Diversity of innate immune recognition mechanism for bacterial polymeric meso-diaminopimelic acid-type peptidoglycan in insects.

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor.

The Lyn kinase C-lobe mediates Golgi export of Lyn through conformation-dependent ACSL3 association.

The Src-family tyrosine kinase Lyn has a role in signal transduction at the cytoplasmic face of the plasma membrane upon extracellular ligand stimulation. After synthesis in the cytoplasm, Lyn accumulates on the Golgi and is subsequently transported to the plasma membrane. However, the mechanism of Lyn trafficking remains elusive.

Thermodynamic characterization of the interaction between prefoldin and group II chaperonin.

Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1).

Topological analysis of the extrinsic PsbO, PsbP and PsbQ proteins in a green algal PSII complex by cross-linking with a water-soluble carbodiimide.

The close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits.

Comparative studies of lepidopteran baculovirus-specific protein FP25K: development of a novel Bombyx mori nucleopolyhedrovirus-based vector with a modified fp25K gene.

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation.

The PsbK subunit is required for the stable assembly and stability of other small subunits in the PSII complex in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

PsbK is a small membrane protein of the PSII core complex and is highly conserved from cyanobacteria to plants. Here, we studied its role in the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, by focusing on a psbK disruptant with hexahistidine-tagged CP47. The psbK disruptant showed photoautotrophic growth comparable with that of the wild type under a wide range of light conditions.

The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site.

RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown.

Identification and assignment of three disulfide bonds in mammalian leukocyte cell-derived chemotaxin 2 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Mammalian leukocyte cell-derived chemotaxin 2 (LECT2) contains six evolutionarily conserved cysteine residues. To date, however, the presence of disulfide linkages between these residues has not been determined. To search for disulfide bonds, the protein was proteolitically digested and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization?time of flight mass spectrometry.

Identification of acrolein-conjugated protein in plasma of patients with brain infarction.

It is known that the level of protein-conjugated acrolein in plasma is a good marker of chronic renal failure and brain infarction. Thus, studies were carried out to determine which kinds of plasma proteins are conjugated with acrolein. It was found that acrolein was mainly conjugated with albumin.

Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells.

Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated.

Crystal structures of the lumazine protein from Photobacterium kishitanii in complexes with the authentic chromophore, 6,7-dimethyl- 8-(1'-D-ribityl) lumazine, and its analogues, riboflavin and flavin mononucleotide, at high resolution.

Lumazine protein (LumP) is a fluorescent accessory protein having 6,7-dimethyl-8-(1'-d-ribityl) lumazine (DMRL) as its authentic chromophore. It modulates the emission of bacterial luciferase to shorter wavelengths with increasing luminous strength. To obtain structural information on the native structure as well as the interaction with bacterial luciferase, we have determined the crystal structures of LumP from Photobacterium kishitanii in complexes with DMRL and its analogues, riboflavin (RBF) and flavin mononucleotide (FMN), at resolutions of 2.

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition.

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase.

Structural basis for catalytic activation of thiocyanate hydrolase involving metal-ligated cysteine modification.

Thiocyanate hydrolase (SCNase) is a member of a family of nitrile hydratase proteins, each of which contains a unique noncorrin cobalt center with two post-translationally modified cysteine ligands, cysteine-sulfenic acid or -sulfenate (Cys-SO(H)), and cysteine-sulfininate (Cys-SO(2)(-)), respectively. We have found that a partially matured recombinant SCNase was activated during storage. The crystal structures of SCNase before and after storage demonstrated that Cys-SO(2)(-) modification of gammaCys131 proceeded to completion prior to storage, while Cys-SO(H) modification of gammaCys133 occurred during storage.

Mitochondrial ubiquitin ligase MITOL ubiquitinates mutant SOD1 and attenuates mutant SOD1-induced reactive oxygen species generation.

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis.

Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells.

In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, to the medium.

Functional expression of three Rieske non-heme iron oxygenases derived from actinomycetes in Rhodococcus species for investigation of their degradation capabilities of dibenzofuran and chlorinated dioxins.

The activity of Rieske non-heme iron oxygenases (aromatic hydrocarbon dioxygenases, AhDOs) is important for the bacterial degradation of aromatic pollutants such as polycyclic aromatic hydrocarbons and dioxins. During our analysis of the role of AhDOs in dioxin bioremediation, some enzymes derived from high G + C Gram-positive actinomycetes were difficult to produce in active form in the Escherichia coli protein expression system. In this study, we constructed a heterologous expression system for AhDOs in Rhodococcus species using a constitutive expression promoter, P(dfdB), and a shuttle vector, pRK401, and analyzed the ability of these enzymes to degrade dibenzofuran and deplete several chlorinated dioxins.

Identification of casein kinase-1 phosphorylation sites on TDP-43.

TAR DNA-binding protein of 43 kDa (TDP-43) is deposited as hyperphosphorylated cytoplasmic and intranuclear inclusions in brains of patients with frontotemporal lobar degeneration with ubiquitinated inclusions and amyotrophic lateral sclerosis. In this study, we identified 29 phosphorylation sites on recombinant TDP-43 that are phosphorylated by casein kinase-1 (CK1). Interestingly, 18 of them were located in the C-terminal glycine-rich region of TDP-43.

The Triacylated ATP Binding Cluster Transporter Substrate-binding Lipoprotein of Staphylococcus aureus Functions as a Native Ligand for Toll-like Receptor 2.

Some synthetic lipopeptides, in addition to native lipoproteins derived from both Gram-negative bacteria and mycoplasmas, are known to activate TLR2 (Toll-like receptor 2). However, the native lipoproteins inherent to Gram-positive bacteria, which function as TLR2 ligands, have not been characterized. Here, we have purified a native lipoprotein to homogeneity from Staphylococcus aureus to study as a native TLR2 ligand.

Conformational transition of Sec machinery inferred from bacterial SecYE structures.

Over 30% of proteins are secreted across or integrated into membranes. Their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the evolutionarily conserved Sec translocon (SecYEG in prokaryotes and Sec61, comprising alpha-, gamma- and beta-subunits, in eukaryotes). The translocon then functions as a protein-conducting channel.

Mg2+-sensing mechanism of Mg2+ transporter MgtE probed by molecular dynamics study.

Proper regulation of the intracellular ion concentration is essential to maintain life and is achieved by ion transporters that transport their substrates across the membrane in a strictly regulated manner. MgtE is a Mg(2+) transporter that may function in the homeostasis of the intracellular Mg(2+) concentration. A recent crystallographic study revealed that its cytosolic domain undergoes a Mg(2+)-dependent structural change, which is proposed to gate the ion-conducting pore passing through the transmembrane domain.

Novel catalytic activity of nitrile hydratase from Rhodococcus sp. N771.

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme catalyzing the hydration of various nitriles to the corresponding amides. We report a novel catalytic activity of NHase.

Schizosaccharomyces pombe Ddb1 recruits substrate-specific adaptor proteins through a novel protein motif, the DDB-box.

DDB1 was isolated as a UV-damaged DNA-binding protein, but recent studies established that it plays a role as a component of cullin 4A ubiquitin ligases. Cullin-RING complexes are the largest known ubiquitin ligase family, with hundreds of substrate-specific adaptor subunits and which are defined by characteristic motifs. A common motif for DDB1/cullin 4 ubiquitin ligases, a WDXR motif, was recently reported.

The identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase I.

Osteoclasts, bone-resorptive multinucleated cells derived from hematopoietic stem cells, are associated with many bone-related diseases, such as osteoporosis. Osteoclast-targeting small-molecule inhibitors are valuable tools for studying osteoclast biology and for developing antiresorptive agents. Here, we have discovered that methyl-gerfelin (M-GFN), the methyl ester of the natural product gerfelin, suppresses osteoclastogenesis.

Characterization of highly purified photosystem I complexes from the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017.

Photochemically active photosystem (PS) I complexes were purified from the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017, and several of their properties were characterized. PS I complexes consist of 11 subunits, including PsaK1 and PsaK2; a new small subunit was identified and named Psa27. The new subunit might replace the function of PsaI that is absent in A.

Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration.

Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides.