Alternative splicing of Mff regulates AMPK-mediated phosphorylation, mitochondrial fission and antiviral response.

Mitochondrial morphology and function change dynamically in response to intracellular signaling and the surrounding environment. The mitochondrial fission factor Mff, which localizes to the outer mitochondrial membrane, mediates not only mitochondrial fission by recruiting the dynamin-related GTPase Drp1 to mitochondrial fission sites but also the double-stranded RNA-induced antiviral response on mitochondria through mitochondrial antiviral signaling (MAVS). Mff is reported to be regulated by AMP-activated protein kinase (AMPK)-mediated protein phosphorylation and alternative pre-mRNA splicing; however, the relationships among RNA splicing, phosphorylation, and multiple functions of Mff have not been fully understood. Here, we showed that mouse Mff has a tissue-specific splicing pattern, and at least eight Mff splice isoforms were expressed in mouse embryonic fibroblasts (MEFs). We introduced single Mff isoforms into Mff knockout MEFs and found that insertion of exon 6 just after the phosphorylation site, by the alternative splicing, reduced its phosphorylation by AMPK and its functions in mitochondrial fission and the antiviral response. In addition, the underlying mechanism repressing these functions was independent of phosphorylation. These results indicate that multiple functions of Mff on mitochondria are regulated by AMPK-mediated phosphorylation and alternative splicing, under the control of energy metabolism and cellular differentiation.

Insights into the regulation of mitochondrial functions by protein kinase A-mediated phosphorylation.

Cyclic AMP (cAMP)-protein kinase A (PKA) signaling is a highly conserved pathway in eukaryotes and plays a central role in cell signaling cascades in response to environmental changes. Elevated cAMP levels promote the activation of PKA, which phosphorylates various downstream proteins. Many cytosolic and nuclear proteins, such as metabolic enzymes and transcriptional factors, have been identified as substrates for PKA, suggesting that PKA-mediated regulation occurs predominantly in the cytosol. Mitochondrial proteins are also phosphorylated by PKA, and PKA-mediated phosphorylation of mitochondrial proteins is considered to control a variety of mitochondrial functions, including oxidative phosphorylation, protein import, morphology and quality control. In this review, we outline PKA mitochondrial substrates and summarize the regulation of mitochondrial functions through PKA-mediated phosphorylation.

Neuronal MML-1/MXL-2 regulates systemic aging via glutamate transporter and cell nonautonomous autophagic and peroxidase activity.

Accumulating evidence has demonstrated the presence of intertissue-communication regulating systemic aging, but the underlying molecular network has not been fully explored. We and others previously showed that two basic helix-loop-helix transcription factors, MML-1 and HLH-30, are required for lifespan extension in several longevity paradigms, including germlineless Caenorhabditis elegans. However, it is unknown what tissues these factors target to promote longevity. Here, using tissue-specific knockdown experiments, we found that MML-1 and its heterodimer partners MXL-2 and HLH-30 act primarily in neurons to extend longevity in germlineless animals. Interestingly, however, the downstream cascades of MML-1 in neurons were distinct from those of HLH-30. Neuronal RNA interference (RNAi)-based transcriptome analysis revealed that the glutamate transporter GLT-5 is a downstream target of MML-1 but not HLH-30. Furthermore, the MML-1-GTL-5 axis in neurons is critical to prevent an age-dependent collapse of proteostasis and increased oxidative stress through autophagy and peroxidase MLT-7, respectively, in long-lived animals. Collectively, our study revealed that systemic aging is regulated by a molecular network involving neuronal MML-1 function in both neural and peripheral tissues.

TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.

PINK1 is activated by autophosphorylation and forms a high-molecular-weight complex, thereby initiating the selective removal of damaged mitochondria by autophagy. Other than translocase of the outer mitochondrial membrane complexes, members of PINK1-containing protein complexes remain obscure. By mass spectrometric analysis of PINK1 co-immunoprecipitates, we identify the inner membrane protein TIM23 as a component of the PINK1 complex. TIM23 downregulation decreases PINK1 levels and significantly delays autophosphorylation, indicating that TIM23 promotes PINK1 accumulation in response to depolarization. Moreover, inactivation of the mitochondrial protease OMA1 not only enhances PINK1 accumulation but also represses the reduction in PINK1 levels induced by TIM23 downregulation, suggesting that TIM23 facilitates PINK1 activation by safeguarding against degradation by OMA1. Indeed, deficiencies of pathogenic PINK1 mutants that fail to interact with TIM23 are partially restored by OMA1 inactivation. These findings indicate that TIM23 plays a distinct role in activating mitochondrial autophagy by protecting PINK1.

Role of Monomer/Tetramer Equilibrium of Rod Visual Arrestin in the Interaction with Phosphorylated Rhodopsin.

The phototransduction cascade in vertebrate rod visual cells is initiated by the photoactivation of rhodopsin, which enables the activation of the visual G protein transducin. It is terminated by the phosphorylation of rhodopsin, followed by the binding of arrestin. Here we measured the solution X-ray scattering of nanodiscs containing rhodopsin in the presence of rod arrestin to directly observe the formation of the rhodopsin/arrestin complex. Although arrestin self-associates to form a tetramer at physiological concentrations, it was found that arrestin binds to phosphorylated and photoactivated rhodopsin at 1:1 stoichiometry. In contrast, no complex formation was observed for unphosphorylated rhodopsin upon photoactivation, even at physiological arrestin concentrations, suggesting that the constitutive activity of rod arrestin is sufficiently low. UV-visible spectroscopy demonstrated that the rate of the formation of the rhodopsin/arrestin complex well correlates with the concentration of arrestin monomer rather than the tetramer. These findings indicate that arrestin monomer, whose concentration is almost constant due to the equilibrium with the tetramer, binds to phosphorylated rhodopsin. The arrestin tetramer would act as a reservoir of monomer to compensate for the large changes in arrestin concentration in rod cells caused by intense light or adaptation.

A phase retrieval algorithm for triply periodic minimal surface like structures.

A method to solve the crystallographic phase problem of materials with triply periodic minimal surface like structures, such as lyotropic liquid crystal bicontinuous cubic phases, is reported. In triply periodic minimal surface like structures, the difference between the maximum and minimum electron densities tends to be the smallest for the true phase combination among the possible combinations [Oka (2022). Acta Cryst. A78, 430-436]. Using this feature, a new iterative phase retrieval algorithm for structure determination was developed. The algorithm modifies electron densities outside the upper and lower thresholds in the iterative Fourier transformation process with fixed amplitudes for the structure factors, and efficiently searches for the structure with the smallest difference between the maximum and minimum electron densities. The proper structure was determined by this algorithm for all tested data for lyotropic liquid crystal bicontinuous cubic phases and mesoporous silicas. Although some cases required constraints such as the volume fraction for structure determination, more than half could be determined without any constraints, including space groups.

Aggregation structure of chiral cubic liquid crystals revealed by X-ray diffraction utilizing a new algorithm.

Chiral aggregation structure spontaneously formed by achiral rodlike molecules, a long-time unsolved problem in liquid crystal science, has been clarified by applying a new crystallographic algorithm recently developed while utilizing aggregation characteristics of this type. Bicontinuously interwoven networks characterize it similarly to the neighboring Gyroid phase in a phase diagram against the alkyl chain length and temperature. However, the network connectivity is significantly different from the bicontinuous networks that have been either known for related compounds or assumed for this phase. The network is compatible with the homochiral arrangement of rodlike molecules with successive twists by a proper angle between adjacent junctions.

Crystallographic phase retrieval method for liquid crystal bicontinuous phases: indicator-based method.

An indicator-based crystallographic phase retrieval method has been developed for diffraction data of bicontinuous cubic phases of lyotropic liquid crystals. Such liquid crystals have large structural disorder; the number of independent Bragg reflections that can be observed is limited. This paper proposes two indicators to identify plausible combination(s) of crystallographic phases, i.e. electron-density distribution. The indicators are based on the characteristics of the liquid crystals: amphiphilic molecules diffuse mainly in the direction parallel to polar-nonpolar interfaces and the electron density in the direction parallel to the interfaces is averaged. One indicator is the difference between the maximum and minimum electron density, and the other is calculated from the Hessian matrix of the electron density. Using test data, the electron densities were calculated for all possible phase combinations and indicators were obtained. The results indicated that the electron densities with the minimum indicators were close to the true electron density. Therefore, this method is effective for phase retrieval. The accuracy of the phase retrieval decreased when the volume fraction of the region including the triply periodic minimal surface increased.

Localization of myoglobin in mitochondria: implication in regulation of mitochondrial respiration in rat skeletal muscle.

Mitochondria play a principal role in metabolism, and mitochondrial respiration is an important process for producing adenosine triphosphate. Recently, we showed the possibility that the muscle-specific protein myoglobin (Mb) interacts with mitochondrial complex IV to augment the respiration capacity in skeletal muscles. However, the precise mechanism for the Mb-mediated upregulation remains under debate. The aim of this study was to ascertain whether Mb is truly integrated into the mitochondria of skeletal muscle and to investigate the submitochondrial localization. Isolated mitochondria from rat gastrocnemius muscle were subjected to different proteinase K (PK) concentrations to digest proteins interacting with the outer membrane. Western blotting analysis revealed that the PK digested translocase of outer mitochondrial membrane 20 (Tom20), and the immunoreactivity of Tom20 decreased with the amount of PK used. However, the immunoreactivity of Mb with PK treatment was better preserved, indicating that Mb is integrated into the mitochondria of skeletal muscle. The mitochondrial protease protection assay experiments suggested that Mb localizes within the mitochondria in the inner membrane from the intermembrane space side. These results strongly suggest that Mb inside muscle mitochondria could be implicated in the regulation of mitochondrial respiration via complex IV.

Polar-Nonpolar Interfaces of Normal Bicontinuous Cubic Phases in Nonionic Surfactant/Water Systems Are Parallel to the Gyroid Surface.

We investigated the structures of normal (type I) bicontinuous cubic phases in hexa-, hepta-, and octaethylene glycol dodecyl ether/water mixtures by small-angle X-ray crystallography of single-crystal domains. Reconstructed electron densities showed that the hydrophilic chains with high electron density are confined to a film centered on the surface of the Gyroid (a triply periodic minimal surface), while hydrophobic chains with low electron density are distributed within the pair of interwoven labyrinths carved out by the Gyroid. Further, the local minimum within the high electron density region, due to bulk water, coincides precisely with the Gyroid. This minimum is less pronounced in mixtures with longer ethylene glycol chains, consistent with their decreased water content. Our analysis clearly shows that the polar-nonpolar interfaces are parallel to the Gyroid surface in all mixtures. The repulsive hydration or overlapping force between the pair of facing monolayers of ethylene glycol chains on either side of the Gyroid surface is the likely origin of the parallel interfaces.

The mitochondrial inner membrane protein LETM1 modulates cristae organization through its LETM domain.

LETM1 is a mitochondrial inner membrane protein that is required for maintaining the mitochondrial morphology and cristae structures, and regulates mitochondrial ion homeostasis. Here we report a role of LETM1 in the organization of cristae structures. We identified four amino acid residues of human LETM1 that are crucial for complementation of the growth deficiency caused by gene deletion of a yeast LETM1 orthologue. Substituting amino acid residues with alanine disrupts the correct assembly of a protein complex containing LETM1 and prevents changes in the mitochondrial morphology induced by exogenous LETM1 expression. Moreover, the LETM1 protein changes the shapes of the membranes of in vitro-reconstituted proteoliposomes, leading to the formation of invaginated membrane structures on artificial liposomes. LETM1 mutant proteins with alanine substitutions fail to facilitate the formation of invaginated membrane structures, suggesting that LETM1 plays a fundamental role in the organization of mitochondrial membrane morphology.

Conformational Differences among Metarhodopsin I, Metarhodopsin II, and Opsin Probed by Wide-Angle X-ray Scattering.

Among the photoproducts of vertebrate rhodopsin, only metarhodopsin II (Meta-II) preferentially adopts the active structure in which transmembrane helices are rearranged. Light-induced helical rearrangement of rhodopsin in membrane-embedded form was directly monitored by wide-angle X-ray scattering (WAXS) using nanodiscs. The change in the WAXS curve for the formation of Meta-II was characterized by a peak at 0.2 Å-1 and a valley at 0.6 Å-1, which were not observed in metarhodopsin I and opsin. However, acid-induced active opsin (Opsin*) showed a 0.2 Å-1 peak, but no 0.6 Å-1 valley. Analyses using the model structures based on the crystal structures of dark state and Meta-II suggest that the outward movement of helix VI occurred in Opsin*. However, the displaced helices III and V in Meta-II resulting from the disruption of cytoplasmic ionic lock were restored in Opsin*, which is likely to destabilize the G-protein-activating structure of opsin.

Functional and structural characterization of HspB1/Hsp27 from Chinese hamster ovary cells.

Small heat shock proteins (sHsps) endow cells with stress tolerance. Of the various sHsps in mammals, HspB1, also known as Hsp27, is the most ubiquitous. To examine the structure and function of HspB1, we expressed, purified, and characterized HspB1 from Chinese hamster (Cricetulus griseus) ovary cells (CgHspB1). CgHspB1 forms a large oligomeric structure. We observed a monodisperse 16-mer with an elongated sphere, but this is affected by changes in various conditions, including temperature. Under dilute conditions, CgHspB1 dissociates into small oligomers at elevated temperatures. The dissociated conformers interacted with the gel filtration column through hydrophobic interactions. In contrast, dissociation of the oligomer was not observed by small-angle X-ray scattering at 55 °C. The result partially coincides with the results of size exclusion chromatography, showing that dissociation did not occur at high protein concentrations. However, a significant structural change in the oligomeric conformations appears to occur between room and higher temperatures. Reflecting their status as homeotherms, mammalian sHsps are regulated by phosphorylation. A phosphorylation mimic mutant of CgHspB1 with the replacement of Ser15 to Asp exhibited relatively lower oligomer stability and greater protective ability against thermal aggregation than the wild-type protein. The result clearly shows a correlation between oligomer dissociation and chaperone activity.

Concentration of mitochondrial DNA mutations by cytoplasmic transfer from platelets to cultured mouse cells.

Accumulation of mutations in mitochondrial DNA (mtDNA) is thought to be responsible for mitochondrial, and other, diseases and biological phenomena, such as diabetes, cancer, neurodegenerative diseases, and aging. Mouse models may elucidate the relationship between mutations in mtDNA and these abnormalities. However, because of the difficulty of mtDNA manipulation, generation of mouse models has not sufficiently progressed to enable such studies. To overcome this difficulty and to establish a source of diverse mtDNA mutations, we here generated cultured mouse cells containing mtDNA derived from an mtDNA mutator mouse that accumulates random mtDNA mutations with age. Mutation analysis of the obtained transmitochondrial cytoplasmic hybrid cells (cybrids) revealed that the cells harbored diverse mtDNA mutations occurring at a higher frequency than in mouse tissues, and exhibited severe respiration defects that would be lethal in tissues or organs. Abnormal respiratory complex formation and high stress on the mitochondrial protein quality control system appeared to be involved in these severe respiration defects. The mutation rates of the majority of highly accumulated mutations converged to either approximately 5%, 10%, or 40%, suggesting that these mutations are linked on the respective mtDNA molecules, and mtDNA in cybrid cells likely consisted of mtDNA molecules clonally expanded from the small population of introduced mtDNAs. Thus, the linked mutations in these cybrid cells cannot be evaluated individually. In addition, mtDNA mutations homologous to confirmed pathogenic mutations in human were rarely observed in our generated cybrids. However, the transmitochondrial cybrids constitute a useful tool for concentrating pathogenic mtDNA mutations and as a source of diverse mtDNA mutations to elucidate the relationship between mtDNA mutations and diseases.

In vitro synthesis of the human calcium transporter Letm1 within cell-sized liposomes and investigation of its lipid dependency.

The human mitochondrion-derived calcium transporter Letm1 was synthesized by reconstituted in vitro transcription-translation (IVTT) in cell-sized liposomes and the dependency of Letm1 on phospholipid composition was investigated. Components for IVTT were encapsulated into cell-sized vesicles together with the DNA encoding Letm1, thereby preparing proteoliposomes. The synthesis of Letm1 and pH-dependent calcium transport activity were confirmed by flow cytometry. Finally, we investigated the effect of phospholipid composition on Letm1 transport activity and found that cardiolipin present in the mitochondrial membrane plays an important role on the transport activity of Letm1.

Polar-Nonpolar Interfaces of Inverse Bicontinuous Cubic Phases in Phytantriol/Water System are Parallel to Triply Periodic Minimal Surfaces.

We investigated two distinct lyotropic liquid crystal inverse bicontinuous cubic phases of phytantriol/water mixtures by small-angle X-ray crystallography of the single-crystal regions. Reconstructed electron density maps revealed hydrophilic head and hydrophobic tail regions of the phytantriol bilayer membranes and water regions. The bilayer membranes are shown to be located on the D and gyroid triply periodic minimal surfaces. To investigate the structures of the polar-nonpolar interfaces, we optimized two models: a parallel surface model and a constant mean curvature surface model. The parallel surface model agreed well with the X-ray data, and the R factors, which show the degree of agreement between those structural models and the data, were less than 0.04. In stark contrast, the constant mean curvature surface model deviated significantly from the data, and the R factors were around 0.15. We therefore conclude that the polar-nonpolar interface of the inverse bicontinuous cubic phase of the phytantriol/water system is close to a parallel surface to a triply periodic minimal surface.

Structural insights into ubiquitin phosphorylation by PINK1.

Mutations of PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin (Ub) ligase parkin can cause familial parkinsonism. These two proteins are essential for ubiquitylation of damaged mitochondria and subsequent degradation. PINK1 phosphorylates Ser65 of Ub and the Ub-like (UBL) domain of parkin to allosterically relieve the autoinhibition of parkin. To understand the structural mechanism of the Ub/UBL-specific phosphorylation by PINK1, we determined the crystal structure of Tribolium castaneum PINK1 kinase domain (TcPINK1) in complex with a nonhydrolyzable ATP analogue at 2.5 Å resolution. TcPINK1 consists of the N- and C-terminal lobes with the PINK1-specific extension. The ATP analogue is bound in the cleft between the N- and C-terminal lobes. The adenine ring of the ATP analogue is bound to a hydrophobic pocket, whereas the triphosphate group of the ATP analogue and two coordinated Mg ions interact with the catalytic hydrophilic residues. Comparison with protein kinases A and C (PKA and PKC, respectively) unveils a putative Ub/UBL-binding groove, which is wider than the peptide-binding groove of PKA or PKC to accommodate the globular head of Ub or UBL. Further crosslinking analyses suggested a PINK1-interacting surface of Ub. Structure-guided mutational analyses support the findings from the present structural analysis of PINK1.

Physicochemical Properties of the Mammalian Molecular Chaperone HSP60.

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

Inactivation of cardiolipin synthase triggers changes in mitochondrial morphology.

Mitochondrial tubular structures are maintained by a balance between membrane fusion and fission that is regulated by various factors, including Drp1 and mitofusin/fzo-1. Here we report the role of cardiolipin (CL) synthase in the regulation of mitochondrial morphology. Knockdown of CL synthase induced mitochondrial elongation in nematode and human cells. Knockdown of both nematode cardiolipin synthase and drp-1 or fzo-1 suggested that knocking down CL synthase decreases mitochondrial division. Mass spectrometric analysis of human CL synthase-knocked down cells revealed a decreased amount of CL and an accumulation of phosphatidylglycerol, a CL precursor. Knockdown of other genes involved in CL synthesis did not influence mitochondrial morphology. Thus, mitochondrial elongation may result from the accumulation of phosphatidylglycerol rather than decreased CL.

Small-Angle X-ray Crystallography on Single-Crystal Regions of Inverse Bicontinuous Cubic Phases: Lipid Bilayer Structures and Gaussian Curvature-Dependent Fluctuations.

I report for the first time an X-ray crystallographic study on single-crystal regions of cubic phases of a lyotropic liquid crystal. The single-crystal regions of three inverse bicontinuous cubic phases of a lipid, monoolein, diffracted X-ray only in small-angle regions, but amplitudes of structure factors were determined from the small-angle X-ray diffraction data with high accuracy. Structure factors from lipid bilayer models with constant thickness were optimized to amplitudes obtained from the X-ray data. By using amplitudes of the structure factors from X-ray data and phases from the models, electron density maps of three cubic phases were reconstructed. Lipid bilayer membranes, consisting of high density head regions and low density tail regions, were clearly distinguished in the electron density maps. Water regions had slightly lower density than that of the lipid head regions and were clearly visible for two of the cubic phases. Centers of bilayer membranes were located on the corresponding triply periodic minimal surfaces in the maps. Electron density data indicated Gaussian curvature-dependent fluctuations of bilayer membranes: the smaller the Gaussian curvature is, the larger the fluctuation becomes. The technique described in this report is expected to bring new knowledge in the structural research of lyotropic liquid crystals.