Victoria Küttner

Nucleocytosolic Depletion of the Energy Metabolite Acetyl-Coenzyme A Stimulates Autophagy and Prolongs Lifespan

Summary Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast. Blocking the mitochondrial route to AcCoA by deletion of the CoA-transferase ACH1 caused cytosolic accumulation of the AcCoA precursor acetate. This led to hyperactivation of nucleocytosolic AcCoA-synthetase Acs2p, triggering histone acetylation, repression of autophagy genes, and an age-dependent defect in autophagic flux, culminating in a reduced lifespan. Inhibition of nutrient signaling failed to restore, while simultaneous knockdown of ACS2 reinstated, autophagy and survival of ach1 mutant. Brain-specific knockdown of Drosophila AcCoA synthetase was sufficient to enhance autophagic protein clearance and prolong lifespan. Since AcCoA integrates various nutrition pathways, our findings may explain diet-dependent lifespan and autophagy regulation.

Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells

Oxidative folding facilitates protein import into the mitochondrial intermembrane space. An analysis of the process in intact mammalian cells reveals the contributions of Mia40, ALR, glutathione, and the membrane potential. Proteins that rely on oxidative folding remain stable and reduced in the cytosol for several minutes.

The Ca2+-Dependent Release of the Mia40-Induced MICU1-MICU2 Dimer from MCU Regulates Mitochondrial Ca2+ Uptake

The essential oxidoreductase Mia40/CHCHD4 mediates disulfide bond formation and protein folding in the mitochondrial intermembrane space. Here, we investigated the interactome of Mia40 thereby revealing links between thiol-oxidation and apoptosis, energy metabolism, and Ca(2+) signaling. Among the interaction partners of Mia40 is MICU1-the regulator of the mitochondrial Ca(2+) uniporter (MCU), which transfers Ca(2+) across the inner membrane. We examined the biogenesis of MICU1 and find that Mia40 introduces an intermolecular disulfide bond that links MICU1 and its inhibitory paralog MICU2 in a heterodimer. Absence of this disulfide bond results in increased receptor-induced mitochondrial Ca(2+) uptake. In the presence of the disulfide bond, MICU1-MICU2 heterodimer binding to MCU is controlled by Ca(2+) levels: the dimer associates with MCU at low levels of Ca(2+) and dissociates upon high Ca(2+) concentrations. Our findings support a model in which mitochondrial Ca(2+) uptake is regulated by a Ca(2+)-dependent remodeling of the uniporter complex.

Global remodelling of cellular microenvironment due to loss of collagen VII

The mammalian cellular microenvironment is shaped by soluble factors and structural components, the extracellular matrix, providing physical support, regulating adhesion and signalling. A global, quantitative mass spectrometry strategy, combined with bioinformatics data processing, was developed to assess proteome differences in the microenvironment of primary human fibroblasts. We studied secreted proteins of fibroblasts from normal and pathologically altered skin and their post‐translational modifications. The influence of collagen VII, an important structural component, which is lost in genetic skin fragility, was used as model. Loss of collagen VII had a global impact on the cellular microenvironment and was associated with proteome alterations highly relevant for disease pathogenesis including decrease in basement membrane components, increase in dermal matrix proteins, TGF‐β and metalloproteases, but not higher protease activity. The definition of the proteome of fibroblast microenvironment and its plasticity in health and disease identified novel disease mechanisms and potential targets of intervention.

Endonuclease G mediates α‐synuclein cytotoxicity during Parkinson's disease

Malfunctioning of the protein α‐synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinsons disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson‐diseased patients, while EndoG depletion largely reduces α‐synuclein‐induced cell death in human neuroblastoma cells. Xenogenic expression of human α‐synuclein in yeast cells triggers mitochondria‐nuclear translocation of EndoG and EndoG‐mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α‐synuclein‐driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α‐synuclein‐expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α‐synuclein cytotoxicity.

Altered MCM protein levels and autophagic flux in aged and systemic sclerosis dermal fibroblasts.

Aging is a common risk factor of many disorders. With age, the level of insoluble extracellular matrix increases leading to increased stiffness of a number of tissues. Matrix accumulation can also be observed in fibrotic disorders, such as systemic sclerosis (SSc). Although the intrinsic aging process in skin is phenotypically distinct from SSc, here we demonstrate similar behavior of aged and SSc skin fibroblasts in culture. We have used quantitative proteomics to characterize the phenotype of dermal fibroblasts from healthy subjects of various ages and from patients with SSc. Our results demonstrate that proteins involved in DNA and RNA processing decrease with age and in SSc, while those involved in mitochondrial and other metabolic processes behave the opposite. Specifically, mini-chromosome maintenance (MCM) helicase proteins are less abundant with age and SSc, and they exhibit an altered subcellular distribution. We observed that lower levels of MCM7 correlate with reduced cell proliferation, lower autophagic capacity and higher intracellular protein expression phenotypes of aged and SSc cells. Additionally, we show that SSc fibroblasts exhibit higher levels of senescence than their healthy counterparts, suggesting further similarities between the fibrotic disorder and the aging process. Hence, at the molecular level, SSc fibroblasts exhibit intrinsic characteristics of fibroblasts from aged skin.

Loss of Collagen VII Is Associated with Reduced Transglutaminase 2 Abundance and Activity

Absence of collagen VII leads to widespread cellular and tissue phenotypes. However, the underlying molecular mechanisms are not well understood. To gain insights into cellular responses to loss of collagen VII, we undertook a quantitative disease proteomics approach. By using recessive dystrophic epidermolysis bullosa (RDEB), a skin blistering disease caused by collagen VII deficiency, as a genetic model, collagen VII-dependent differences in cellular protein abundances and protein-protein interactions were analyzed. Absence of collagen VII led to alterations of intracellular protein compositions and to perturbations in cell adhesion, protein trafficking, and the turnover pathway autophagy. A potential linker of the different cellular phenotypes is transglutaminase 2 (TGM2), a multifunctional enzyme important for protein cross-linking. TGM2 was identified as a stable interaction partner of collagen VII. In RDEB, both abundance and activity of TGM2 were reduced, accounting not only for diminished adhesion and perturbed autophagy but also for reduced cross-linking of the extracellular matrix and for decreased epidermal-dermal integrity in RDEB.

The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier

Significance Loss of podocyte adhesion is a hallmark of glomerular disease progression. Here we unravel the in vivo composition of the podocyte adhesion machinery by the use of quantitative proteomics and identify the FERM domain protein EPB41L5 as a selectively enriched novel podocyte focal adhesion protein. EPB41L5 is essential to maintaining podocyte adhesion in vivo by recruiting the Rho GEF ARHGEF18, initiating a signaling cascade and ultimately resulting in increased actomyosin activity and focal adhesion stabilization. As EPB41L5 is down-regulated in various glomerular pathologies, these findings offer a perspective for interventions aiming to prevent loss of podocytes in glomerular disease. Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

ABSTRACT Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.

A Multi-layered Quantitative In Vivo Expression Atlas of the Podocyte Unravels Kidney Disease Candidate Genes

Summary Damage to and loss of glomerular podocytes has been identified as the culprit lesion in progressive kidney diseases. Here, we combine mass spectrometry-based proteomics with mRNA sequencing, bioinformatics, and hypothesis-driven studies to provide a comprehensive and quantitative map of mammalian podocytes that identifies unanticipated signaling pathways. Comparison of the in vivo datasets with proteomics data from podocyte cell cultures showed a limited value of available cell culture models. Moreover, in vivo stable isotope labeling by amino acids uncovered surprisingly rapid synthesis of mitochondrial proteins under steady-state conditions that was perturbed under autophagy-deficient, disease-susceptible conditions. Integration of acquired omics dimensions suggested FARP1 as a candidate essential for podocyte function, which could be substantiated by genetic analysis in humans and knockdown experiments in zebrafish. This work exemplifies how the integration of multi-omics datasets can identify a framework of cell-type-specific features relevant for organ health and disease.