Supercomplex Research Articles

Inhibition mechanism of potential antituberculosis compound lansoprazole sulfide

Tuberculosis is one of the most common causes of death worldwide, with a rapid emergence of multi-drug-resistant strains underscoring the need for new antituberculosis drugs. Recent studies indicate that lansoprazole-a known gastric proton pump inhibitor and its intracellular metabolite, lansoprazole sulfide (LPZS)-are potential antituberculosis compounds. Yet, their inhibitory mechanism and site of action still remain unknown. Here, we combine biochemical, computational, and structural approaches to probe the interaction of LPZS with the respiratory chain supercomplex III2IV2 of Mycobacterium smegmatis, a close homolog of Mycobacterium tuberculosis supercomplex. We show that LPZS binds to the Qo cavity of the mycobacterial supercomplex, inhibiting the quinol substrate oxidation process and the activity of the enzyme. We solve high-resolution (2.6 Å) cryo-electron microscopy (cryo-EM) structures of the supercomplex with bound LPZS that together with microsecond molecular dynamics simulations, directed mutagenesis, and functional assays reveal key interactions that stabilize the inhibitor, but also how mutations can lead to the emergence of drug resistance. Our combined findings reveal an inhibitory mechanism of LPZS and provide a structural basis for drug development against tuberculosis.

HADHA Regulates Respiratory Complex Assembly and Couples FAO and OXPHOS.

Oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) are key bioenergetics pathways. The machineries for both processes are localized in mitochondria. Secondary OXPHOS defects have been documented in patients with primary FAO deficiencies, and vice versa. However, the underlying mechanisms remain unclear. Intrigued by the observations that regulation of supercomplexes (SCs) assembly in a mouse OXPHOS deficient cell line and its derivatives is associated with the changes in lipid metabolism, a proteomics analysis is carried out and identified mitochondrial trifunctional protein (MTP) subunit alpha (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, HADHA) as a potential regulatory factor for SCs assembly. HADHA-Knockdown cells and mouse embryonic fibroblasts (MEFs) derived from HADHA-Knockout mice displayed both reduced SCs assembly and defective OXPHOS. Stimulation of OXPHOS induced in cell culture by replacing glucose with galactose and of lipid metabolism in mice with a high-fat diet (HFD) both exhibited increased HADHA expression. HADHA Heterozygous mice fed with HFD showed enhanced steatosis associated with a reduction of SCs assembly and OXPHOS function. The results indicate that HADHA participates in SCs assembly and couples FAO and OXPHOS.

Inhibition of the FOXO1–ROCK1 axis mitigates cardiomyocyte injury under chronic hypoxia in Tetralogy of Fallot by maintaining mitochondrial quality control

IntroductionPersistent chronic myocardial hypoxia causes disturbances in mitochondrial quality control (MQC), ultimately leading to increased cardiomyocyte injury in patients with Tetralogy of Fallot (TOF). The present study aimed to identify the key effector molecules of cardiomyocyte injury under chronic hypoxia in TOF. MethodsClinical data from TOF patients were collected and whole transcriptome sequencing was performed on myocardial samples. Chronic hypoxia models were established in cardiac-specific knockout mice and cardiomyocytes, and a series of molecular experiments were used to determine the specific mechanisms involved. ResultsClinical cohort data and whole-transcriptome sequencing analysis of myocardial samples from TOF patients revealed that forkhead box O1 (FOXO1) plays an important role in chronic hypoxic cardiomyocyte injury. In a model of chronic hypoxia established in FOXO1 cardiac-specific knockout mice and FOXO1 gene-deficient cardiomyocytes, the AMPK signaling pathway regulates the expression of FOXO1, which in turn disrupts MQC by regulating the transcriptional activation of Rho-associated protein kinase 1 (ROCK1), and increasing the production of mitochondrial ROS, thereby exacerbating damage to cardiomyocytes. Excessive reactive oxygen species (ROS) production during MQC dysfunction further activates Cox7a2L to increase the assembly of the respiratory chain supercomplex. In addition, we found that miR-27b-3p partially binds to the 3′ untranslated region of FOXO1 to exert a protective effect. ConclusionsMaintenance of MQC under chronic hypoxia is achieved through a series of injury-protection mechanisms, suggesting that FOXO1 inhibition may be crucial for future mitigation of chronic hypoxic cardiomyocyte injury in TOF.

Structural basis of respiratory complex adaptation to cold temperatures.

In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain. Yet, the structural basis of respiratory complex adaptation upon cold exposure remains elusive. Herein, we combined thermoregulatory physiology and cryoelectron microscopy (cryo-EM) to study endogenous respiratory supercomplexes from mice exposed to different temperatures. A cold-induced conformation of CI:III2 (termed type 2) supercomplex was identified with a ∼25° rotation of CIII2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting catalytic states that favor electron transfer. Large-scale supercomplex simulations in mitochondrial membranes reveal how lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations, and biochemical analyses unveil the thermoregulatory mechanisms and dynamics of increased respiratory capacity in brown fat at the structural and energetic level.

Characterization of Shy1, the Schizosaccharomyces pombe homolog of human SURF1

Cytochrome c oxidase (complex IV) is the terminal enzyme in the mitochondrial respiratory chain. As a rare neurometabolic disorder caused by mutations in the human complex IV assembly factor SURF1, Leigh Syndrome (LS) is associated with complex IV deficiency. In this study, we comprehensively characterized Schizosaccharomyces pombe Shy1, the homolog of human SURF1. Bioinformatics analysis revealed that Shy1 contains a conserved SURF1 domain that links to the biogenesis of complex IV and shares high structural similarity with its homologs in Saccharomyces cerevisiae and humans. Our study showed that Shy1 is required for the expression of mtDNA-encoded genes and physically interacts with structural subunits and assembly factors of complex IV. Interestingly, Rip1, the subunit of ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex (complex III), can also co-immunoprecipitate with Shy1, suggesting Shy1 may be involved in the assembly of the mitochondrial respiratory chain supercomplexes. This conclusion is further corroborated by our BN-PAGE analysis. Unlike its homologs, deletion of shy1 does not critically disrupt respiratory chain assembly, indicating the presence of the compensatory mechanism(s) within S. pombe that ensure mitochondrial functionality. Collectively, our investigation elucidates that Shy1 plays a pivotal role in the sustainability of the regular function of mitochondria by participating in the assembly of complex IV in S. pombe.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Plant supercomplex I + III2 structure and function: implications for the growing field.

Mitochondrial respiration is major source of chemical energy for all free-living eukaryotes. Nevertheless, the mechanisms of the respiratory complexes and supercomplexes remain poorly understood. Here, I review recent structural and functional investigations of plant supercomplex I + III2 from Arabidopsis thaliana and Vigna radiata. I discuss commonalities, open questions and implications for complex I, complex III2 and supercomplexes in plants and non-plants. Studies across further clades will enhance our understanding of respiration and the potential universal mechanisms of its complexes and supercomplexes.

Respiratory complexes and supercomplexes of the model yeast S. cerevisiae

Respiratory complexes and supercomplexes of the model yeast S. cerevisiae

Inhibition and substrate binding site analysis of the obligate respiratory chain supercomplex of Actinobacteria

Inhibition and substrate binding site analysis of the obligate respiratory chain supercomplex of Actinobacteria

Cooperative assembly of respiratory supercomplexes

Cooperative assembly of respiratory supercomplexes

Respiratory supercomplexes act early to support complex I assembly

Respiratory supercomplexes act early to support complex I assembly

Respiratory supercomplexes act early to support complex I assembly

Respiratory supercomplexes act early to support complex I assembly

Detailed analysis of Mdivi-1 effects on complex I and respiratory supercomplex assembly

Several human diseases, including cancer and neurodegeneration, are associated with excessive mitochondrial fragmentation. In this context, mitochondrial division inhibitor (Mdivi-1) has been tested as a therapeutic to block the fission-related protein dynamin-like protein-1 (Drp1). Recent studies suggest that Mdivi-1 interferes with mitochondrial bioenergetics and complex I function. Here we show that the molecular mechanism of Mdivi-1 is based on inhibition of complex I at the IQ site. This leads to the destabilization of complex I, impairs the assembly of N- and Q-respirasomes, and is associated with increased ROS production and reduced efficiency of ATP generation. Second, the calcium homeostasis of cells is impaired, which for example affects the electrical activity of neurons. Given the results presented here, a potential therapeutic application of Mdivi-1 is challenging because of its potential impact on synaptic activity. Similar to the Complex I inhibitor rotenone, Mdivi-1 may lead to neurodegenerative effects in the long term.

EARLY NODULIN93 acts via cytochrome c oxidase to alter respiratory ATP production and root growth in plants.

EARLY NODULIN 93 (ENOD93) has been genetically associated with biological nitrogen fixation in legumes and nitrogen use efficiency in cereals, but its precise function is unknown. We show that hidden Markov models define ENOD93 as a homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 regulates cytochrome oxidase (CIV), influencing the generation of a mitochondrial proton motive force in yeast (Saccharomyces cerevisiae). Knockout of ENOD93 in Arabidopsis (Arabidopsis thaliana) causes a short root phenotype and early flowering. ENOD93 is associated with a protein complex the size of CIV in mitochondria, but neither CIV abundance nor its activity changed in ruptured organelles of enod93. However, a progressive loss of ADP-dependent respiration rate was observed in intact enod93 mitochondria, which could be recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 in a CIV-dependent manner, but ATP synthesis and ADP depletion rates progressively decreased. The respiration rate of whole enod93 seedlings was elevated, and root ADP content was nearly double that in wild type without a change in ATP content. We propose that ENOD93 and HYPOXIA-INDUCED GENE DOMAIN 2 (HIGD2) are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded by two distinct genes.

MIRO1 controls energy production and proliferation of smooth muscle cells.

The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown. An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC). MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I. Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.

Upstream alternative polyadenylation in SCN5A produces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration.

SCN5A encodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials. SCN5A gene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the first SCN5A coding exon. This yields a SCN5A-short transcript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-length SCN5A mRNA ratios in failing hearts. Knock-in of the human SCN5A APA sequence into mice is sufficient to enable expression of SCN5A -short transcript, while significantly decreasing expression of full-length SCN5A mRNA. Notably, SCN5A -short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation of SCN5A produces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.

Interactome of the HIV-1 proteome and human host RNA

The human immunodeficiency virus (HIV-1) is highly dependent on a variety of host factors. Beside proteins, host RNA molecules are reported to aid HIV-1 replication and latency maintenance. Here, we implement multiple workflows of native RNA immunoprecipitation and sequencing (nRIPseq) to determine direct host RNA interaction partners of all 18 HIV-1 (poly)proteins. We identify 1,727 HIV-1 protein – human RNA interactions in the Jurkat cell line and 1,558 interactions in SupT1 cells for a subset of proteins, and discover distinct cellular pathways that seem to be used or controlled by HIV-1 on the RNA level: Tat binds mRNAs of proteins involved in the super elongation complex (AFF1-4, Cyclin-T1). Correlation of the interaction scores (based on binding abundancy) allows identifying the highest confidence interactions, for which we perform a small-scale knockdown screen that leads to the identification of three HIV-1 protein binding RNA interactors involved in HIV-1 replication (AFF2, H4C9 and RPLP0).

Life cycle assessment of carbon emissions for cross-sea tunnel: A case study of Shenzhen-Zhongshan Bridge and Tunnel in China

Due to significant population concentration and capital influx in Guangdong-Hong Kong-Macao Greater Bay Area, the construction of cross-sea tunnels with significant consumption of various resources and materials, has been frequently witnessed. However, there is a lack of knowledge regarding how carbon emissions of cross-sea transportation infrastructure are generated across its life-cycle stages. This study proposes a life cycle assessment (LCA) approach for quantifying the carbon emissions and exploring the carbon reduction potentials with a case study of a world-renowned cross-sea tunnel project in Guangdong-Hong Kong-Macao Greater Bay Area. The results find that this project contributes approximately 849 kilotons CO2eq of carbon emissions with an emission intensity of 1.1 kilotons CO2eq per meter. The materialization stage is the largest contributor of carbon emissions (474.9 kilotons CO2eq), followed by service stage (248.3 kilotons CO2eq, accounting for 29.2 %). Some carbon emissions of raw materials can be offset by using recycled materials. The discarded concrete, block, stone, and sand, occupying over 90 % of the total recycled waste in weight could achieve a 93.5 % of carbon reduction potentially. It provides the opportunity to reveal the engineering details and carbon emission for a world-class super complex cross-sea transportation infrastructure. This study makes one of the first attempts to quantify life-cycle carbon emissions of cross-sea transportation infrastructure, which enriches foundational dataset for environmental impact assessment in this emerging field. The findings of this study can provide scientific references for formulating targeted low-carbon strategies for cross-sea transportation infrastructure across its different life-cycle stages.

MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription.

Loss-of-function mutations in methyl-CpG binding protein 2 ( MECP2 ) cause Rett syndrome, a postnatal neurodevelopmental disorder that occurs in ∼1/10,000 live female births. MeCP2 binds to methylated cytosines across genomic DNA and recruits various partners to regulate gene expression. MeCP2 has been shown to repress transcription in vitro and interacts with co-repressors such as the Sin3A and NCoR complexes. Based on these observations, MeCP2 has been largely considered as a repressor of transcription. However, a mouse model of RTT displays many down-regulated genes, and those same genes are up-regulated in a MECP2 duplication mouse model. Furthermore, TCF20, which has been associated with transcriptional activation, have recently been identified as a protein interactor of MeCP2. These data broaden the potential functions of MeCP2 as a regulator of gene expression. Yet, the molecular mechanisms underlying MeCP2-dependent gene regulation remain largely unknown. Here, using a human MECP2 gain-of-function Drosophila model, we screened for genetic modifiers of MECP2 -induced phenotypes. Our approach identified several subunits of the Drosophila super elongation complex, a P-TEFb containing RNA polymerase II (RNA pol II) elongation factor required for the release of promoter-proximally paused RNA pol II, as genetic interactors of MECP2 . We discovered that MeCP2 physically interacts with the SEC in human cells and in the mouse brain. Furthermore, we found that MeCP2 directly binds AFF4, the scaffold of the SEC, via the transcriptional repression domain. Finally, loss of MeCP2 in the mouse cortex caused reduced binding of AFF4 specifically on a subset of genes involved in the regulation of synaptic function, which also displayed the strongest decrease in RNA pol II binding in the genebody. Taken together, our study reveals a previously unrecognized mechanism through which MeCP2 regulates transcription, providing a new dimension to its regulatory role in gene expression.

High-intensity interval training using electrical stimulation ameliorates muscle fatigue in chronic kidney disease-related cachexia by restoring mitochondrial respiratory dysfunction.

Exercise, especially high-intensity interval training (HIIT), can increase mitochondrial respiratory capacity and enhance muscular endurance, but its systemic burden makes it difficult to safely and continuously prescribe for patients with chronic kidney disease (CKD)-related cachexia who are in poor general condition. In this study, we examined whether HIIT using electrical stimulation (ES), which does not require whole-body exercise, improves muscle endurance in the skeletal muscle of 5/6 nephrectomized rats, a widely used animal model for CKD-related cachexia. Male Wistar rats (10weeks old) were randomly assigned to a group of sham-operated (Sham) rats and a group of 5/6 nephrectomy (Nx) rats. HIIT was performed on plantar flexor muscles in vivo with supramaximal ES every other day for 4weeks to assess muscle endurance, myosin heavy-chain isoforms, and mitochondrial respiratory function in Nx rats. A single session was also performed to identify upstream signaling pathways altered by HIIT using ES. In the non-trained plantar flexor muscles from Nx rats, the muscle endurance was significantly lower than that in plantar flexor muscles from Sham rats. The proportion of myosin heavy chain IIa/x, mitochondrial content, mitochondrial respiratory capacity, and formation of mitochondrial respiratory supercomplexes in the plantaris muscle were also significantly decreased in the non-trained plantar flexor muscles from Nx rats than compared to those in plantar flexor muscles from Sham rats. Treatment with HIIT using ES for Nx rats significantly improved these molecular and functional changes to the same degrees as those in Sham rats. Furthermore, a single session of HIIT with ES significantly increased the phosphorylation levels of AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK), pathways that are essential for mitochondrial activation signaling by exercise, in the plantar muscles of both Nx and Sham rats. The findings suggest that HIIT using ES ameliorates muscle fatigue in Nx rats via restoration of mitochondrial respiratory dysfunction with activation of AMPK and p38 MAPK signaling. Our ES-based HIIT protocol can be performed without placing a burden on the whole body and be a promising intervention that is implemented even in conditions of reduced general performance status such as CKD-related cachexia.