Adenine nucleotide carrier protein dysfunction in human disease.

Adenine nucleotide translocase (ANT) is the prototypical member of the mitochondrial carrier protein family, primarily involved in ADP/ATP exchange across the inner mitochondrial membrane. Several carrier proteins evolutionarily related to ANT, including SLC25A24 and SLC25A25, are believed to promote the exchange of cytosolic ATP-Mg2+ with phosphate in the mitochondrial matrix. They allow a net accumulation of adenine nucleotides inside mitochondria, which is essential for mitochondrial biogenesis and cell growth. In the last two decades, mutations in the heart/muscle isoform 1 of ANT (ANT1) and the ATP-Mg2+ transporters have been found to cause a wide spectrum of human diseases by a recessive or dominant mechanism. Although loss-of-function recessive mutations cause a defect in oxidative phosphorylation and an increase in oxidative stress which drives the pathology, it is unclear how the dominant missense mutations in these proteins cause human diseases. In this review, we focus on how yeast was productively used as a model system for the understanding of these dominant diseases. We also describe the relationship between the structure and function of ANT and how this may relate to various pathologies. Particularly, mutations in Aac2, the yeast homolog of ANT, were recently found to clog the mitochondrial protein import pathway. This leads to mitochondrial precursor overaccumulation stress (mPOS), characterized by the toxic accumulation of unimported mitochondrial proteins in the cytosol. We anticipate that in coming years, yeast will continue to serve as a useful model system for the mechanistic understanding of mitochondrial protein import clogging and related pathologies in humans.

Mitochondrial protein import clogging as a mechanism of disease.

Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog ADP/ATP carrier 2 (Aac2), cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis, and cell viability independent of ANT1's nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 causes severe clogging primarily at the translocase of the outer membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.

Inside our cells, compartments known as mitochondria generate the chemical energy required for life processes to unfold. Most of the proteins found within mitochondria are manufactured in another part of the cell (known as the cytosol) and then imported with the help of specialist machinery. For example, the TOM and TIM22 channels provide a route for the proteins to cross the two membrane barriers that separate the cytosol from the inside of a mitochondrion. ANT1 is a protein that is found inside mitochondria in humans, where it acts as a transport system for the cell's energy currency. Specific mutations in the gene encoding ANT1 have been linked to degenerative conditions that affect the muscles and the brain. However, it remains unclear how these mutations cause disease. To address this question, Coyne et al. recreated some of the mutations in the gene encoding the yeast equivalent of ANT1 (known as Aac2). Experiments in yeast cells carrying these mutations showed that the Aac2 protein accumulated in the TOM and TIM22 channels, creating a 'clog' that prevented other essential proteins from reaching the mitochondria. As a result, the yeast cells died. Mutant forms of the human ANT1 protein also clogged up the TOM and TIM22 channels of human cells in a similar way. Further experiments focused on mice genetically engineered to produce a "super-clogger" version of the mouse equivalent of ANT1. The animals soon developed muscle and neurological conditions similar to those observed in human diseases associated with ANT1. The findings of Coyne et al. suggest that certain genetic mutations in the gene encoding the ANT1 protein cause disease by blocking the transport of other proteins to the mitochondria, rather than by directly affecting ANT1's nucleotide trnsport role in the cell. This redefines our understanding of diseases associated with mitochondrial proteins, potentially altering how treatments for these conditions are designed.

Clinical Features of Genetic Resilience in Chronic Obstructive Pulmonary Disease.

Introduction: In the personalized risk quantification of chronic obstructive pulmonary disease (COPD), genome-wide association studies and polygenic risk scores (PRS) complement traditional risk factors, such as age and cigarette smoking. However, despite being at considerable levels of risk, some individuals do not develop COPD. Research on COPD resilience remains largely unexplored. Methods: We applied the previously published COPD PRS to whole genome sequencing data from non-Hispanic white and African American individuals in the COPDGene study. We defined genetic resilience as individuals unaffected by COPD with a polygenic risk score above the 90 th percentile. We defined risk-matched case individuals as those with COPD (i.e., FEV 1 /FVC < 0.70) and a PRS above the 90 th percentile. We defined low risk individuals without COPD (i.e., FEV 1 /FVC > 0.70) as a polygenic risk score below the 10 th percentile. We compared genetically resilient individuals to risk-matched individuals with COPD and low risk individuals by demographics, lung function, respiratory symptoms, co-morbidities, and chest CT scan measurements. We also performed survival analyses, differential expression analysis, and matching for sensitivity analyses. Results: We identified 211 resilient individuals without COPD, 605 genetic risk-matched individuals with COPD, and 527 low-risk individuals without COPD. Resilient individuals had higher FEV 1 % predicted and lower percent emphysema. In contrast, resilient individuals had higher airway wall thickness compared to low-risk unaffected individuals. While there was no difference in survival between low-risk and resilient individuals, resilient individuals had higher survival compared to risk matched cases. We also identified two genes that were differentially expressed between low-risk unaffected individuals and resilient individuals. Conclusion: Genetically resilient individuals had a reduced burden of COPD disease-related measures compared to risk-matched cases but had subtly increased measures compared to low-risk unaffected individuals. Further genetic studies will be needed to illuminate the underlying pathobiology of our observations.

Consequences of inner mitochondrial membrane protein misfolding.

Proteins embedded in the inner mitochondrial membrane (IMM) perform essential cellular functions. Maintaining the folding state of these proteins is therefore of the utmost importance, and this is ensured by IMM chaperones and proteases that refold and degrade unassembled and misfolded proteins. However, the physiological consequences specific to IMM protein misfolding remain obscure because deletion of these chaperones/proteases (the typical experimental strategy) often affects many mitochondrial processes other than protein folding and turnover. Thus, novel experimental systems are needed to evaluate the direct effects of misfolded protein on the membrane. Such a system has been developed in recent years. Studies suggest that numerous pathogenic mutations in isoform 1 of adenine nucleotide translocase (Ant1) cause its misfolding on the IMM. In this review, we first discuss potential mechanisms by which dominant Ant1 mutations may cause disease, highlighting IMM protein misfolding, per se, as a likely pathological factor. Then we discuss the intramitochondrial effects of Ant1 misfolding such as IMM proteostatic stress, respiratory chain dysfunction, and mtDNA instability. Finally, we summarize the mounting evidence that IMM proteostatic stress can perturb mitochondrial protein import to cause the toxic accumulation of mitochondrial proteins in the cytosol: a cell stress mechanism termed mitochondrial Precursor Overaccumulation Stress (mPOS).

Mitochondrial carrier protein overloading and misfolding induce aggresomes and proteostatic adaptations in the cytosol.

Previous studies in yeast showed that mitochondrial stressors not directly targeting the protein import machinery can cause mitochondrial precursor overaccumulation stress (mPOS) in the cytosol independent of bioenergetics. Here, we demonstrate mPOS and stress responses in human cells. We show that overloading of mitochondrial membrane carrier, but not matrix proteins, is sufficient to induce cytosolic aggresomes and apoptosis. The aggresomes appear to triage unimported mitochondrial proteins. Interestingly, expression of highly unstable mutant variants of the mitochondrial carrier protein, Ant1, also induces aggresomes despite a greater than 20-fold reduction in protein level compared to wild type. Thus, overloading of the protein import machinery, rather than protein accumulation, is critical for aggresome induction. The data suggest that the import of mitochondrial proteins is saturable and that the cytosol is limited in degrading unimported mitochondrial proteins. In addition, we found that EGR1, eEF1a, and ubiquitin C are up-regulated by Ant1 overloading. These proteins are known to promote autophagy, protein targeting to aggresomes, and the processing of protein aggregates, respectively. Finally, we found that overexpression of the misfolded variants of Ant1 induces additional cytosolic responses including proteasomal activation. In summary, our work captured a profound effect of unimported mitochondrial proteins on cytosolic proteostasis and revealed multiple anti-mPOS mechanisms in human cells.

Mitochondria-cytosol-nucleus crosstalk: learning from Saccharomyces cerevisiae.

Mitochondria are key cell organelles with a prominent role in both energetic metabolism and the maintenance of cellular homeostasis. Since mitochondria harbor their own genome, which encodes a limited number of proteins critical for oxidative phosphorylation and protein translation, their function and biogenesis strictly depend upon nuclear control. The yeast Saccharomyces cerevisiae has been a unique model for understanding mitochondrial DNA organization and inheritance as well as for deciphering the process of assembly of mitochondrial components. In the last three decades, yeast also provided a powerful tool for unveiling the communication network that coordinates the functions of the nucleus, the cytosol and mitochondria. This crosstalk regulates how cells respond to extra- and intracellular changes either to maintain cellular homeostasis or to activate cell death. This review is focused on the key pathways that mediate nucleus-cytosol-mitochondria communications through both transcriptional regulation and proteostatic signaling. We aim to highlight yeast that likely continues to serve as a productive model organism for mitochondrial research in the years to come.

mPOS is a novel mitochondrial trigger of cell death - implications for neurodegeneration.

In addition to its central role in energy metabolism, the mitochondrion has many other functions essential for cell survival. When stressed, the multifunctional mitochondria are expected to engender multifaceted cell stress with complex physiological consequences. Potential extra-mitochondrial proteostatic burdens imposed by inefficient protein import have been largely overlooked. Accumulating evidence suggests that a diverse range of pathogenic mitochondrial stressors, which do not directly target the core protein import machinery, can reduce cell fitness by disrupting the proteostatic network in the cytosol. The resulting stress, named mitochondrial precursor overaccumulation stress (mPOS), is characterized by the toxic accumulation of unimported mitochondrial proteins in the cytosol. Here, we review our current understanding of how mitochondrial dysfunction can impact the cytosolic proteome and proteostatic signaling. We also discuss the intriguing possibility that the mPOS model may help untangle the cause-effect relationship between mitochondrial dysfunction and cytosolic protein aggregation, which are probably the two most prominent molecular hallmarks of neurodegenerative disease.