Cryo-EM structures of engineered active bc1-cbb3 type CIII2CIV super-complexes and electronic communication between the complexes

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.

Identification of Key Pathways and Genes in Polycystic Ovary Syndrome via Integrated Bioinformatics Analysis and Prediction of Small Therapeutic Molecules

To add a enhance understanding of polycystic ovary syndrome (PCOS) at the molecular level, this investigation aimed to find the genes and crucial pathways linked with PCOS by using integrated bioinformatics analysis. Based on the expression profiling by high throughput sequencing data GSE84958 derived from the Gene Expression Omnibus, the differentially expressed genes (DEGs) between PCOS samples and normal controls were identified. With DEGs, we performed a series of functional enrichment analyses. Then, a protein–protein interaction (PPI) network, miRNA - target genes and TF - target gene networks were constructed and visualized, with which the hub gene nodes were screened out. Finally, validation of hub genes was performed by using receiver operating characteristic (ROC) and RT-PCR. Molecular docking studies performed. A total of 739 DEGs were screened out, among which 360 genes were up regulated and 379 genes were down regulated. GO enrichment analysis indicated that up regulated genes were mainly involved in peptide metabolic process, organelle envelope and RNA binding and the down regulated genes were significantly enriched in plasma membrane bounded cell projection organization, neuron projection and DNA-binding transcription factor activity, RNA polymerase II-specific. REACTOME pathway enrichment analysis showed that the up regulated genes were mainly enriched in translation and respiratory electron transport and the down regulated genes were mainly enriched in generic transcription pathway and transmembrane transport of small molecules. The top 10 hub genes in the constructed PPI network, miRNA - target gene network and TF - target gene network were SAA1, ADCY6, POLR2K, RPS15, RPS15A, CTNND1, ESR1, NEDD4L, KNTC1 and NGFR. The modules analysis showed that genes in the top 2 significant modules of PPI network were mainly associated with respiratory electron transport and signalling by NGF, respectively. We find a series of crucial genes along with the pathways that were most closely related with PCOS initiation and advancement. Our investigations provide a more detailed molecular mechanism for the progression of PCOS, detail information on the potential biomarkers and therapeutic targets.

Post-Translational Modifications of Cytochrome c in Cell Life and Disease

Mitochondria are the powerhouses of the cell, whilst their malfunction is related to several human pathologies, including neurodegenerative diseases, cardiovascular diseases, and various types of cancer. In mitochondrial metabolism, cytochrome c is a small soluble heme protein that acts as an essential redox carrier in the respiratory electron transport chain. However, cytochrome c is likewise an essential protein in the cytoplasm acting as an activator of programmed cell death. Such a dual role of cytochrome c in cell life and death is indeed fine-regulated by a wide variety of protein post-translational modifications. In this work, we show how these modifications can alter cytochrome c structure and functionality, thus emerging as a control mechanism of cell metabolism but also as a key element in development and prevention of pathologies.

Cryo-EM Structures of Respiratory bc1-cbb3 type CIII2CIV Supercomplex and Electronic Communication Between the Complexes

The respiratory electron transport complexes convey electrons from nutrients to oxygen and generate a proton-motive force used for energy (ATP) production in cells. These enzymes are conserved among organisms, and organized as individual complexes or combined forming large super-complexes (SC). Bacterial electron transport pathways are more branched than those of mitochondria and contain multiple variants of such complexes depending on their growth modes. The Gram-negative species deploy a mitochondrial-like cytochrome bc1 (Complex III, CIII2), and may have bacteria-specific cbb3-type cytochrome c oxidases (Complex IV, CIV) in addition to, or instead of, the canonical aa3-type CIV. Electron transfer between these complexes is mediated by two different carriers: the soluble cytochrome c2 which is similar to mitochondrial cytochrome c and the membrane-anchored cytochrome cy which is unique to bacteria. Here, we report the first cryo-EM structure of a respiratory bc1-cbb3 type SC (CIII2CIV, 5.2Å resolution) and several conformers of native CIII2 (3.3Å resolution) from the Gram-negative bacterium Rhodobacter capsulatus. The SC contains all catalytic subunits and cofactors of CIII2 and CIV, as well as two extra transmembrane helices attributed to cytochrome cy and the assembly factor CcoH. Remarkably, some of the native CIII2 are structural heterodimers with different conformations of their [2Fe-2S] cluster-bearing domains. The unresolved cytochrome c domain of cy suggests that it is mobile, and it interacts with CIII2CIV differently than cytochrome c2. Distance requirements for electron transfer suggest that cytochrome cy and cytochrome c2 donate electrons to heme cp1 and heme cp2 of CIV, respectively. For the first time, the CIII2CIV architecture and its electronic connections establish the structural features of two separate respiratory electron transport pathways (membrane-confined and membrane-external) between its partners in Gram-negative bacteria.

Roles of Alanine Dehydrogenase and Induction of Its Gene inMycobacterium smegmatisunder Respiration-Inhibitory Conditions

ABSTRACTHere we demonstrated that the inhibition of electron flux through the respiratory electron transport chain (ETC) by either the disruption of the gene for the major terminal oxidase (aa3cytochromecoxidase) or treatment with KCN resulted in the induction ofaldencoding alanine dehydrogenase inMycobacterium smegmatis. A decrease in functionality of the ETC shifts the redox state of the NADH/NAD+pool toward a more reduced state, which in turn leads to an increase in cellular levels of alanine by Ald catalyzing the conversion of pyruvate to alanine with the concomitant oxidation of NADH to NAD+. The induction ofaldexpression under respiration-inhibitory conditions inM. smegmatisis mediated by the alanine-responsive AldR transcriptional regulator. The growth defect ofM. smegmatisby respiration inhibition was exacerbated by inactivation of thealdgene, suggesting that Ald is beneficial toM. smegmatisin its adaptation and survival under respiration-inhibitory conditions by maintaining NADH/NAD+homeostasis. The low susceptibility ofM. smegmatistobcc1complex inhibitors appears to be, at least in part, attributable to the high expression level of thebdquinol oxidase inM. smegmatiswhen thebcc1-aa3branch of the ETC is inactivated.IMPORTANCEWe demonstrated that the functionality of the respiratory electron transport chain is inversely related to the expression level of thealdgene encoding alanine dehydrogenase inMycobacterium smegmatis. Furthermore, the importance of Ald in NADH/NAD+homeostasis during the adaptation ofM. smegmatisto severe respiration-inhibitory conditions was demonstrated in this study. On the basis of these results, we propose that combinatory regimens including both an Ald-specific inhibitor and respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline are likely to enable a more efficient therapy for tuberculosis.