Hana Nůsková

Pyruvate dehydrogenase (PDH) complex is a key regulator of metabolism, controlling flux from glycolysis into the tricarboxylic acid (TCA) cycle. PDH is often dysregulated in metabolic diseases and cancer. We discover here that in addition to its well characterized regulation by phosphorylation, PDH activity is also regulated via inhibitory oxidation of three cysteines of the DLAT subunit - Cys488, Cys586 and Cys618. Oxidation of these cysteines increases under oxidative stress, and is highly prevalent in mouse and human tissues. This mechanism enables cells to efficiently fight oxidative stress. Under oxidative stress, cells execute a metabolic shift, blocking glycolytic flux into the TCA cycle to shunt glycolytic carbons toward the pentose phosphate pathway. This generates NADPH which cells use to detoxify H ₂ O ₂ and to reduce oxidized cysteines. We find that DLAT oxidation is required for cells to mount an effective antioxidant response. Mutations preventing DLAT oxidation render PDH resistant to oxidative inhibition, impair NADPH generation, reduce peroxide detoxification, and sensitize cells to oxidative damage and death. DLAT oxidation also drives a Warburg-like metabolic phenotype of increased glucose uptake, elevated lactate secretion, and reduced TCA cycle activity. Thus DLAT oxidation is a redox-sensing mechanism that regulates PDH activity and central carbon metabolism.

Small cell lung cancer (SCLC) patients frequently experience a remarkable response to first-line therapy. Follow up maintenance treatments aim to control residual tumor cells, but generally fail due to cross-resistance, inefficient targeting of tumor vulnerabilities, or dose-limiting toxicity, resulting in relapse and disease progression. Here we show that SCLC cells, similar to their cells of origin, pulmonary neuroendocrine cells, exhibit low activity in pathways protecting against reactive oxygen species (ROS). When exposed to a thioredoxin reductase 1 (TXNRD1) inhibitor, these cells quickly exhaust their ROS-scavenging capacity, regardless of their molecular subtype or resistance to first-line therapy. Importantly, unlike non-cancerous cells, SCLC cells cannot adapt to drug-induced ROS stress due to the suppression of ROS defense mechanisms by multiple layers of gene regulation. By exploiting this difference in oxidative stress management, we safely increase the therapeutic dose of TXNRD1 inhibitors in vivo by pharmacological activation of the NRF2 stress response pathway. This results in improved tumor control without added toxicity to healthy tissues. These findings underscore the therapeutic potential of TXNRD1 inhibitors for maintenance therapy in SCLC.

Chemical inducers of proximity have transformed small-molecule pharmacology, but the large, bifunctional architectures they often employ introduce new and poorly understood off-target risks. During a targeted protein degrader synthesis project, we identified a subset of compounds that cause rapid and unexpected ATP depletion in cells. Mechanistic studies traced this effect to inhibition of mitochondrial complex I, a central component of oxidative phosphorylation. This inhibition does not stem from off-target binding by either of the two target ligands, but from the overall long, linear architecture of the bifunctional molecules, which renders them effective ligands of the narrow ubiquinone binding tunnel of complex I. Strikingly, this liability extends to structurally unrelated bifunctional molecules, including six androgen receptor PROTACs including the clinical candidate ARV-110, which inhibits complex I at low nanomolar concentrations. To mitigate complex I inhibition, we established a generalizable design strategy to disrupt linear molecular geometry through the introduction of structural “bumps” or “kinks”. In a proof-of-concept study, we successfully apply this strategy to the ARV-110 scaffold, discovering potent AR-degrading ARV-110 analogs that do not inhibit complex I. These findings uncover a previously underappreciated structural determinant of off-target mitochondrial toxicity and establish new design principles for safer proximity-inducing therapeutics.

Despite a strong rationale for why cancer cells are susceptible to redox-targeting drugs, such drugs often face tumor resistance or dose-limiting toxicity in preclinical and clinical studies. An important reason is the lack of specific biomarkers to better select susceptible cancer entities and stratify patients. Using a large panel of lung cancer cell lines, we identified a set of “antioxidant-capacity” biomarkers (ACB), which were tightly repressed, partly by STAT3 and STAT5A/B in sensitive cells, rendering them susceptible to multiple redox-targeting and ferroptosis-inducing drugs. Contrary to expectation, constitutively low ACB expression was not associated with an increased steady state level of reactive oxygen species (ROS) but a high level of nitric oxide, which is required to sustain high replication rates. Using ACBs, we identified cancer entities with a high percentage of patients with favorable ACB expression pattern, making it likely that more responders to ROS-inducing drugs could be stratified for clinical trials.

Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.

Background: The pleomorphic clinical presentation makes the diagnosis of desminopathy difficult. We aimed to describe the prevalence, phenotypic expression, and mitochondrial function of individuals with putative disease-causing desmin (DES) variants identified in patients with an unexplained etiology of cardiomyopathy. Methods: A total of 327 Czech patients underwent whole exome sequencing and detailed phenotyping in probands harboring DES variants. Results: Rare, conserved, and possibly pathogenic DES variants were identified in six (1.8%) probands. Two DES variants previously classified as variants of uncertain significance (p.(K43E), p.(S57L)), one novel DES variant (p.(A210D)), and two known pathogenic DES variants (p.(R406W), p.(R454W)) were associated with characteristic desmin-immunoreactive aggregates in myocardial and/or skeletal biopsy samples. The individual with the novel DES variant p.(Q364H) had a decreased myocardial expression of desmin with absent desmin aggregates in myocardial/skeletal muscle biopsy and presented with familial left ventricular non-compaction cardiomyopathy (LVNC), a relatively novel phenotype associated with desminopathy. An assessment of the mitochondrial function in four probands heterozygous for a disease-causing DES variant confirmed a decreased metabolic capacity of mitochondrial respiratory chain complexes in myocardial/skeletal muscle specimens, which was in case of myocardial succinate respiration more profound than in other cardiomyopathies. Conclusions: The presence of desminopathy should also be considered in individuals with LVNC, and in the differential diagnosis of mitochondrial diseases.

The biogenesis of mammalian ATP synthase is complex process believed to proceed via several modules. It starts with the formation of F1 catalytic part, which is in the later steps connected with the membranous subcomplex. The final phase is represented by incorporation of the two mtDNA-encoded subunits Fo-a and A6L. However, little is known about the position of two newly described Fo accessory subunits DAPIT (also termed Usmg5) and MLQ (also known as c14orf2) in the assembly scheme and about their role in regulation of ATP synthase biogenesis. To resolve this, we have utilised several model systems, namely rho0 cells lacking mtDNA and thus both subunits Fo-a and A6L, cells harbouring 9205delTA microdeletion, which results in the absence of the subunit Fo-a, HEK293 cells with knockdown of DAPIT protein and HEK293 cells with knockout of MLQ protein and followed the assembly state of ATP synthase among them. Contrary to previously reported data, we observed normal levels of assembled ATP synthase in DAPIT knockdown and MLQ knockout cells. Our results indicate that lack of DAPIT protein leads to the assembly of more labile, but complete and functional holoenzyme. Absence of either Fo-a alone or Fo-a and A6L results into the normal levels of structurally altered, labile, and ~60 kDa smaller vestigial enzyme complex, which also lacks DAPIT and MLQ. This complex retains the ATP hydrolytic activity but is unable to synthesize ATP. Cells with the MLQ knockout presented with the phenotype similar to the lack of Fo-a: normal content of smaller and labile complex. In the absence of MLQ, vestigial ATP synthase did not contain also subunits Fo-a and A6L. This complex also retained ATP hydrolytic activity, while its phosphorylating capacity was affected. In all the cell lines tested, the individual subunits seemed to be associated only with assembled ATP synthase complex, indicating that once subunits dissociate from the complex, they are degraded in the cell. This hypothesis is supported by the fact, that in the cells lacking subunit MLQ the biosynthesis of both mtDNA-encoded subunits Fo-a and A6L is normal, but they are degraded at faster pace than the rest of the complex. Based on our data, we conclude that MLQ and Fo-a closely associate and their incorporation into the enzyme complex depends on each another. On the contrary, DAPIT protein seems to be incorporated at the very last step and its presence stabilises the holoenzyme.

Biogenesis of F ₁ F _o ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP‐synthase deficiency and leads to a neonatal mitochondrial encephalo‐cardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F ₁ domain and peripheral stalk but lacking F _o proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70 ^‐/‐ cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F ₁ ‐deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate‐limiting step of ATP‐synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.—Kovalčíkova, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nůsková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliáš, J., Čunátová, K., Kořínek, V., Sedlacek, R., Mráček, T., Houštěk, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme. FASEB J. 33, 14103‐14117 (2019). www.fasebj.org