechanisms.

Adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+), the hexameric ring-shaped motor complexes located in all ATP-driven proteolytic machines, are involved in many cellular processes. Powered by cycles of ATP binding and hydrolysis, conformational changes in AAA+ ATPases can generate mechanical work that unfolds a substrate protein inside the central axial channel of ATPase ring for degradation. Three-dimensional visualizations of several AAA+ ATPase complexes in the act of substrate processing for protein degradation have been resolved at the atomic level thanks to recent technical advances in cryogenic electron microscopy (cryo-EM). Here, we summarize the resulting advances in structural and biochemical studies of AAA+ proteases in the process of proteolysis reactions, with an emphasis on cryo-EM structural analyses of the 26S proteasome, Cdc48/p97 and FtsH-like mitochondrial proteases. These studies reveal three highly conserved patterns in the structure-function relationship of AAA+ ATPase hexamers that were observed in the human 26S proteasome, thus suggesting common dynamic models of mechanochemical coupling during force generation and substrate translocation.

Determination of Aconitase Activity: A Substrate of the Mitochondrial Lon Protease.

Mitochondrial aconitase is a reversible enzyme that catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid cycle. Mitochondrial aconitase is very sensitive to oxidative inactivation and can aggregate and accumulate in the mitochondrial matrix causing mitochondrial dysfunction. Lon protease, one of the major quality control proteases in mitochondria, degrades oxidized aconitase maintaining mitochondrial homeostasis. This chapter describes a step-by-step protocol for a simple and reliable measurement of mitochondrial aconitase, as well as citrate synthase activity, using isolated mitochondria from cells. The protocol is simple and fast, and it is optimized for a 96-well plate using a microplate reader.

Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy.

Mitophagy, the autophagic removal of mitochondria, occurs through a highly selective mechanism. In the yeast Saccharomyces cerevisiae, the mitochondrial outer membrane protein Atg32 confers selectivity for mitochondria sequestration as a cargo by the autophagic machinery through its interaction with Atg11, a scaffold protein for selective types of autophagy. The activity of mitophagy in vivo must be tightly regulated considering that mitochondria are essential organelles that produce most of the cellular energy, but also generate reactive oxygen species that can be harmful to cell physiology. We found that Atg32 was proteolytically processed at its C terminus upon mitophagy induction. Adding an epitope tag to the C terminus of Atg32 interfered with its processing and caused a mitophagy defect, suggesting the processing is required for efficient mitophagy. Furthermore, we determined that the mitochondrial i-AAA protease Yme1 mediated Atg32 processing and was required for mitophagy. Finally, we found that the interaction between Atg32 and Atg11 was significantly weakened in yme1∆ cells. We propose that the processing of Atg32 by Yme1 acts as an important regulatory mechanism of cellular mitophagy activity.