Elucidation of the interaction proteome of mitochondrial chaperone Hsp78 highlights its role in protein aggregation during heat stress.

Chaperones of the Hsp100/Clp family represent major components of protein homeostasis, conferring maintenance of protein activity under stress. The ClpB-type members of the family, present in bacteria, fungi, and plants, are able to resolubilize aggregated proteins. The mitochondrial member of the ClpB family in Saccharomyces cerevisiae is Hsp78. Although Hsp78 has been shown to contribute to proteostasis in elevated temperatures, the biochemical mechanisms underlying this mitochondria-specific thermotolerance are still largely unclear. To identify endogenous chaperone substrate proteins, here, we generated an Hsp78-ATPase mutant with stabilized substrate-binding behavior. We used two stable isotope labeling-based quantitative mass spectrometry approaches to analyze the role of Hsp78 during heat stress-induced mitochondrial protein aggregation and disaggregation on a proteomic level. We first identified the endogenous substrate spectrum of the Hsp78 chaperone, comprising a wide variety of proteins related to metabolic functions including energy production and protein synthesis, as well as other chaperones, indicating its crucial functions in mitochondrial stress resistance. We then compared these interaction data with aggregation and disaggregation processes in mitochondria under heat stress, which revealed specific aggregation-prone protein populations and demonstrated the direct quantitative impact of Hsp78 on stress-dependent protein solubility under different conditions. We conclude that Hsp78, together with its cofactors, represents a recovery system that protects major mitochondrial metabolic functions during heat stress as well as restores protein biogenesis capacity after the return to normal conditions.

Role of Mitochondrial Protein Import in Age-Related Neurodegenerative and Cardiovascular Diseases.

Mitochondria play a critical role in providing energy, maintaining cellular metabolism, and regulating cell survival and death. To carry out these crucial functions, mitochondria employ more than 1500 proteins, distributed between two membranes and two aqueous compartments. An extensive network of dedicated proteins is engaged in importing and sorting these nuclear-encoded proteins into their designated mitochondrial compartments. Defects in this fundamental system are related to a variety of pathologies, particularly engaging the most energy-demanding tissues. In this review, we summarize the state-of-the-art knowledge about the mitochondrial protein import machinery and describe the known interrelation of its failure with age-related neurodegenerative and cardiovascular diseases.

Proteomic analysis demonstrates the role of the quality control protease LONP1 in mitochondrial protein aggregation.

The mitochondrial matrix protease LONP1 is an essential part of the organellar protein quality control system. LONP1 has been shown to be involved in respiration control and apoptosis. Furthermore, a reduction in LONP1 level correlates with aging. Up to now, the effects of a LONP1 defect were mostly studied by utilizing transient, siRNA-mediated knockdown approaches. We generated a new cellular model system for studying the impact of LONP1 on mitochondrial protein homeostasis by a CRISPR/Cas-mediated genetic knockdown (gKD). These cells showed a stable reduction of LONP1 along with a mild phenotype characterized by absent morphological differences and only small negative effects on mitochondrial functions under normal culture conditions. To assess the consequences of a permanent LONP1 depletion on the mitochondrial proteome, we analyzed the alterations of protein levels by quantitative mass spectrometry, demonstrating small adaptive changes, in particular with respect to mitochondrial protein biogenesis. In an additional proteomic analysis, we determined the temperature-dependent aggregation behavior of mitochondrial proteins and its dependence on a reduction of LONP1 activity, demonstrating the important role of the protease for mitochondrial protein homeostasis in mammalian cells. We identified a significant number of mitochondrial proteins that are affected by a reduced LONP1 activity especially with respect to their stress-induced solubility. Taken together, our results suggest a very good applicability of the LONP1 gKD cell line as a model system for human aging processes.

Accessing Mitochondrial Protein Import in Living Cells by Protein Microinjection.

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

The Mitochondrial Lon Protease: Novel Functions off the Beaten Track?

To maintain organellar function, mitochondria contain an elaborate endogenous protein quality control system. As one of the two soluble energy-dependent proteolytic enzymes in the matrix compartment, the protease Lon is a major component of this system, responsible for the degradation of misfolded proteins, in particular under oxidative stress conditions. Lon defects have been shown to negatively affect energy production by oxidative phosphorylation but also mitochondrial gene expression. In this review, recent studies on the role of Lon in mammalian cells, in particular on its protective action under diverse stress conditions and its relationship to important human diseases are summarized and commented.

Mitochondria as Potential Targets in Alzheimer Disease Therapy: An Update.

Alzheimer disease (AD) is a progressive and deleterious neurodegenerative disorder that affects mostly the elderly population. At the moment, no effective treatments are available in the market, making the whole situation a compelling challenge for societies worldwide. Recently, novel mechanisms have been proposed to explain the etiology of this disease leading to the new concept that AD is a multifactor pathology. Among others, the function of mitochondria has been considered as one of the intracellular processes severely compromised in AD since the early stages and likely represents a common feature of many neurodegenerative diseases. Many mitochondrial parameters decline already during the aging, reaching an extensive functional failure concomitant with the onset of neurodegenerative conditions, although the exact timeline of these events is still unclear. Thereby, it is not surprising that mitochondria have been already considered as therapeutic targets in neurodegenerative diseases including AD. Together with an overview of the role of mitochondrial dysfunction, this review examines the pros and cons of the tested therapeutic approaches targeting mitochondria in the context of AD. Since mitochondrial therapies in AD have shown different degrees of progress, it is imperative to perform a detailed analysis of the significance of mitochondrial deterioration in AD and of a pharmacological treatment at this level. This step would be very important for the field, as an effective drug treatment in AD is still missing and new therapeutic concepts are urgently needed.

γ-Oryzanol Improves Cognitive Function and Modulates Hippocampal Proteome in Mice.

Rice (Oryza sativa L.) is the richest source of γ-oryzanol, a compound endowed with antioxidant and anti-inflammatory properties. γ-Oryzanol has been demonstrated to cross the blood-brain barrier in intact form and exert beneficial effects on brain function. This study aimed to clarify the effects of γ-oryzanol in the hippocampus in terms of cognitive function and protein expression. Adult mice were administered with γ-oryzanol 100 mg/kg or vehicle (control) once a day for 21 consecutive days following which cognitive behavior and hippocampal proteome were investigated. Cognitive tests using novel object recognition and Y-maze showed that long-term consumption of γ-oryzanol improves cognitive function in mice. To investigate the hippocampal proteome modulated by γ-oryzanol, 2D-difference gel electrophoresis (2D-DIGE) was performed. Interestingly, we found that γ-oryzanol modulates quantitative changes of proteins involved in synaptic plasticity and neuronal trafficking, neuroprotection and antioxidant activity, and mitochondria and energy metabolism. These findings suggested γ-oryzanol as a natural compound able to maintain and reinforce brain function. Although more intensive studies are needed, we propose γ-oryzanol as a putative dietary phytochemical for preserving brain reserve, the ability to tolerate age-related changes, thereby preventing clinical symptoms or signs of neurodegenerative diseases.

Analysis of heat-induced protein aggregation in human mitochondria.

Proteins in mammalian cells exhibit optimal stability at physiological temperatures, and even small temperature variations may cause unfolding and nonspecific aggregation. Because this process leads to a loss of function of the affected polypeptides and to cytotoxic stress, formation of protein aggregates has been recognized as a major pathogenic factor in human diseases. In this study, we determined the impact of physiological heat stress on mitochondria isolated from HeLa cells. We found that the heat-stressed mitochondria had lower membrane potential and ATP level and exhibited a decreased production of reactive oxygen species. An analysis of the mitochondrial proteome by 2D PAGE showed that the overall solubility of endogenous proteins was only marginally affected by elevated temperatures. However, a small subset of polypeptides exhibited an high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat-stress conditions. Moreover, mitochondrial translation and the import of cytosolic proteins were defective in the heat-stressed mitochondria. Both types of nascent polypeptides, produced by translation or imported into the mitochondria, exhibited a strong tendency to aggregate in the heat-exposed mitochondria. We propose that a fast and specific inactivation of elongation factors may prevent the accumulation of misfolded nascent polypeptides and may thereby attenuate proteotoxicity under heat stress.

IMiQ: a novel protein quality control compartment protecting mitochondrial functional integrity.

Aggregation processes can cause severe perturbations of cellular homeostasis and are frequently associated with diseases. We performed a comprehensive analysis of mitochondrial quality and function in the presence of aggregation-prone polypeptides. Despite a significant aggregate formation inside mitochondria, we observed only a minor impairment of mitochondrial function. Detoxification of aggregated reporter polypeptides as well as misfolded endogenous proteins inside mitochondria takes place via their sequestration into a specific organellar deposit site we termed intramitochondrial protein quality control compartment (IMiQ). Only minor amounts of endogenous proteins coaggregated with IMiQ deposits and neither resolubilization nor degradation by the mitochondrial protein quality control system were observed. The single IMiQ aggregate deposit was not transferred to daughter cells during cell division. Detoxification of aggregates via IMiQ formation was highly dependent on a functional mitochondrial fission machinery. We conclude that the formation of an aggregate deposit is an important mechanism to maintain full functionality of mitochondria under proteotoxic stress conditions.

Mitochondrial quality control by the Pink1/Parkin system.

Mitochondrial dysfunction represents a prominent pathological feature in many neurodegenerative diseases, particularly in Parkinson's disease (PD). Mutations in the genes encoding the proteins Pink1 and Parkin have been identified as genetic risk factors in familiar cases of PD. Research during the last decade has identified both proteins as crucial components of an organellar quality control system that contributes to the maintenance of mitochondrial function in healthy cells. The Pink1/Parkin system acts as a sensor for mitochondrial quality and is activated, in particular, after the loss of the electric potential across the inner mitochondrial membrane. Pink1 molecules accumulate at the surface of damaged mitochondria to recruit and activate Parkin, which, in turn, elicits a signaling pathway eventually leading to the autophagic removal of the damaged organelles. This review summarizes recent advances in our knowledge of the functional role of the Pink1/Parkin system in preventing the accumulation of damaged mitochondria by mitophagy.

Protein quality control at the mitochondrion.

Mitochondria are essential constituents of a eukaryotic cell by supplying ATP and contributing to many mayor metabolic processes. As endosymbiotic organelles, they represent a cellular subcompartment exhibiting many autonomous functions, most importantly containing a complete endogenous machinery responsible for protein expression, folding and degradation. This article summarizes the biochemical processes and the enzymatic components that are responsible for maintaining mitochondrial protein homoeostasis. As mitochondria lack a large part of the required genetic information, most proteins are synthesized in the cytosol and imported into the organelle. After reaching their destination, polypeptides must fold and assemble into active proteins. Under pathological conditions, mitochondrial proteins become misfolded or damaged and need to be repaired with the help of molecular chaperones or eventually removed by specific proteases. Failure of these protein quality control mechanisms results in loss of mitochondrial function and structural integrity. Recently, novel mechanisms have been identified that support mitochondrial quality on the organellar level. A mitochondrial unfolded protein response allows the adaptation of chaperone and protease activities. Terminally damaged mitochondria may be removed by a variation of autophagy, termed mitophagy. An understanding of the role of protein quality control in mitochondria is highly relevant for many human pathologies, in particular neurodegenerative diseases.

Amyloid ß-peptides interfere with mitochondrial preprotein import competence by a coaggregation process.

Aß peptides play a central role in the etiology of Alzheimer disease (AD) by exerting cellular toxicity correlated with aggregate formation. Experimental evidence has shown intraneuronal accumulation of Aß peptides and interference with mitochondrial functions. Nevertheless, the relevance of intracellular Aß peptides in the pathophysiology of AD is controversial. Here we found that the two major species of Aß peptides, in particular Aß42, exhibited a strong inhibitory effect on the preprotein import reactions essential for mitochondrial biogenesis. However, Aß peptides interacted only weakly with mitochondria and did not affect the inner membrane potential or the structure of the preprotein translocase complexes. Aß peptides significantly decreased the import competence of mitochondrial precursor proteins via an extramitochondrial coaggregation mechanism. Coaggregation and import inhibition were significantly stronger for the longer peptide Aß42, correlating with its importance in AD pathology. Our results demonstrate that direct interference of aggregation-prone Aß peptides with mitochondrial protein biogenesis represents a crucial aspect of the pathobiochemical mechanisms contributing to cellular damage in AD.

Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy.

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient's fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease.

Role of Mitochondrial Protein Quality Control in Oxidative Stress-induced Neurodegenerative Diseases.

Proteins are constantly exposed to environmental stressors such as free radicals and heat shock leading to their misfolding and later to aggregation. In particular mitochondrial proteins are challenged by reactive oxygen species (ROS) due to the oxidative metabolism of the organelle. Protein aggregation has been associated with a wide variety of pathological conditions called proteopathies. However, for the maintenance of protein and cellular homeostasis, mitochondria have developed an elaborate protein quality control system consisting of chaperones and ATP-dependent proteases, specifically employed to rescue this organelle from damage due to the accumulation of misfolded proteins and toxic aggregates. Aging is characterized by a general decline of mitochondrial functions, correlating with a decrease in mitochondrial protein quality control activity and an increase of free radical production. In particular in age-related diseases like neurodegeneration, a correlation between mitochondrial damage and disease onset has been established. In this review we summarize the current knowledge about mitochondrial protein quality control mechanisms in mammalian cells, with a special emphasis on the role in oxidative stress and in neurodegenerative diseases.

Biochemical properties of the kinase PINK1 as sensor protein for mitochondrial damage signalling.

Defects of mitochondrial functions have been implicated in many different human diseases, in particular neurodegenerative diseases. The kinase PINK1 [phosphatase and tensin homologue (PTEN)-induced kinase 1] has been identified as a crucial player in a specific damage signalling pathway, eliminating defective mitochondria by an autophagic process. Mutations in PINK1 have been shown to cause familial cases of Parkinson's disease. In this review, we summarize the biochemical mechanisms that underlie the association of PINK1 with mitochondria under normal and pathological conditions. This unconventional mitochondrial localization pathway is discussed in the context of the role of PINK1 as a sensor of mitochondrial damage and a causative factor in Parkinson's disease.

In vitro analysis of the mitochondrial preprotein import machinery using recombinant precursor polypeptides.

The import of proteins into mitochondria represents an essential process for the survival of eukaryotic cells. Most mitochondrial proteins are synthesized as cytosolic precursor proteins. A complex chain of reactions needs to be followed to achieve a successful transport of these precursors from the cytosol through the double membrane system to their final destination inside the mitochondria. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Using this basic system, most of the protein components of the import machinery have been identified and functionally characterized. However, a detailed definition of the molecular mechanisms requires more specialized assay techniques. Here we describe modifications of the standard in vitro import assay technique that are based on the utilization of large amounts of recombinant preprotein constructs. The application of saturating amounts of substrate preproteins is a prerequisite for the determination of translocation kinetics and energy requirements of the import process. Accumulation of preproteins as membrane-spanning translocation intermediates further provides a basis for the functional and structural characterization of the active translocation machinery.

α-Synuclein is localized to mitochondria-associated ER membranes.

Familial Parkinson disease is associated with mutations in α-synuclein (α-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type α-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human α-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type α-syn in mutant α-syn-expressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that α-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type α-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in α-syn. We believe that our results have far-reaching implications for both our understanding of α-syn biology and the treatment of synucleinopathies.

Cytosolic cleaved PINK1 represses Parkin translocation to mitochondria and mitophagy.

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson's disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK152, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.

The functional interaction of mitochondrial Hsp70s with the escort protein Zim17 is critical for Fe/S biogenesis and substrate interaction at the inner membrane preprotein translocase.

The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.

The role of AAA+ proteases in mitochondrial protein biogenesis, homeostasis and activity control.

Mitochondria are specialised organelles that are structurally and functionally integrated into cells in the vast majority of eukaryotes. They are the site of numerous enzymatic reactions, some of which are essential for life. The double lipid membrane of the mitochondrion, that spatially defines the organelle and is necessary for some functions, also creates a physical but semi-permeable barrier to the rest of the cell. Thus to ensure the biogenesis, regulation and maintenance of a functional population of proteins, an autonomous protein handling network within mitochondria is required. This includes resident mitochondrial protein translocation machinery, processing peptidases, molecular chaperones and proteases. This review highlights the contribution of proteases of the AAA+ superfamily to protein quality and activity control within the mitochondrion. Here they are responsible for the degradation of unfolded, unassembled and oxidatively damaged proteins as well as the activity control of some enzymes. Since most knowledge about these proteases has been gained from studies in the eukaryotic microorganism Saccharomyces cerevisiae, much of the discussion here centres on their role in this organism. However, reference is made to mitochondrial AAA+ proteases in other organisms, particularly in cases where they play a unique role such as the mitochondrial unfolded protein response. As these proteases influence mitochondrial function in both health and disease in humans, an understanding of their regulation and diverse activities is necessary.