Structural Basis of Mitochondrial Scaffolds by Prohibitin Complexes: Insight into a Role of the Coiled-Coil Region.

The coiled-coil motif mediates subunit oligomerization and scaffolding and underlies several fundamental biologic processes. Prohibitins (PHBs), mitochondrial inner membrane proteins involved in mitochondrial homeostasis and signal transduction, are predicted to have a coiled-coil motif, but their structural features are poorly understood. Here we solved the crystal structure of the heptad repeat (HR) region of PHB2 at 1.7-Å resolution, showing that it assembles into a dimeric, antiparallel coiled-coil with a unique negatively charged area essential for the PHB interactome in mitochondria. Disruption of the HR coiled-coil abolishes well-ordered PHB complexes and the mitochondrial tubular networks accompanying PHB-dependent signaling. Using a proximity-dependent biotin identification (BioID) technique in live cells, we mapped a number of mitochondrial intermembrane space proteins whose association with PHB2 relies on the HR coiled-coil region. Elucidation of the PHB complex structure in mitochondria provides insight into essential PHB interactomes required for mitochondrial dynamics as well as signal transduction.

RLR-mediated antiviral innate immunity requires oxidative phosphorylation activity.

Mitochondria act as a platform for antiviral innate immunity, and the immune system depends on activation of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) signaling pathway via an adaptor molecule, mitochondrial antiviral signaling. We report that RLR-mediated antiviral innate immunity requires oxidative phosphorylation (OXPHOS) activity, a prominent physiologic function of mitochondria. Cells lacking mitochondrial DNA or mutant cells with respiratory defects exhibited severely impaired virus-induced induction of interferons and proinflammatory cytokines. Recovery of the OXPHOS activity in these mutants, however, re-established RLR-mediated signal transduction. Using in vivo approaches, we found that mice with OXPHOS defects were highly susceptible to viral infection and exhibited significant lung inflammation. Studies to elucidate the molecular mechanism of OXPHOS-coupled immune activity revealed that optic atrophy 1, a mediator of mitochondrial fusion, contributes to regulate the antiviral immune response. Our findings provide evidence for functional coordination between RLR-mediated antiviral innate immunity and the mitochondrial energy-generating system in mammals.

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES.

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro(103). A molecular dynamics simulation and mutational analyses revealed that Arg(40) in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.

Interaction between Viral Proteins and Mitochondria.

Recent advances reveal that mitochondria are not limited to functioning only as the cellular powerhouse and in apoptosis, but that they act as central hubs for multiple signal transductions. Studies over the last decade indicate that mitochondria in vertebrates are involved in the front line of host defense, especially against RNA viruses. Mitochondrial-mediated antiviral innate immunity depends on activation of the retinoic acid-inducible gene I (RIG-I)-like receptors signal transduction pathway, and the mitochondrial surface acts as a platform for the assembly of signaling molecules, including mitochondrial antiviral signaling (MAVS) during the process. Some viral encoded proteins target to the mitochondria post-infection, however, thereby evading the cellular immune response. Here we review specific interactions between mitochondria and viral proteins and discuss their physiologic effects on the host cells.

Influenza A virus protein PB1-F2 translocates into mitochondria via Tom40 channels and impairs innate immunity.

Mitochondria contribute to cellular innate immunity against RNA viruses. Mitochondrial-mediated innate immunity is regulated by signalling molecules that are recruited to the mitochondrial membrane, and depends on the mitochondrial inner membrane potential (Δψm). Here we examine the physiological relevance of Δψm and the mitochondrial-associating influenza A viral protein PB1-F2 in innate immunity. When expressed in host cells, PB1-F2 completely translocates into the mitochondrial inner membrane space via Tom40 channels, and its accumulation accelerates mitochondrial fragmentation due to reduced Δψm. By contrast, PB1-F2 variants lacking a C-terminal polypeptide, which is frequently found in low pathogenic subtypes, do not affect mitochondrial function. PB1-F2-mediated attenuation of Δψm suppresses the RIG-I signalling pathway and activation of NLRP3 inflammasomes. PB1-F2 translocation into mitochondria strongly correlates with impaired cellular innate immunity, making this translocation event a potential therapeutic target.

A structural perspective of the MAVS-regulatory mechanism on the mitochondrial outer membrane using bioluminescence resonance energy transfer.

In most eukaryotic cells, mitochondria have various essential roles for proper cell function, such as energy production, and in mammals mitochondria also act as a platform for antiviral innate immunity. Mitochondrial-mediated antiviral immunity depends on the activation of the cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathway, and on the participation of mitochondrial antiviral signaling (MAVS), which is localized on the mitochondrial outer membrane. After RNA virus infection, RLRs translocate to the mitochondrial surface to interact with MAVS, and the adaptor protein undergoes a conformational change that is essential for downstream signaling, although its structural features are poorly understood. Here we examined the MAVS-regulatory mechanism on the mitochondrial outer membrane using bioluminescence resonance energy transfer (BRET) in live cells. Using a combination of BRET and functional analysis, we found that the activated MAVS conformation is a highly ordered oligomer, at least more than three molecules per complex unit on the membrane. Hepatitis C virus NS3/4A protease and mitofusin 2, which are known MAVS inhibitors, interfere with MAVS homotypic oligomerization in a distinct manner, each differentially altering the active conformation of MAVS. Our results reveal structural features underlying the precise regulation of MAVS signaling on the mitochondrial outer membrane, and may provide insight into other signaling systems involving organelles.