The gateway reflex regulates tissue-specific autoimmune diseases.

The dynamic interaction and movement of substances and cells between the central nervous system (CNS) and peripheral organs are meticulously controlled by a specialized vascular structure, the blood-brain barrier (BBB). Experimental and clinical research has shown that disruptions in the BBB are characteristic of various neuroinflammatory disorders, including multiple sclerosis. We have been elucidating a mechanism termed the "gateway reflex" that details the entry of immune cells, notably autoreactive T cells, into the CNS at the onset of such diseases. This process is initiated through local neural responses to a range of environmental stimuli, such as gravity, electricity, pain, stress, light, and joint inflammation. These stimuli specifically activate neural pathways to open gateways at targeted blood vessels for blood immune cell entry. The gateway reflex is pivotal in managing tissue-specific inflammatory diseases, and its improper activation is linked to disease progression. In this review, we present a comprehensive examination of the gateway reflex mechanism.

Computer model of IL-6-dependent rheumatoid arthritis in F759 mice.

The interleukin-6 (IL-6) amplifier, which describes the simultaneous activation of signal transducer and activator of transcription 3 (STAT3) and NF-κb nuclear factor kappa B (NF-κB), in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-κB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NF-κB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 (interleukin 17) signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-κB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and C-C motif chemokine ligand 2 (CCL2), phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of T helper 17 (Th17) cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.

GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation.

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common ß chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

Zoobiquity experiments show the importance of the local MMP9-plasminogen axis in inflammatory bowel diseases in both dogs and patients.

Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD) and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2 and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G > T and c.478A>T, and found that ICRPs with the T/T SNP risk alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk alleles but no differences in serum. Moreover, we show that MMP9, which is an NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were co-localized with epithelial cells expressing MMP9 in normal colons with the risk alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn's disease also co-localized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in the intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.

Structural Insights into Methylated DNA Recognition by the Methyl-CpG Binding Domain of MBD6 from Arabidopsis thaliana.

Cytosine methylation is an epigenetic modification essential for formation of mature heterochromatin, gene silencing, and genomic stability. In plants, methylation occurs not only at cytosine bases in CpG but also in CpHpG and CpHpH contexts, where H denotes A, T, or C. Methyl-CpG binding domain (MBD) proteins, which recognize symmetrical methyl-CpG dinucleotides and act as gene repressors in mammalian cells, are also present in plant cells, although their structural and functional properties still remain poorly understood. To fill this gap, in this study, we determined the solution structure of the MBD domain of the MBD6 protein from Arabidopsis thaliana and investigated its binding properties to methylated DNA by binding assays and an in-depth NMR spectroscopic analysis. The AtMBD6 MBD domain folds into a canonical MBD structure in line with its binding specificity toward methyl-CpG and possesses a DNA binding interface similar to mammalian MBD domains. Intriguingly, however, the binding affinity of the AtMBD6 MBD domain toward methyl-CpG-containing DNA was found to be much lower than that of known mammalian MBD domains. The main difference arises from the absence of positively charged residues in AtMBD6 that supposedly interact with the DNA backbone as seen in mammalian MBD/methyl-CpG-containing DNA complexes. Taken together, we have established a structural basis for methyl-CpG recognition by AtMBD6 to develop a deeper understanding how MBD proteins work as mediators of epigenetic signals in plant cells.

Room-temperature hyperpolarization of polycrystalline samples with optically polarized triplet electrons: pentacene or nitrogen-vacancy center in diamond?

We demonstrate room-temperature 13C hyperpolarization by dynamic nuclear polarization (DNP) using optically polarized triplet electron spins in two polycrystalline systems: pentacene-doped [carboxyl-13C] benzoic acid and microdiamonds containing nitrogen-vacancy (NV-) centers. For both samples, the integrated solid effect (ISE) is used to polarize the 13C spin system in magnetic fields of 350-400 mT. In the benzoic acid sample, the 13C spin polarization is enhanced by up to 0.12 % through direct electron-to-13C polarization transfer without performing dynamic 1H polarization followed by 1H-13C cross-polarization. In addition, the ISE has been successfully applied to polarize naturally abundant 13C spins in a microdiamond sample to 0.01 %. To characterize the buildup of the 13C polarization, we discuss the efficiencies of direct polarization transfer between the electron and 13C spins as well as that of 13C-13C spin diffusion, examining various parameters which are beneficial or detrimental for successful bulk dynamic 13C polarization.

Backbone and side-chain resonance assignments of the methyl-CpG-binding domain of MBD6 from Arabidopsis thaliana.

Epigenetic regulation is essential to various biological phenomena such as cell differentiation and cancer. DNA methylation is one of the most important epigenetic signals, as it is directly involved in gene silencing of transposable elements, genomic imprinting, and chromosome X inactivation. To mediate these processes, methyl-CpG-binding domain (MBD) proteins recognize specific signals encoded in the form of DNA methylation patterns. AtMBD6, one of the 12 MBD proteins in Arabidopsis thaliana, shares a high sequential homology in the MBD domain with mammalian MBD proteins, but a detailed characterization of its structural and functional properties remains elusive. Here, we report the 1H, 13C, and 15N resonance assignments of the isolated MBD domain of AtMBD6. Analysis of the chemical shift data implied that the MBD domain of AtMBD6 has a secondary structure similar to that of mammalian MeCP2, while the ß-strands ß1 and ß3 of AtMBD6 were found to be longer than those of MeCP2. The structural differences provide insight into the different recognition mechanisms of methylated DNA by plant and mammalian MBDs. The assignments reported here will aid further analyses such as titration experiments and three-dimensional structure determination using NMR to yield a detailed characterization of the interaction between AtMBD6 and methylated DNAs.

Cooperative Domain Formation by Homologous Motifs in HOIL-1L and SHARPIN Plays A Crucial Role in LUBAC Stabilization.

The linear ubiquitin chain assembly complex (LUBAC) participates in inflammatory and oncogenic signaling by conjugating linear ubiquitin chains to target proteins. LUBAC consists of the catalytic HOIP subunit and two accessory subunits, HOIL-1L and SHARPIN. Interactions between the ubiquitin-associated (UBA) domains of HOIP and the ubiquitin-like (UBL) domains of two accessory subunits are involved in LUBAC stabilization, but the precise molecular mechanisms underlying the formation of stable trimeric LUBAC remain elusive. We solved the co-crystal structure of the binding regions of the trimeric LUBAC complex and found that LUBAC-tethering motifs (LTMs) located N terminally to the UBL domains of HOIL-1L and SHARPIN heterodimerize and fold into a single globular domain. This interaction is resistant to dissociation and plays a critical role in stabilizing trimeric LUBAC. Inhibition of LTM-mediated HOIL-1L/SHARPIN dimerization profoundly attenuated the function of LUBAC, suggesting LTM as a superior target of LUBAC destabilization for anticancer therapeutics.

TFL1-Like Proteins in Rice Antagonize Rice FT-Like Protein in Inflorescence Development by Competition for Complex Formation with 14-3-3 and FD.

Hd3a, a rice homolog of FLOWERING LOCUS T (FT), is a florigen that induces flowering. Hd3a forms a ternary 'florigen activation complex' (FAC) with 14-3-3 protein and OsFD1 transcription factor, a rice homolog of FD that induces transcription of OsMADS15, a rice homolog of APETALA1 (AP1), which leads to flowering. TERMINAL FLOWER 1 (TFL1) represses flowering and controls inflorescence architecture. However, the molecular basis for floral repression by TFL1 remains poorly understood. Here we show that RICE CENTRORADIALIS (RCN), rice TFL1-like proteins, compete with Hd3a for 14-3-3 binding. All four RCN genes are predominantly expressed in the vasculature, and RCN proteins are transported to the shoot apex to antagonize florigen activity and regulate inflorescence development. The antagonistic function of RCN to Hd3a is dependent on its 14-3-3 binding activity. Our results suggest a molecular basis for regulation of the balance between florigen FT and anti-florigen TFL1.

The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase.

Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.

Purification, crystallization and preliminary X-ray crystallographic analysis of a rice Rac/Rop GTPase, OsRac1.

Small GTPases regulate a large variety of key cellular processes. Plant small Rac/Rop GTPases have recently received broad attention as it is becoming clear that these enzymes regulate various plant cellular processes. OsRac1, a rice Rac/Rop protein, is a key regulator of reactive oxygen species (ROS) production and induces immune responses. Although four structures of plant small GTPases have been reported, all of these were of the inactive form. Here, OsRac1 was purified and co-crystallized with the GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP). The crystal belonged to space group P2(1)2(1)2(1) and a complete data set was collected to 1.9 Šresolution.

MtnBD is a multifunctional fusion enzyme in the methionine salvage pathway of Tetrahymena thermophila.

To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and ¹H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.

Structure and function of florigen and the receptor complex.

In the 1930s, the flowering hormone, florigen, was proposed to be synthesized in leaves under inductive day length and transported to the shoot apex, where it induces flowering. More recently, generated genetic and biochemical data suggest that florigen is a protein encoded by the gene, FLOWERING LOCUS T (FT). A rice (Oryza sativa) FT homolog, Hd3a, interacts with the rice FD homolog, OsFD1, via a 14-3-3 protein. Formation of this tri-protein complex is essential for flowering promotion by Hd3a in rice. In addition, the multifunctionality of FT homologs, other than for flowering promotion, is an emerging concept. Here we review the structural and biochemical features of the florigen protein complex and discuss the molecular basis for the multifunctionality of FT proteins.

Utilization of lysine ¹³C-methylation NMR for protein-protein interaction studies.

Chemical modification is an easy way for stable isotope labeling of non-labeled proteins. The reductive (13)C-methylation of the amino group of the lysine side-chain by (13)C-formaldehyde is a post-modification and is applicable to most proteins since this chemical modification specifically and quickly proceeds under mild conditions such as 4 °C, pH 6.8, overnight. (13)C-methylation has been used for NMR to study the interactions between the methylated proteins and various molecules, such as small ligands, nucleic acids and peptides. Here we applied lysine (13)C-methylation NMR to monitor protein-protein interactions. The affinity and the intermolecular interaction sites of methylated ubiquitin with three ubiquitin-interacting proteins were successfully determined using chemical-shift perturbation experiments via the (1)H-(13)C HSQC spectra of the (13)C-methylated-lysine methyl groups. The lysine (13)C-methylation NMR results also emphasized the importance of the usage of side-chain signals to monitor the intermolecular interaction sites, and was applicable to studying samples with concentrations in the low sub-micromolar range.

14-3-3 proteins act as intracellular receptors for rice Hd3a florigen.

'Florigen' was proposed 75 years ago to be synthesized in the leaf and transported to the shoot apex, where it induces flowering. Only recently have genetic and biochemical studies established that florigen is encoded by FLOWERING LOCUS T (FT), a gene that is universally conserved in higher plants. Nonetheless, the exact function of florigen during floral induction remains poorly understood and receptors for florigen have not been identified. Here we show that the rice FT homologue Hd3a interacts with 14-3-3 proteins in the apical cells of shoots, yielding a complex that translocates to the nucleus and binds to the Oryza sativa (Os)FD1 transcription factor, a rice homologue of Arabidopsis thaliana FD. The resultant ternary 'florigen activation complex' (FAC) induces transcription of OsMADS15, a homologue of A. thaliana APETALA1 (AP1), which leads to flowering. We have determined the 2.4 Å crystal structure of rice FAC, which provides a mechanistic basis for florigen function in flowering. Our results indicate that 14-3-3 proteins act as intracellular receptors for florigen in shoot apical cells, and offer new approaches to manipulate flowering in various crops and trees.

Surface plasmon resonance study on functional significance of clustered organization of lectin-like oxidized LDL receptor (LOX-1).

Lectin-like oxidized low-density lipoprotein (OxLDL) receptor 1 (LOX-1) is the major OxLDL receptor of vascular endothelial cells and is involved in an early step of atherogenesis. LOX-1 exists as a disulfide-linked homodimer on the cell surface, which contains a pair of the ligand-binding domains (CTLD; C-type lectin-like domain). Recent research using living cells has suggested that the clustered state of LOX-1 dimer on the cell is functionally required. These results questioned how LOX-1 exists on the cell to achieve OxLDL binding. In this study, we revealed the functional significance of the clustered organization of the ligand-binding domain of LOX-1 with surface plasmon resonance. Biotinylated CTLD was immobilized on a streptavidin sensor chip to make CTLD clusters on the surface. In this state, the CTLD had high affinity for OxLDL with a dissociation constant (K(D)) in the nanomolar range. This value is comparable to the K(D) measured for LOX-1 on the cell. In contrast, a single homodimeric LOX-1 extracellular domain had lower affinity for OxLDL in the supra-micromolar range of K(D). Monomeric CTLD showed marginal binding to OxLDL. In combination with the analyses on the loss-of-binding mutant W150A, we concluded that the clustered organization of the properly formed homodimeric CTLD is essential for the strong binding of LOX-1 to OxLDL.

Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1).

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca(2+)-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K(d) approximately 7-12 microm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1(+)NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.

Kinetic and thermodynamic evidence for flipping of a methyl-CpG binding domain on methylated DNA.

The methyl-CpG binding domain (MBD) is a conserved domain in transcriptional factors that binds to methylated CpG dinucleotide DNA sequences in vertebrates. The complex is comprised of an asymmetric MBD monomer and a symmetric DNA duplex. Therefore, in the complex, each strand of the duplex DNA is in contact with the protein at a distinct surface and thus exhibits a different chemical shift in NMR spectra. Two-dimensional chemical exchange spectroscopy revealed the presence of a stochastic exchange of the two strands of the duplex DNA in the complex at a rate of 4 s (-1) at 25 degrees C, which indicates the existence of a motion of the MBD such that the orientation of the MBD becomes reversed with respect to the DNA duplex. Kinetic and thermodynamic analyses using surface plasmon resonance, quartz crystal microbalance, and isothermal titration calorimetry suggest that the reversal of MBD with respect to the DNA duplex takes place without its complete dissociation from DNA, indicating the presence of an intermediate protein-DNA binding state that allows the protein to undergo a flip motion upon DNA.

Chimeric structural stabilities in the coiled-coil structure of the NECK domain in human lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1).

LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) is the major oxidized LDL (OxLDL) receptor on endothelial cells. The extracellular part of LOX-1 comprises an 82-residue stalk region (NECK) and a C-type lectin-like ligand-binding domain (CTLD). The NECK displays sequence similarity to the coiled-coil region of myosin, having been suggested it adopts a rod-like structure. In this article, we report the structural analyses of human LOX-1 NECK using a variety of approaches including limited proteolysis, chemical cross-linking, circular dichroism (CD) and NMR. Our analysis reveals a unique structural feature of the LOX-1 NECK. Despite significant sequence similarity with the myosin coiled-coil, LOX-1 NECK does not form a uniform rod-like structure. Although not random, one-third of the N-terminal NECK is less structured than the remainder of the protein and is highly sensitive to cleavage by a variety of proteases. The coiled-coil structure is localized at the C-terminal part of the NECK, but is in dynamic equilibrium among multiple conformational states on a mus-ms time scale. This chimeric structural property of the NECK region may enable clustered LOX-1 on the cell surface to recognize OxLDL.