Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Protein homeostasis - i.e., proteostasis - is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In this work, we show that the Rqc1p protein is necessary for the thermoadaptation of S. cerevisiae to heat shock, as RQC1-deficient yeast is sensitive to elevated temperatures. In silico approaches - such as multiple sequence alignment, structural analysis, and molecular dynamics simulations - confirmed earlier predictions that Rqc1p shares characteristics with the bHLH family of proteins. We also verified, through computational prediction of sub-cellular localization, that S. cerevisiae Rqc1p contains nuclear localization signals, suggesting that this protein can potentially be translocated toward the nucleus, thereby broadening its current range of recognized biological functions in this organism. Also, analysis of yeast transcriptomes subjected to heat shock showed that Rqc1p mRNA levels do not fluctuate in response to heat shock, suggesting that cellular concentrations of Rqc1p are already at optimal levels to elicit a rapid and effective response during thermal stress in S. cerevisiae.

TAT (Twin-Arginine Translocation) is a preprotein translocation system dedicated to membrane translocation of prefolded proteins in plants and bacteria. The TAT translocon, consisting of the TatABC subunits, drives translocation using proton motive force. However, there have been no reports on the successful reconstitution of the TAT system. In this report, we show that MPIase, a glycolipid that has known to catalyze membrane protein integration, is essential for the TAT system. Our findings in recombinant Escherichia coli demonstrate that overproducing TatABC increases MPIase levels and that depleting MPIase results in TAT precursor accumulation in the cytosol. Furthermore, co-reconstitution of MPIase with TatABC revealed the translocation activities of TAT substrates in a proton motive force-dependent manner. This is the first successful reconstitution of the TAT system and will be advantageous for understanding its mechanisms.

Ribosome biogenesis is a highly coordinated pathway that involves the assembly of ribosomal RNAs (rRNAs) with ribosomal proteins (r-proteins) to generate functional ribosomal subunits (r-subunits). The Saccharomyces cerevisiae (yeast) large 60S r-subunit consists of three rRNA molecules and 46 r-proteins. The contributions of nearly all r-proteins of the yeast large r-subunit have been characterized; however, a few non-essential proteins remain poorly understood. Although non-essential, human eL22 has been identified as a key player in p53 regulation during ribosomal stress and as a highly mutated target in cancers. Despite this function, the role of eL22 in ribosome maturation is still ill-defined. In this study, we characterized yeast eL22 r-protein. Our results show that eL22 assembles into intermediate nucleolar pre-60S ribosomal particles. Loss of eL22 impairs cell growth and reduces 60S r-subunit accumulation, phenotypes that are exacerbated at low temperatures. Analysis of pre-rRNA processing by pulse-chase labeling, northern blot hybridization, and primer extension reveals a defect in 27S pre-rRNA maturation, specifically at the level of 27SB pre-rRNA processing. Consequently, nuclear export of eL22-deficient pre-60S particles is mildly impaired. Furthermore, we identify genetic interactions between eL22 and neighboring r-proteins, eL38 and eL31. We conclude that eL22 assembly is required for optimal pre-60S maturation during middle nucleolar stages, particularly at low temperatures, a function likely supported by the cooperative action of other r-proteins associated with common elements of 25S rRNA. HighlightsO_LIWe have studied the role of r-protein eL22 in yeast ribosome assembly. C_LIO_LIeL22 is required for 60S ribosomal subunit production. C_LIO_LIThe absence of eL22 is critical at low temperatures. C_LIO_LIeL22 is important for 27SB pre-rRNA processing and nuclear export of pre-ribosomes. C_LIO_LIeL22 functionally interacts with r-proteins eL38 and eL31 in domain III of 25S rRNA. C_LI

Conflicting roles have been proposed for the E. coli protein RatA. Initially described as a ribosome targeting toxin, a later report pronounced it the bacterial homologue to the inner mitochondrial membrane protein Coq10. Coq10 proteins are conserved from prokaryotes to human and implicated to serve a lipid chaperone role in the biosynthesis of ubiquinone, a crucial electron carrier during aerobic respiration. We recently identified that the contradictory results published for RatA can be attributed to a mis-annotation of the gene in the reference genome. Here, we further elucidate the molecular function of RatA. We clarify that RatA is not a toxin but serves as a lipid shuttle for ubiquinone from its cytosolic biosynthesis complex to the inner membrane. Furthermore, we show that the loss of RatA results in an impaired, but not abolished electron transport chain and demonstrate broad metabolic adaptations of the cells as a consequence. Therefore, we propose to rename RatA to UbiM to reflect its function and to be in accordance with the naming convention of other ubiquinone biosynthesis proteins.

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

Old Yellow Enzymes (OYEs) are a widely distributed family of ene-reductases that were first described in a Saccharomyces cerevisiae ferment. In plants, cis-12-oxo-phytodienoic acid (cis-OPDA) reductase (OPR) is the best studied OYE. In Arabidopsis thaliana, the peroxisomal AtOPR3 was characterized as the major OPDA reductase, which generates 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid in the jasmonic acid (JA) biosynthesis. In Atopr3 lines, only small amounts of JA are detectable after wounding. Here, we describe an OPR-like enzyme (named BnOPR) from the gram-positive Brevibacillus nitrificans. The sequence was identified in an early version of the Physcomitrium patens genome and is assumed to be a contamination by a bacterium growing in association with P. patens. In complementation experiments with an Atopr3 line, we demonstrate that expression of BnOPR, fused with a peroxisomal targeting signal, rescues the male infertile phenotype and increases JA and JA-Ile levels. The catalytic parameters of BnOPR were determined for a set of substrates, including cis-OPDA and prednisone. Interestingly, B. nitrificans, B. brevis, and Paenibacillus physcomitrellae were shown to have a positive effect on P. patens growth. HighlightThe bacterial enzyme BnOPR rescues the male infertile phenotype of Atopr3 plants.

Cancer, one of the worlds leading causes of death, is characterized by a complex metabolic reprogramming that features the Warburg effect as one of its hallmarks. The Warburg effect involves increased glucose and amino acid metabolism, which promotes tumor proliferation and progression. Although cancer has historically been attributed to genetic mutations, recent studies suggest a possible metabolic origin. However, a key characteristic of cancer cells is their greater adaptability than normal cells, as evidenced by their resistance to chemotherapy, which stems from their high mutability. This underscores the need to examine the relationship between metabolic reprogramming and cancer development from both metabolic and evolutionary perspectives. In this context, Saccharomyces cerevisiae snf1{Delta} strain has emerged as an ideal cellular model for studying the Warburg effect. This study aimed to determine whether deletion of the SNF1 gene in S. cerevisiae affects its chronological aging and competitiveness in a glucose and amino acid-dependent manner. Herein, we provide evidence that the snf1{Delta} strain changes the chronological aging depending on nutrimental condition, under low-nutrient levels shortens (0.1% glucose + 0.1x amino acids), and increases under high-nutrient levels (5% glucose + 3x amino acids). Competitiveness of the snf1{Delta} strain in co-cultivation with wild-type was also improved in 5% glucose + 3x amino acids, by approximately 2 Log10. These results indicate that snf1{Delta} strain aging and competitiveness are also sensitive to nutrimental status, as was observed in cancer cells.

NRF1 is a key mediator of the proteasome recovery pathway, yet its regulation by ER-resident factors is not fully elucidated. Here, we demonstrate that selenoproteins SELS and SELK are critical regulators for NRF1 protein dynamics. SELS stabilizes NRF1, while SELK induces its insolubilization. Their deficiency leads to a hyper-accumulation and increased nuclear localization of NRF1 under proteasome inhibition condition. This results in an augmented transcriptional response of proteasome subunits. These results indicate that SELS and SELK cooperatively gate NRF1 activity by controlling its retrotranslocation and solubility, highlighting a novel layer of selenoprotein-mediated quality control in the proteostasis network.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Ergosterol has multiple functions in filamentous fungi and yeasts, although only a part of the functions seems to be understood. An antifungal peptide, theonellamide A (TNM-A) induces drastic morphological changes in fission yeast cells by targeting plasma membrane ergosterol. TNM-A induces overproduction and ectopic accumulation of cell wall glucan at both growing tips and septum through a yet unknown mechanism. Here we show that TNM-A treatment causes accumulation of 1,3-{beta}-glucan at cell-polarity sites, not by increased activity of 1,3-{beta}-glucan synthase, but by an increased, persistent localization of the glucan synthase enzymes. Screening based on subcellular localization of proteins at periphery or polarity sites suggested the involvement of the Rho family GTPase Cdc42. In agreement, TNM-A induced both activation of Cdc42 and enhancement of membrane trafficking of glucan synthase enzymes. In conclusion, our chemical genetics analyses using TNM-A suggest that membrane ergosterol regulates the activity of Cdc42, which further regulates the localization of glucan synthases and cell wall biosynthesis. Highlights (four sentences)- Thenoellamide A (TNM-A) induces an ectopic overproduction of cell wall glucan. - TNM-A treatment causes increased, persistent localization of glucan synthases at the cell tips and septum. - TNM-A activates Cdc42 and upregulates membrane trafficking of glucan synthases. - Ergosterol is involved in proper activation/inactivation of Cdc42.

Mitochondrial homeostasis, governed by the balance between biogenesis and mitophagy, is essential for steroidogenesis in adrenocortical cells. While the requirement of active mitochondria for steroid synthesis is well-established, the hormonal regulation of genes governing mitochondrial function remains poorly understood. This study investigated whether angiotensin II (Ang II) and the cAMP/PKA pathway modulate the expression of key regulatory factors involved in mitochondrial biogenesis and redox status in the human adrenocortical H295R cell line. Using real-time qPCR and Western blot, we show that Ang II and 8Br-cAMP --a permeant analogue of cAMP-- modulate NRF-1, Nrf2, UCP2, and ANT1 impacting on mitochondrial biogenesis, antioxidant defense, and respiratory activity. These molecular changes correlated with increased mitochondrial membrane polarization, as confirmed by MitoTracker red staining. Interestingly, Ang II stimulation promoted a time-dependent increase in TFAM levels, a key transcription factor in mitochondria, which correlates with the increase in mitochondrial DNA (mtDNA) content. The rate of oxygen consumption (OCR) and mitochondrial parameters were determined, with results showing that Ang II led to a significant increase in basal and maximum respiration, ATP production, and proton leak. These findings suggest that hormone stimulation favors mitochondrial activity, thereby enhancing the bioenergetic capacity of adrenocortical cells. Furthermore, treatment with the uncoupler CCCP triggered a retrograde signaling response, upregulating nuclear-encoded mitochondrial genes to counteract mitochondrial membrane depolarization. Our findings demonstrate for the first time that hormonal signals directly modulate the mitochondrial genetic program in H295R human adrenocortical cells, optimizing the bioenergetic platform required for efficient steroidogenic function.

Photosynthetic membranes undergo structural remodeling in response to environmental stress by altering fatty acid composition and desaturation levels. These changes, mediated by fatty acid desaturases (FADs), are essential for maintaining photosynthetic performance and adaptation. In this study, we demonstrate that copper-deficient Chlamydomonas reinhardtii cells upregulate the expression of the gene encoding stearoyl-ACP desaturase (SAD/FAB2). We propose that this four-fold induction reflects an increased physiological demand for its primary product, oleic acid (18:1{Delta}9), and its subsequent downstream derivatives. The sad mutants exhibit a significant reduction in 18:1{Delta}9 content compared to wild-type cells, which correlates with diminished growth rates. Although SAD abundance increases under Cu deficiency, loss of SAD strongly alters C18 fatty acid composition across Cu conditions, while the growth defect is most apparent under Cu-replete conditions. This suggests that SAD activity may be a limiting factor in copper-depleted environments, leading to slower growth and reduced 18:1{Delta}9 levels in the uncharged galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), both of which are critical for photosynthetic function. The desaturation reaction catalyzed by SAD requires molecular oxygen and electrons supplied by ferredoxin (Fd). Using reciprocal IP-MS, we identified FDX5 as a Cu-deficiency specific SAD interacting protein. However, fdx5 mutants retained wild-type fatty acid profiles, indicating that FDX5 is not strictly required for SAD-dependent lipid desaturation and that another ferredoxin, likely FDX1, can compensate.

Familial Amyotrophic Lateral Sclerosis (fALS) is a fatal neurodegenerative disease, mainly caused by mutations in the superoxide dismutase 1 (SOD1) protein. Mitochondrial dysfunction is a primary hallmark of ALS pathogenesis. However, the molecular mechanism by which SOD1 mutants impair organellar health remains enigmatic. This study demonstrates that mutant SOD1 associates with the TIM23 complex in Saccharomyces cerevisiae via its intermembrane space (IMS) domain. In ALS-associated SOD1 mutants, both binding and expression of TIM23 complex proteins were downregulated, leading to altered translocation of the substrate protein Sdh3, a component of the electron transport chain (ETC) complex II. Disrupted Sdh3 translocation leads to mitochondrial dysfunction, evidenced by decreased ETC complex II activity, reduced functional mass, and compromised organelle integrity. Overexpression of Tim23 partially rescued mitochondrial integrity by increasing ETC complex activity and functional mass and restoring reticular morphology. Strikingly, the improved mitochondrial homeostasis in Tim23-overexpressing cells partially rescued the growth defects caused by mutant SOD1. Collectively, these findings reveal a previously unrecognized regulatory axis between mutant SOD1 and the mitochondrial pre-sequence translocase machinery, highlighting this pathway as a promising target for future ALS therapies and opening new avenues for mechanistic and translational research. Author SummaryFamilial Amyotrophic Lateral Sclerosis (fALS) is a progressive, fatal neuromuscular disorder marked by motor neuron degeneration. The exact cause of ALS remains unclear. Previous research links familial ALS to mutations in the superoxide dismutase 1 (SOD1) gene. SOD1 mutants in ALS disrupt mitochondrial protein translocation, a key mitochondrial process. The mechanism by which SOD1 mutants affect mitochondrial function and integrity by modulating presequence translocase (TIM23 complex) import is not yet understood. The current study addresses a critical gap in ALS research by demonstrating a novel, direct interaction between SOD1 and Tim23 that regulates mitochondrial function in yeast. We found that SOD1 binds Tim23 via Tim23 IMS domain, stabilizes the Tim23CORE complex, enabling Sdh3 import. Loss of SOD1, Tim23, or Tim50 destabilizes the TIM23CORE complex, leading to impaired Sdh3 import and decreased ETC complex-II activity. These changes disrupt mitochondrial structure, causing fragmentation and a loss of functional mitochondrial mass in {Delta}sod1. ALS-linked SOD1 mutants show similar effects: they diminish Sdh3 import by weakening SOD1-Tim23 interaction and lowering TIM23 complex stability, resulting in punctate mitochondria and reduced mitochondrial mass. Collectively, our study identifies the SOD1-TIM23 interaction as a key regulator of mitochondrial health through Sdh3 import via the TIM23CORE complex and indicates this pathway as a potential early intervention target for ALS therapy.

The excitability of afferents involved in nociceptive signaling reflects the interaction of several co-expressed membrane receptors. Current studies have shown that Toll-like receptor-4 (TLR-4) signaling can exacerbate excitation evoked by transient receptor potential vanilloid type 1 (TRPV1) activity, and this interaction plays a key role in driving and sustaining facilitated pain states. The mechanism by which this potentiated TRPV1 activity secondary to TLR-4 agonism occurs in sensory neurons remains unknown, although intracellular kinase activity is a strong candidate. To address this hypothesized linkage, neuronal cell cultures prepared from dorsal root ganglia (DRG) of male wildtype (WT) and Tlr4-/- mice were used to evaluate calcium transients of neurons after capsaicin administration in culture, pre-treated for 30 minutes with the TLR-4 agonist, lipopolysaccharide (LPS). TRPV1 protein expression at the neuron surface in cultured DRG cells with or without LPS treatment was quantified by flow cytometry assay. The roles of protein kinase A (PKA) and C were assessed using selective inhibitors (KT5720 for PKA and Chelerythrine chloride for PKC) applied to WT-DRG neurons or administered in vivo by intraplantar or intrathecal injection, prior to LPS and capsaicin administration. Behavioral effects of in vivo TRPV1 activation were assessed through paw flinch responses evoked by intraplantar capsaicin injection and by hind paw tactile thresholds measured by von Frey filaments. LPS incubation in cultured DRG neurons enhances the intensity of calcium influx following TRPV1 activation in WT but not Tlr4-/ cells. The augmented calcium influx evoked by capsaicin was prevented by the inhibition of PKA but not PKC. Similarly, mice treated with LPS in the hind paw displayed greater nociceptive responding after capsaicin and increased tactile allodynia. The facilitated component was prevented by the local pre-treatment with the PKA inhibitor. Correspondingly, lumbar spinal blockade of PKA resulted in temporary reversal of hyperalgesia induced by intrathecal LPS injection in mice. Together, these results demonstrate the relevance of TLR-4 in modulating the excitability of nociceptor signaling by regulating TRPV1, thereby influencing pain transmission through PKA signaling.

Protein dimerization plays a central role in regulating enzymatic activity, signal transduction, and transcription factor function. Within the PI3K family, different modes of oligomerization have been reported. However, the oligomerization of lipid 5-phosphatases and its functional consequences have not been described. Here, we show for the first time that the lipid 5-phosphatase SHIP2 exists as a homodimer in cells, with its N-terminal region serving as the primary dimerization domain. We further demonstrate that SHIP2 dimerization has no major impact on its catalytic activity but instead profoundly affects its interactome. Interestingly, we identify the stress granule marker G3BP1 as one of the SHIP2 interactors whose association is moderately influenced by SHIP2 oligomerization states. Furthermore, we show that changes in SHIP2 protein levels, enzymatic activity, and oligomeric state alter the cellular response to sodium arsenate-induced stress. In addition, variation in SHIP2 levels and activity affects stress granule size and dynamics. Together, these findings identify SHIP2 oligomerization as a previously unrecognized regulatory mechanism linking phosphoinositide signaling to stress granule biology.

O_LIIn this study, we demonstrate that Benzo[a]pyrene (B[a]P) induces keratinocyte senescence and p21Cip1-dependent keratinocyte differentiation. Atmospheric and environmental pollution are known to induce senescence and promote terminal differentiation in human primary keratinocytes, thus driving skin aging. However, much is still unknown about the underlying molecular mechanisms. We observed that B[a]P, a common atmospheric pollutant, induced senescence in primary keratinocytes in both two-dimensional and three-dimensional (reconstructed human epidermis) culture. This was accompanied by signs of DNA damage in B[a]P-treated cells. B[a]P-treated cells also underwent accelerated late-stage terminal differentiation, indicated by increased IVL and FLG expression from 48 to 96 hours post-exposure. While pharmacological and genetic attenuation of p21Cip1 did not rescue cellular senescence, it prevented the expression of IVL and FLG, suggesting that the late-stage terminal differentiation induced by B[a]P exposure was p21-dependent. Our data thus suggest a key role for the p21Cip1 in the keratinocyte response to pollution-induced damage, where p21Cip1 induces terminal differentiation to maintain skin barrier homeostasis. C_LI

Bacterial genotoxins, Cytolethal Distending Toxin (CDT) and colibactin, cause DNA damage in intoxicated epithelial cells of the host. DNA damage influences {beta}-catenin signaling, altering its stability and nuclear translocation, potentially contributing to cancer development. Using non-transformed hepatocytes and cancer-derived intestinal and hepatic epithelial cell lines, we showed that CDT/CdtB induces the phosphorylation of {beta}-catenin at serine 552, along with the loss of {beta}-catenin from adherens junctions. This leads to the subsequent cytoplasmic accumulation and nuclear translocation of {beta}-catenin, ultimately driving TCF/LEF transcription, the crucial downstream event of Wnt/{beta}-catenin signaling, as well as the transcription of some {beta}-catenin target genes. Colibactin induces similar effects. MK-2206, a direct AKT inhibitor, and metformin, an AMP-activated protein kinase activator that indirectly inhibits AKT, both protected cells against various effects induced by CdtB exposure. These effects include {beta}-catenin phosphorylation at Ser552, the disassembly of cell-cell junctions and the subsequent nuclear accumulation of phosphorylated {beta}-catenin, leading to reduced TCF/LEF-mediated transcription. Additionally, MK-2206 and metformin protected from CdtB-induced epithelial-mesenchymal transition (EMT), including increased nuclear accumulation of SNAIL, enhanced matrix degradation and motility. Overall, these data show that infection with genotoxin-producing bacteria controls some EMT features through {beta}-catenin and AKT-dependent signaling.

We have recently demonstrated that treatment of aged mice with a pan-ERR agonist reverses age-related increase in urinary albumin, decrease in podocyte density, impaired mitochondrial function, and inflammation. The contribution of individual isoforms of ERRs however has not been determined. Since the aging kidney showed a possible compensatory increased expression of ERR{gamma} in the podocytes, in the face of decreased ERR expression, in the present study we aimed to determine the role of ERR{gamma} in aging podocyte. To this end, we cross bred ERR{gamma} floxed mice with podocin-Cre mice to achieve a podocyte-specific ERR{gamma} deletion. While these mice at 3 months of age showed no effect on albuminuria compared to the wild type, when the mice were aged to 21 months of age, there was a significant increase in albuminuria and decrease in podocyte density. Furthermore, we found that the podocyte deletion of ERR{gamma} primarily targeted the expression of mitochondrial biogenesis regulator PGC-1, and mitochondrial fatty acid oxidation enzymes CPT1a and MCAD in the kidney. Electron Microscopy (EM) revealed thickened glomerular basement membrane and diffuse podocyte foot process effacement, as well as severe mitochondrial damage including cristae abnormalities, fragmentation, and changes indicative of altered fusion and fission dynamics. Fluorescence Lifetime Imaging Microscopy (FLIM) to determine NADH and FAD lifetimes indicate a metabolic shift from mitochondrial oxidative phosphorylation towards glycolysis, and decrease in mitochondrial redox capacity. Considering a significantly decreased expression of ERR in aging podocytes plus its traditional role in mitochondrial function, these studies using podocyte ERR{gamma} deletion suggested an overlapping mechanism for ERR/ERR{gamma} to act as modulators of age-related mitochondrial dysfunction and age-related kidney disease.

Statins, widely used cholesterol-lowering drugs, inhibit the mevalonate pathway and reduce coenzyme Q (CoQ) biosynthesis, potentially impairing mitochondrial function. Because astrocytes are essential for maintaining brain redox homeostasis, statin-induced mitochondrial dysfunction in these cells may contribute to CNS pathology. We examined the effects of a six-day statin exposure on mitochondrial bioenergetics in rat astrocytes, focusing on mitochondrial CoQ (mtCoQ) deficiency. Treatment with 200 nM atorvastatin or simvastatin decreased the total mtCoQ pool (mtCoQ9 + mtCoQ10) by 30-35% and decreased the antioxidant pool mtCoQH2 by 40%, whereas the levels of mitochondrial antioxidant proteins, including superoxide dismutase 2 and uncoupling proteins, remained unchanged. Mitochondria of statin-treated astrocytes showed decreased respiratory activity, membrane potential, and ATP synthesis, and increased mtCoQ reduction leading to increased H2O2 production during the oxidation of complex I (CI) and CII substrates. Statin treatment also altered the organization of the respiratory chain, leading to a downregulation of the CI+CIII2+CIV and CIII2+CIV supercomplexes and decreased protein levels and activity of all respiratory chain complexes. Furthermore, a decrease in cytochrome a + a3 content was accompanied by a reduction in the maximum activity of CIV. CoQ10 supplementation elevated mtCoQ levels, restored respiratory function, and decreased H2O2 production in the mitochondria of statin-treated astrocytes. Prolonged statin exposure alters mtCoQ redox homeostasis and impairs mitochondrial bioenergetic function in astrocytes. CoQ10 supplementation attenuates these changes, supporting its potential role in protecting astrocyte mitochondria from statin-induced dysfunction.

Angiotensin-converting enzyme 2 (ACE2) functions as the receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus utilizes the cellular endocytic machinery for entry by binding to defined residues on ACE2 with its spike protein (S protein), whose activation requires a priming process by another transmembrane protease, the transmembrane protease serine 2 (TMPRSS2). In addition, ACE2 itself is cleaved by TMPRSS2, which has been shown to be critical for viral pathology. This study aimed to elucidate the relationship between ACE2 and TMPRSS2 and the mechanism of ACE2 processing under normal cellular conditions. It is shown that interaction of ACE2 with TMPRSS2 results in altered processing, modification and cellular localization. Glycosylation of ACE2 has a major impact on TMPRSS2 interaction, trafficking and shedding of the enzyme. Studies in newly generated TMPRSS2-knockout rats reveal increased ACE2 levels in tissues supporting an important role of TMPRSS2 in ACE2 shedding also in vivo.