Autoimmune disorders associated with gain of function of the intracellular sensor MDA5.

MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5. These findings provide insight into the association between disorders of the innate immune system and autoimmunity.

Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes.

Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-ß induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.

Single-Molecule/Cell Analyses Reveal Principles of Genome-Folding Mechanisms in the Three Domains of Life.

Comparative structural/molecular biology by single-molecule analyses combined with single-cell dissection, mass spectroscopy, and biochemical reconstitution have been powerful tools for elucidating the mechanisms underlying genome DNA folding. All genomes in the three domains of life undergo stepwise folding from DNA to 30-40 nm fibers. Major protein players are histone (Eukarya and Archaea), Alba (Archaea), and HU (Bacteria) for fundamental structural units of the genome. In Euryarchaeota, a major archaeal phylum, either histone or HTa (the bacterial HU homolog) were found to wrap DNA. This finding divides archaea into two groups: those that use DNA-wrapping as the fundamental step in genome folding and those that do not. Archaeal transcription factor-like protein TrmBL2 has been suggested to be involved in genome folding and repression of horizontally acquired genes, similar to bacterial H-NS protein. Evolutionarily divergent SMC proteins contribute to the establishment of higher-order structures. Recent results are presented, including the use of Hi-C technology to reveal that archaeal SMC proteins are involved in higher-order genome folding, and the use of single-molecule tracking to reveal the detailed functions of bacterial and eukaryotic SMC proteins. Here, we highlight the similarities and differences in the DNA-folding mechanisms in the three domains of life.

N-terminal dual lipidation-coupled molecular targeting into the primary cilium.

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders.

Visualization of conformational variability in the domains of long single-stranded RNA molecules.

We demonstrate an application of atomic force microscopy (AFM) for the structural analysis of long single-stranded RNA (>1 kb), focusing on 28S ribosomal RNA (rRNA). Generally, optimization of the conditions required to obtain three-dimensional (3D) structures of long RNA molecules is a challenging or nearly impossible process. In this study, we overcome these limitations by developing a method using AFM imaging combined with automated, MATLAB-based image analysis algorithms for extracting information about the domain organization of single RNA molecules. We examined the 5 kb human 28S rRNA since it is the largest RNA molecule for which a 3D structure is available. As a proof of concept, we determined a domain structure that is in accordance with previously described secondary structural models. Importantly, we identified four additional small (200-300 nt), previously unreported domains present in these molecules. Moreover, the single-molecule nature of our method enabled us to report on the relative conformational variability of each domain structure identified, and inter-domain associations within subsets of molecules leading to molecular compaction, which may shed light on the process of how these molecules fold into the final tertiary structure.

DNA looping-dependent autorepression of LEE1 P1 promoters by Ler in enteropathogenic Escherichia coli (EPEC).

Ler, a homolog of H-NS in enteropathogenic Escherichia coli (EPEC), plays a critical role in the expression of virulence genes encoded by the pathogenic island, locus of enterocyte effacement (LEE). Although Ler acts as an antisilencer of multiple LEE operons by alleviating H-NS-mediated silencing, it represses its own expression from two LEE1 P1 promoters, P1A and P1B, that are separated by 10 bp. Various in vitro biochemical methods were used in this study to elucidate the mechanism underlying transcription repression by Ler. Ler acts through two AATT motifs, centered at position -111.5 on the coding strand and at +65.5 on the noncoding strand, by simultaneously repressing P1A and P1B through DNA-looping. DNA-looping was visualized using atomic force microscopy. It is intriguing that an antisilencing protein represses transcription, not by steric exclusion of RNA polymerase, but by DNA-looping. We propose that the DNA-looping prevents further processing of open promoter complex (RPO) at these promoters during transcription initiation.

Transcriptional Repressor TrmBL2 from Thermococcus kodakarensis Forms Filamentous Nucleoprotein Structures and Competes with Histones for DNA Binding in a Salt- and DNA Supercoiling-dependent Manner.

Architectural DNA proteins play important roles in the chromosomal DNA organization and global gene regulation in living cells. However, physiological functions of some DNA-binding proteins from archaea remain unclear. Recently, several abundant DNA-architectural proteins including histones, Alba, and TrmBL2 have been identified in model euryarchaeon Thermococcus kodakarensis. Although histones and Alba proteins have been previously characterized, the DNA binding properties of TrmBL2 and its interplay with the other major architectural proteins in the chromosomal DNA organization and gene transcription regulation remain largely unexplored. Here, we report single-DNA studies showing that at low ionic strength (<300 mM KCl), TrmBL2 binds to DNA largely in non-sequence-specific manner with positive cooperativity, resulting in formation of stiff nucleoprotein filamentous patches, whereas at high ionic strength (>300 mM KCl) TrmBL2 switches to more sequence-specific interaction, suggesting the presence of high affinity TrmBL2-filament nucleation sites. Furthermore, in vitro assays indicate the existence of DNA binding competition between TrmBL2 and archaeal histones B from T. kodakarensis, which can be strongly modulated by DNA supercoiling and ionic strength of surrounding solution. Overall, these results advance our understanding of TrmBL2 DNA binding properties and provide important insights into potential functions of architectural proteins in nucleoid organization and gene regulation in T. kodakarensis.

Probing in vivo dynamics of mitochondria and cortical actin networks using high-speed atomic force/fluorescence microscopy.

The dynamics of the cell membrane and submembrane structures are closely linked, facilitating various cellular activities. Although cell surface research and cortical actin studies have shown independent mechanisms for the cell membrane and the actin network, it has been difficult to obtain a comprehensive understanding of the dynamics of these structures in live cells. Here, we used a combined atomic force/optical microscope system to analyze membrane-based cellular events at nanometer-scale resolution in live cells. Imaging the COS-7 cell surface showed detailed structural properties of membrane invagination events corresponding to endocytosis and exocytosis. In addition, the movement of mitochondria and the spatiotemporal dynamics of the cortical F-actin network were directly visualized in vivo. Cortical actin microdomains with sizes ranging from 1.7×10(4) to 1.4×10(5) nm2 were dynamically rearranged by newly appearing actin filaments, which sometimes accompanied membrane invaginations, suggesting that these events are integrated with the dynamic regulation of submembrane organizations maintained by actin turnovers. These results provide novel insights into the structural aspects of the entire cell membrane machinery which can be visualized with high temporal and spatial resolution.

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure.

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.

Interaction of branch migration translocases with the Holliday junction-resolving enzyme and their implications in Holliday junction resolution.

Double-strand break repair involves the formation of Holliday junction (HJ) structures that need to be resolved to promote correct replication and chromosomal segregation. The molecular mechanisms of HJ branch migration and/or resolution are poorly characterized in Firmicutes. Genetic evidence suggested that the absence of the RuvAB branch migration translocase and the RecU HJ resolvase is synthetically lethal in Bacillus subtilis, whereas a recU recG mutant was viable. In vitro RecU, which is restricted to bacteria of the Firmicutes phylum, binds HJs with high affinity. In this work we found that RecU does not bind simultaneously with RecG to a HJ. RuvB by interacting with RecU bound to the central region of HJ DNA, loses its nonspecific association with DNA, and re-localizes with RecU to form a ternary complex. RecU cannot stimulate the ATPase or branch migration activity of RuvB. The presence of RuvB·ATPγS greatly stimulates RecU-mediated HJ resolution, but the addition of ATP or RuvA abolishes this stimulatory effect. A RecU·HJ·RuvAB complex might be formed. RecU does not increase the RuvAB activities but slightly inhibits them.

Spatial control of proton pump H,K-ATPase docking at the apical membrane by phosphorylation-coupled ezrin-syntaxin 3 interaction.

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser-66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. Here we show that phosphorylation of Ser-66 induces a conformational change of ezrin that enables its association with syntaxin 3 (Stx3) and provides a spatial cue for H,K-ATPase trafficking. This conformation-dependent association is specific for Stx3, and the binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser-66 prevents ezrin-Stx3 association and insertion of H,K-ATPase into the apical plasma membrane of parietal cells. Using atomic force microscopic analyses, our study revealed that phosphorylation of Ser-66 induces unfolding of ezrin molecule to allow Stx3 binding to its N terminus. Given the essential role of Stx3 in polarized secretion, our study presents the first evidence in which phosphorylation-induced conformational rearrangement of the ezrin molecule provides a spatial cue for polarized membrane trafficking in epithelial cells.

Intermolecular disulfide bonds between nucleoporins regulate karyopherin-dependent nuclear transport.

Disulfide (S-S) bonds play important roles in the regulation of protein function and cellular stress responses. In this study, we demonstrate that distinct sets of nucleoporins (Nups), components of the nuclear pore complex (NPC), form S-S bonds and regulate nuclear transport through the NPC. Kinetic analysis of importin ß demonstrated that the permeability of the NPC was increased by dithiothreitol treatment and reduced by oxidative stress. The permeability of small proteins such as GFP was not affected by either oxidative stress or a reducing reagent. Immunoblot analysis revealed that the oxidative stress significantly induced S-S bond formation in Nups 358, 155, 153 and 62 but not 88 and 160. The direct involvement of cysteine residues in the formation of S-S bonds was confirmed by mutating conserved cysteine residues in Nup62, which abolished the formation of S-S bonds and enhanced the permeability of the NPC. Knocking down Nup62 reduced the stress-inducible S-S bonds of Nup155, suggesting that Nup62 and Nup155 are covalently coupled via S-S bonds. From these results, we propose that the inner channel of the NPC is somehow insulated from the cytoplasm and is more sensitive than the cytoplasm to the intracellular redox state.

Caprice/MISP is a novel F-actin bundling protein critical for actin-based cytoskeletal reorganizations.

Caprice [C19orf21 actin-bundling protein in characteristic epithelial cells, also called mitotic interactor and substrate of Plk1 (MISP)] is a novel actin-related protein identified in the highly-insoluble subcellular scaffold proteins. This protein contains multiple actin-binding sites, forms characteristic mesh-like F-actin bundles in vitro, and exhibits capricious localization and expression patterns in vivo. Overexpression or knock-down of Caprice resulted in a dramatic effect on cellular morphology by inducing stress fiber-like thick filaments or filopodial formations, respectively. Caprice is expressed and localized in distinct cells and tissues with specialized actin-based structures, such as growth cones of migrating neurons and stereocilia of inner ear hair cells. However, Caprice gene expression is varied among different cell types; especially enriched in several epithelial cells whereas relatively suppressed in a subset of epithelial cells, fibroblasts, and neuroblastoma cells at the transcriptional level. Thus, this protein is expected to be an effector for cell type-specific actin reorganization with its direct actin-binding properties and provides a novel model of cell morphology regulation by a non-ubiquitous single actin-bundling protein.

An alternative beads-on-a-string chromatin architecture in Thermococcus kodakarensis.

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone-like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle-positioning code and are excluded from gene-regulatory DNA suggesting a functional organization. Beads-on-a-string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone-fold proteins in a variety of multimeric forms.

Human G-proteins, ObgH1 and Mtg1, associate with the large mitochondrial ribosome subunit and are involved in translation and assembly of respiratory complexes.

The bacterial homologues of ObgH1 and Mtg1, ObgE and RbgA, respectively, have been suggested to be involved in the assembly of large ribosomal subunits. We sought to elucidate the functions of ObgH1 and Mtg1 in ribosome biogenesis in human mitochondria. ObgH1 and Mtg1 are localized in mitochondria in association with the inner membrane, and are exposed on the matrix side. Mtg1 and ObgH1 specifically associate with the large subunit of the mitochondrial ribosome in GTP-dependent manner. The large ribosomal subunit stimulated the GTPase activity of Mtg1, whereas only the intrinsic GTPase activity was detectable with ObgH1. The knockdown of Mtg1 decreased the overall mitochondrial translation activity, and caused defects in the formation of respiratory complexes. On the other hand, the depletion of ObgH1 led to the specific activation of the translation of subunits of Complex V, and disrupted its proper formation. Our results suggested that Mtg1 and ObgH1 function with the large subunit of the mitochondrial ribosome, and are also involved in both the translation and assembly of respiratory complexes. The fine coordination of ribosome assembly, translation and respiratory complex formation in mammalian mitochondria is affirmed.

Bacillus subtilis DprA recruits RecA onto single-stranded DNA and mediates annealing of complementary strands coated by SsbB and SsbA.

Naturally transformable bacteria recombine internalized ssDNA with a homologous resident duplex (chromosomal transformation) or complementary internalized ssDNAs (plasmid or viral transformation). Bacillus subtilis competence-induced DprA, RecA, SsbB, and SsbA proteins are involved in the early processing of the internalized ssDNA, with DprA physically interacting with RecA. SsbB and SsbA bind and melt secondary structures in ssDNA but limit RecA loading onto ssDNA. DprA binds to ssDNA and facilitates partial dislodging of both single-stranded binding (SSB) proteins from ssDNA. In the absence of homologous duplex DNA, DprA does not significantly increase RecA nucleation onto protein-free ssDNA. DprA facilitates RecA nucleation and filament extension onto SsbB-coated or SsbB plus SsbA-coated ssDNA. DprA facilitates RecA-mediated DNA strand exchange in the presence of both SSB proteins. DprA, which plays a crucial role in plasmid transformation, anneals complementary strands preferentially coated by SsbB to form duplex circular plasmid molecules. Our results provide a mechanistic framework for conceptualizing the coordinated events modulated by SsbB in concert with SsbA and DprA that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.

Visualization of structural changes accompanying activation of N-methyl-D-aspartate (NMDA) receptors using fast-scan atomic force microscopy imaging.

NMDA receptors are widely expressed in the central nervous system and play a major role in excitatory synaptic transmission and plasticity. Here, we used atomic force microscopy (AFM) imaging to visualize activation-induced structural changes in the GluN1/GluN2A NMDA receptor reconstituted into a lipid bilayer. In the absence of agonist, AFM imaging revealed two populations of particles with heights above the bilayer surface of 8.6 and 3.4 nm. The taller, but not the shorter, particles could be specifically decorated by an anti-GluN1 antibody, which recognizes the S2 segment of the agonist-binding domain, indicating that the two populations represent the extracellular and intracellular regions of the receptor, respectively. In the presence of glycine and glutamate, there was a reduction in the height of the extracellular region to 7.3 nm. In contrast, the height of the intracellular domain was unaffected. Fast-scan AFM imaging combined with UV photolysis of caged glutamate permitted the detection of a rapid reduction in the height of individual NMDA receptors. The reduction in height did not occur in the absence of the co-agonist glycine or in the presence of the selective NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid, indicating that the observed structural change was caused by receptor activation. These results represent the first demonstration of an activation-induced effect on the structure of the NMDA receptor at the single-molecule level. A change in receptor size following activation could have important functional implications, in particular by affecting interactions between the NMDA receptor and its extracellular synaptic partners.

Dynamics of WD-repeat containing proteins in SSU processome components.

Nine WD-repeat containing proteins in human SSU processome components have been found in a HeLa cell nuclear matrix fraction. In these proteins, t-UTP sub-complex components, i.e., CIRH1A, UTP15, and WDR43, were shown to be immobilized in the fibrillar centers of nucleoli in living cells. In this study, the dynamics of the remaining six proteins fused with green fluorescent protein (GFP), i.e., PWP2-GFP, TBL3-GFP, GFP-UTP18, GFP-NOL10, GFP-WDR46, and GFP-WDSOF1, were examined in living cells. The findings were as follows. (i) The majority of UTP-B sub-complex components, i.e., PWP2-GFP, TBL3-GFP, and GFP-UTP18, are localized to the dense fibrillar component and granular component regions in nucleoli; (ii) When rRNA transcription is suppressed, the majority of GFP-fused UTP-B sub-complex components are localized in the cap and body regions of nucleoli. (iii) The mobility of these proteins except for GFP-WDSOF1, and half of GFP-UTP18 and GFP-WDR46, respectively, is very low in living cells. (iv) When rRNA transcription is suppressed, the mobility of these proteins except for GFP-WDSOF1 is accelerated but still slow. These findings and others suggest that these WD-repeat proteins other than GFP-WDSOF1 found in the nuclear matrix fraction bind tightly to some macro-protein complexes and act as a scaffold or a core for the complexes in nucleoli.

Karyopherin-independent spontaneous transport of amphiphilic proteins through the nuclear pore.

Highly selective nucleocytoplasmic molecular transport is critical to eukaryotic cells, which is illustrated by size-filtering diffusion and karyopherin-mediated passage mechanisms. However, a considerable number of large proteins without nuclear localization signals are localized to the nucleus. In this paper, we provide evidence for the spontaneous migration of large proteins in a karyopherin-independent manner. Time-lapse observation of a nuclear transport assay revealed that several large molecules spontaneously and independently pass through the nuclear pore complex (NPC). The amphiphilic motifs were sufficient to overcome the selectivity barrier of the NPC. Furthermore, the amphiphilic property of these proteins enables altered local conformation in hydrophobic solutions so that elevated surface hydrophobicity facilitates passage through the nuclear pore. The molecular dynamics simulation revealed the conformational change of the amphiphilic structure that exposes the hydrophobic amino acid residues to the outer surface in a hydrophobic solution. These results contribute to the understanding of nucleocytoplasmic molecular sorting and the nature of the permeability barrier.

Nucleolar scaffold protein, WDR46, determines the granular compartmental localization of nucleolin and DDX21.

The nuclear scaffold is an insoluble nuclear structure that contributes to the inner nuclear organization. In this study, we showed that one of the nuclear scaffold proteins, WDR46, plays a role as a fundamental scaffold component of the nucleolar structure. WDR46 is a highly insoluble nucleolar protein, and its subcellular localization is dependent on neither DNA nor RNA. The N- and C-terminal regions of WDR46 are predicted to be intrinsically disordered, and both regions are critical for the nucleolar localization of WDR46 and the association with its binding partners. When WDR46 was knocked down, two of its binding partners, nucleolin and DDX21 (involved in 18S rRNA processing), were mislocalized from the granular component to the edges of the nucleoli, whereas other binding partners, NOP2 and EBP2 (involved in 28S rRNA processing), were not affected. This is because the proper recruitment of nucleolin and DDX21 to the nucleoli in daughter cells after cell division is ensured by WDR46. These findings suggest a structural role for WDR46 in organizing the 18S ribosomal RNA processing machinery. This role of WDR46 is enabled by its interaction property via intrinsically disordered regions.