DJ-1 is a multifunctional protein that plays essential roles in tissues with higher order biological functions such as the testis and brain. DJ-1 is related to male fertility, and its level in sperm decreases in response to exposure to sperm toxicants. DJ-1 has also been identified as a hydroperoxide-responsive protein. Recently, a mutation of DJ-1 was found to be responsible for familial Parkinson's disease. Here, we present the crystal structure of DJ-1 refined to 1.95-Å resolution. DJ-1 forms a dimer in the crystal, and the monomer takes a flavodoxin-like Rossmann-fold. DJ-1 is structurally most similar to the monomer subunit of protease I, the intracellular cysteine protease from Pyrococcus horikoshii, and belongs to the Class I glutamine amidotransferaselike superfamily. However, DJ-1 contains an additional ␣-helix at the C-terminal region, which blocks the putative catalytic site of DJ-1 and appears to regulate the enzymatic activity. DJ-1 may induce conformational changes to acquire catalytic activity in response to oxidative stress.DJ-1 was initially identified as a novel oncogene product that transforms mouse NIH3T3 cells in cooperation with activated Ras. DJ-1 is an ϳ20-kDa protein comprising 189 amino acid residues ubiquitously expressed in various human tissues and with a particularly high level of expression in the testes (1). SP22 1 or CAP1, a rat homologue of human DJ-1, was subsequently identified as a key protein related to infertility in male rats exposed to sperm toxicants such as ornidazole and epichlorohydrin where DJ-1/CAP1/SP22 levels in the sperm and epididymis decreased with increased rat infertility (2-4). With the exception of DJ-1, no other protein decreased in response to exposure to sperm toxicants, supporting the close relationship between DJ-1 function and male fertility. Recently, Klinefelter et al. (5) revealed that DJ-1/CAP1/SP22 was located on the equatorial segment of the matured sperm head and anti-SP22

Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

Transcription factor IRF-3 is post-translationally activated by Toll-like receptor (TLR) signaling and has critical roles in the regulation of innate immunity. Here we present the X-ray crystal structure of the C-terminal regulatory domain of IRF-3(175-427) (IRF-3 175C) at a resolution of 2.3 A. IRF-3 175C is structurally similar to the Mad homology domain 2 of the Smad family. Structural and functional analyses reveal phosphorylation-induced IRF-3 dimerization, which generates an extensive acidic pocket responsible for binding with p300/CBP. Although TLR and Smad signaling are evolutionarily independent, our results suggest that IRF-3 originates from Smad and acquires its function downstream of TLR.

The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.

A complex of atypical PKC and Par6 is a common regulator for cell polarity-related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKC and Par6␣ PB1 domains to a resolution of 1.5 Å. Both PB1 domains adopt a ubiquitin fold. PKC PB1 presents an OPR, PC, and AID (OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6␣ PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKC PB1 and the conserved lysine residue on the Par6␣ PB1. Structural comparison of the PKC and Par6␣ PB1 complex with the p40 phox and p67 phox PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.

Nitric oxide (NO) has multiple important actions that contribute to the maintenance of vascular homeostasis. NO is synthesized by three different isoforms of NO synthase (NOS), all of which have been reported to be expressed in human atherosclerotic vascular lesions. Although the regulatory roles of endothelial NOS (eNOS) and inducible NOS (iNOS) on the development of atherosclerosis have been described, little is known about the role of neuronal NOS (nNOS). Here, we show that nNOS also exerts important vasculoprotective effects in vivo. In a carotid artery ligation model, nNOS gene-deficient (nNOS-KO) mice exhibited accelerated neointimal formation and constrictive vascular remodeling caused by blood flow disruption. In a rat balloon injury model, the selective inhibition of nNOS activity potently enhanced vasoconstrictor responses to a variety of calcium-mobilizing stimuli, suppressed tissue cGMP concentrations, a marker of vascular NO production, and exacerbated neointimal formation. In both models, nNOS was absent before injury and was up-regulated only after the injury, and was predominantly expressed in the neointima and medial smooth muscle cells. These results provide the first direct evidence that nNOS plays important roles in suppressing arteriosclerotic vascular lesion formation in vivo.

The ␤-1,3-glucan recognition protein (␤GRP)/Gram-negative bacteriabinding protein 3 (GNBP3) is a crucial pattern-recognition receptor that specifically binds ␤-1,3-glucan, a component of fungal cell walls. It evokes innate immunity against fungi through activation of the prophenoloxidase (proPO) cascade and Toll pathway in invertebrates. The ␤GRP consists of an N-terminal ␤-1,3-glucan-recognition domain and a C-terminal glucanase-like domain, with the former reported to be responsible for the proPO cascade activation. This report shows the solution structure of the N-terminal ␤-1,3-glucan recognition domain of silkworm ␤GRP. Although the N-terminal domain of ␤GRP has a ␤-sandwich fold, often seen in carbohydrate-binding modules, both NMR titration experiments and mutational analysis showed that ␤GRP has a binding mechanism which is distinct from those observed in previously reported carbohydarate-binding domains. Our results suggest that ␤GRP is a ␤-1,3-glucan-recognition protein that specifically recognizes a triple-helical structure of ␤-1,3-glucan.phenol oxidase ͉ innate immunity ͉ pattern recognition

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5-triphosphated single-stranded RNA (5ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.A variety of pathogen-associated molecular patterns, including microbial peptidoglycan, lipopolysaccharide, ␤-1,3-glucan, and viral DNA or RNA are recognized by pattern recognition receptors that evoke the innate immune responses of host cells. In viral infections, double-stranded RNA (dsRNA) 2 is recognized by Toll-like receptor-3 in the early endosome and by RIG-I like receptors (RLRs) in the cytoplasm. These two receptors initiate the innate immune responses including the production of cytokines and type-I interferon, which are critical for the subsequent adaptive immune response (1).The RLR comprises three homologs: RIG-I (retinoic acidinducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2) (see Fig. 1A) (2), and they sense a viral infection by recognizing replicating viral RNA in the cytoplasm. The RIG-I and MDA5 consist of three functional domains: tandem-CARDs (caspase activation and recruitment domain), a DEAD box helicase-like domain, and a well conserved C-terminal domain (CTD), whereas LGP2 has only the DEAD box helicase like domain and well conserved CTD. The three RLRs are considered to play different roles in the recognition of pathogen-associated molecular patterns and to be activated by different viruses and different viral RNAs. RIG-I is activated by a variety of viruses, including paramyxovirus, rhabdovirus, and orthomyxovirus, recognizing not only dsRNA but also 5Ј-triphosphated single-stranded RNA (5Јppp-ssRNA) (3, 4), and MDA5 is mainly activated by picornavirus (5...