The Emerging Role of Proteolysis in Mitochondrial Quality Control and the Etiology of Parkinson's Disease

Mitochondria are highly dynamic organelles that are important for many diverse cellular processes, such as energy metabolism, calcium buffering, and apoptosis. Mitochondrial biology and dysfunction have recently been linked to different types of cancers and neurodegenerative diseases, most notably Parkinson's disease. Thus, a better understanding of the quality control systems that maintain a healthy mitochondrial network can facilitate the development of effective treatments for these diseases. In this perspective, we will discuss recent advances on two mitochondrial quality control pathways: the UPS and mitophagy, highlight how new players may be contributing to regulate these pathways. We believe the proteases involved will be key and novel regulators of mitochondrial quality control, and this knowledge will provide insights into future studies aimed to combat neurodegenerative diseases

Caenorhabditis elegans is a useful model for anthelmintic discovery

Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery

Chemical–Genetic Profiling of Imidazo[1,2-a]pyridines and -Pyrimidines Reveals Target Pathways Conserved between Yeast and Human Cells

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships

Matrix metalloproteinase processing of monocyte chemokines

Extracellular matrix degradation occurs in chronic inflammatory diseases such as arthritis and disturbances in connective tissue homeostasis contribute to various lung, neurological and cardiovascular diseases and is pivotal in tumor metastasis. The matrix metalloproteinase (MMP) family of endoproteinases is implicated in these processes by a general ability to degrade the structural components of the extracellular matrix. To understand the biological role of MMP proteolysis in physiological and pathological processes, it is necessary to identify biologically relevant substrates. I initiated yeast two-hybrid screens to identify novel substrates of gelatinase A using the hemopexin C domain of the enzyme as bait. Initial screens of a cDNA library constructed from Concanavalin A-treated human gingival fibroblasts identified the chemokine monocyte chemoattractant protein (MCP)-3 as a hemopexin C domain binding protein. Incubation of MCP-3 with gelatinase A resulted in cleavage of MCP-3 at Gly⁴-lle⁵ a preferred scissile bond sequence for the enzyme. The turnover rate, k[sub cat]/K[sub m] was determined to be 8,000 M⁻¹s⁻¹, more efficient than gelatinase A cleavage of gelatin. Indeed, cleaved MCP-3 was identified in human rheumatoid arthritis synovial fluids. Gelatinase A (and other MMP)-mediated cleavage of MCP-3 resulted in conversion of chemokine receptor agonist activity to a general acting antagonist, impairing the activity of several related chemokines. The mechanistic importance of the hemopexin C domain in gelatinase A cleavage of MCP-3 was determined. The turnover rate was reduced to 500 M⁻¹s⁻¹ upon removal of the hemopexin C domain from the enzyme. Exogenous hemopexin C domain competed for cleavage whereas the collagen binding domain of gelatinase A did not. Specificity of MCP cleavage could be attributed to unique binding of both the gelatinase A and membrane-type (MT)-1 hemopexin C domains to only MCP-3 and not MCP-1, -2, or -4. Chemokine chimeras further demonstrated the importance of hemopexin C domain exosites in catalysis. My results provide evidence that MMP activity can contribute toward the reparative process in inflammation and that interactions of MMPs with chemokines provide a self-attenuating network to dissipate pro-inflammatory activities. I propose that MMP processing of chemokines is a new paradigm in chemokine and MMP biology in the regulation of inflammation.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

The Mitochondrial Rhomboid Protease PARL Is Regulated by PDK2 to Integrate Mitochondrial Quality Control and Metabolism

Mitochondrial quality control (MQC) systems are essential for mitochondrial health and normal cellular function. Dysfunction of MQC is emerging as a central mechanism for the pathogenesis of various diseases, including Parkinson’s disease. The mammalian mitochondrial rhomboid protease, PARL, has been proposed as a regulator of PINK1/PARKIN-mediated mitophagy, which is an essential component of MQC. PARL undergoes an N-terminal autocatalytic cleavage (β cleavage), which is required for efficient mitophagy. We demonstrate that β cleavage responds to mitochondrial stress, triggered by the depletion of mitochondrial ATP. Furthermore, we show that PDK2, a key regulator in metabolic plasticity, phosphorylates PARL and regulates β cleavage. Through regulating β cleavage and the production of a less active enzyme, PACT, PDK2 negatively regulates PINK1/PARKIN-mediated mitophagy. Taken together, we propose that PDK2/PARL senses defects in mitochondrial bioenergetics, integrating mitochondrial metabolism to mitophagy and MQC in human health and disease

USP30: protector of peroxisomes and mitochondria

In our recent publication, we describe a mechanism by which peroxisomes are protected from degradation by autophagy under basal conditions. Taking a page from mitophagy, peroxisomes also recruit the mitochondria deubiquitinating enzyme USP30 to counter the action of PEX2, the peroxisomal E3 ubiquitin ligase to regulate pexophagy

Protocol for evaluating mitochondrial morphology changes in response to CCCP-induced stress through open-source image processing software

Summary: Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses.For complete details on the use and execution of this protocol, please refer to Nag et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Cardiolipin synthesizing enzymes form a complex that interacts with cardiolipin-dependent membrane organizing proteins.

The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multiple cardiolipin-binding mitochondrial membrane proteins, including prohibitins, stomatin-like protein 2 and the MICOS components MIC60 and MIC19. We further mapped the protein-protein interaction sites between PGS1 and itself, CLS1, MIC60 and PHB. Overall, this study provides evidence for the presence of a cardiolipin synthesis structure that transiently interacts with cardiolipin-dependent protein complexes

PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics

Summary: Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase