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Pyruvate metabolism defects lead to severe neuropathies such as the Leigh syndrome (LS) but the molecular mechanisms underlying neuronal cell death remain poorly understood. Here, we unravel a connection between pyruvate metabolism and the regulation of the epitranscriptome that plays an essential role during brain development. Using genetically engineered mouse model and primary neuronal cells, we identify the transcription factor E4F1 as a key coordinator of AcetylCoenzyme A (AcCoA) production by the pyruvate dehydrogenase complex (PDC) and its utilization as an essential co-factor by the Elongator complex to acetylate tRNAs at the wobble position uridine 34 (U 34 ). E4F1-mediated direct transcriptional regulation of Dlat and Elp3, two genes encoding key subunits of the PDC and of the Elongator complex, respectively, ensures proper translation fidelity and cell survival in the central nervous system (CNS) during mouse embryonic development. Furthermore, analysis of PDH-deficient cells highlight a crosstalk linking the PDC to ELP3 expression that is perturbed in LS patients.

Cuproptosis caused by copper overload is mediated by a novel regulatory mechanism that differs from previously documented mechanisms regulating cell death. Cells dependent on mitochondrial respiration showed increased sensitivity to a copper ionophore elesclomol that induced cuproptosis. Maternal embryonic leucine zipper kinase(MELK) promotes tumorigenesis and tumor progression through the PI3K/mTOR pathway, which exerts its effects partly by targeting the pyruvate dehydrogenase complex(PDHc) and reprogramming the morphology and function of mitochondria. However, the role of MELK in cuproptosis remains unclear. Here, we validated that elevated MELK expression enhanced the activity of PI3K/mTOR signaling and subsequently promoted Dihydrolipoamide S-Acetyltransferase (DLAT) expression and stabilized mitochondrial function. This regulatory effect helped to improve mitochondrial respiration, eliminate excessive intracellular reactive oxygen species (ROS), reduce intracellular oxidative stress/damage and the possibility of mitochondria-induced cell fate alternations, and ultimately promote the progression of HCC. Meanwhile, elesclomol reduced translocase of outer mitochondrial membrane 20(TOM 20) expression and increased DLAT oligomers. Moreover, the above changes of MELK to HCC were abolished by elesclomol. In conclusion, MELK enhanced the levels of the cuproptosis-related signature(CRS) gene DLAT (especially the proportion of DLAT monomer) by activating the PI3K/mTOR pathway, thereby promoting elesclomol drug resistance, altering mitochondrial function, and ultimately promoting HCC progression.

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site. The pyruvate dehydrogenase complex (PDHc) is a large multienzyme complex that converts pyruvate into acetyl-coenzyme A and in E. coli the core of the PDHc is formed by 24 copies of dihydrolipoyl transacetylase. Here, the authors present the cryo-EM structure of the E. coli dihydrolipoyl transacetylase 24-mer core in a native resting state including lipoyl domains, and discuss the mechanism of substrate shuttling by the lipoyl domains.

Copper is a trace element essential for numerous biological activities, whereas the mitochondria serve as both major sites of intracellular copper utilization and copper reservoir. Here, we investigated the impact of mitochondrial copper overload on the tricarboxylic acid cycle, renal senescence and fibrosis. We found that copper ion levels are significantly elevated in the mitochondria in fibrotic kidney tissues, which are accompanied by reduced pyruvate dehydrogenase (PDH) activity, mitochondrial dysfunction, cellular senescence and renal fibrosis. Conversely, lowering mitochondrial copper levels effectively restore PDH enzyme activity, improve mitochondrial function, mitigate cellular senescence and renal fibrosis. Mechanically, we found that mitochondrial copper could bind directly to lipoylated dihydrolipoamide acetyltransferase (DLAT), the E2 component of the PDH complex, thereby changing the interaction between the subunits of lipoylated DLAT, inducing lipoylated DLAT protein dimerization, and ultimately inhibiting PDH enzyme activity. Collectively, our study indicates that mitochondrial copper overload could inhibit PDH activity, subsequently leading to mitochondrial dysfunction, cellular senescence and renal fibrosis. Reducing mitochondrial copper overload might therefore serve as a strategy to rescue renal fibrosis.

The role of copper in cancer treatment is multifaceted, with copper homeostasis-related genes associated with both breast cancer prognosis and chemotherapy resistance. Interestingly, both elimination and overload of copper have been reported to have therapeutic potential in cancer treatment. Despite these findings, the exact relationship between copper homeostasis and cancer development remains unclear, and further investigation is needed to clarify this complexity. The pan-cancer gene expression and immune infiltration analysis were performed using the Cancer Genome Atlas Program (TCGA) dataset. The R software packages were employed to analyze the expression and mutation status of breast cancer samples. After constructing a prognosis model to separate breast cancer samples by LASSO-Cox regression, we examined the immune statement, survival status, drug sensitivity and metabolic characteristics of the high- and low-copper related genes scoring groups. We also studied the expression of the constructed genes using the human protein atlas database and analyzed their related pathways. Finally, copper staining was performed with the clinical sample to investigate the distribution of copper in breast cancer tissue and paracancerous tissue. Pan-cancer analysis showed that copper-related genes are associated with breast cancer, and the immune infiltration profile of breast cancer samples is significantly different from that of other cancers. The essential copper-related genes of LASSO-Cox regression were ATP7B (ATPase Copper Transporting Beta) and DLAT (Dihydrolipoamide S-Acetyltransferase), whose associated genes were enriched in the cell cycle pathway. The low-copper related genes scoring group presented higher levels of immune activation, better probabilities of survival, enrichment in pathways related to pyruvate metabolism and apoptosis, and higher sensitivity to chemotherapy drugs. Immunohistochemistry staining showed high protein expression of ATP7B and DLAT in breast cancer samples. The copper staining showed copper distribution in breast cancer tissue. This study displayed the potential impacts of copper-related genes on the overall survival, immune infiltration, drug sensitivity and metabolic profile of breast cancer, which could predict patients' survival and tumor statement. These findings may serve to support future research efforts aiming at improving the management of breast cancer.

Chemotherapy is often a primary treatment for cancer. However, resistance leads to therapeutic failure. Acetylation dynamics play important regulatory roles in cancer cells, but the mechanisms by which acetylation mediates therapy resistance remain poorly understood. Here, using acetylome-focused RNA interference (RNAi) screening, we find that acetylation induced by mitochondrial dihydrolipoyl transacetylase (DLAT), independent of the pyruvate dehydrogenase complex, is pivotal in promoting resistance to chemotherapeutics, such as cisplatin. Mechanistically, DLAT acetylates methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) at lysine 44 and promotes 10-formyl-tetrahydrofolate (10-formyl-THF) and consequent mitochondrially encoded cytochrome c oxidase II (MT-CO2) induction. DLAT signaling is elevated in cancer patients refractory to chemotherapy or chemoimmunotherapy. A decoy peptide DMp39, designed to target DLAT signaling, effectively sensitizes cancer cells to cisplatin in patient-derived xenograft models. Collectively, our study reveals the crucial role of DLAT in shaping chemotherapy resistance, which involves an interplay between acetylation signaling and metabolic reprogramming, and offers a unique decoy peptide technology to overcome chemotherapy resistance. Chemotherapy is commonly used for the treatment of cancer but resistance often occurs. Here, the authors identify mitochondrial DLAT as driver of chemotherapy resistance via interaction with (MTHFD2) and develop a decoy peptide which blocks this interaction, restoring sensitivity to chemotherapy.

Recent advances in cryo-electron microscopy (cryo-EM) have facilitated the high-resolution structural determination of macromolecular complexes in their native states, providing valuable insights into their dynamic behaviors. However, insufficient understanding or experience with the cryo-EM image processing parameters can result in the loss of biological meaning. In this paper, we investigate the dihydrolipoyl acetyltransferase (E2) inner core complex of the pyruvate dehydrogenase complex (PDC) and reconstruct the 3D maps using five different symmetry parameters. The results demonstrate that the reconstructions yield structurally identical 3D models even at a near-atomic structure. This finding underscores a crucial message for researchers engaging in single-particle analysis (SPA) with relatively user-friendly and convenient image processing software. This approach helps reduce the risk of missing critical biological details, such as the dynamic properties of macromolecules.

The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis.

The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC.

The study of protein-protein interactions is an essential process to understand the biological functions of proteins and the underlying mechanisms. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) is one of the most extensively used high-throughput techniques to discover novel protein-protein interactions. However, the traditional CoIP process uses whole cell lysate, disrupts cellular organization, and leads to potential false positives by inducing artificial protein-protein interactions. Here, we have developed a strategy by combining subcellular fractionation with CoIP-MS to study the interacting proteins of the complement component 1, q subcomponent binding protein (C1QBP) in the mitochondria. Using this method, a novel C1QBP interacting protein, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (DLAT) was identified and validated. Furthermore, the activity of the pyruvate dehydrogenase (PDH) was found to be affected by the expression level of C1QBP. These results provide novel insights regarding the mitochondrial function of C1QBP in the regulation of cellular energy metabolism. This method could also be used to analyze the subcellular protein-protein interactions for other proteins of interest.