'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.     

Novel scaffold for cathepsin K inhibitors

Pyrrolopyrimidine, a novel scaffold, allows to adjust interactions within the S3 subsite of cathepsin K. The core intermediate 10 facilitated the P3 optimization and identified highly potent and selective cathepsin K inhibitors 11-20.     

On-chip identification and interaction analysis of gel-resolved proteins using a diamond-like carbon-coated plate

We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect protein-protein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip.     

Valproic Acid-Induced Anxiety and Depression Behaviors are Ameliorated in p39 Cdk5 Activator-Deficient Mice

Neurochemical Research - Valproic acid (VPA) is a drug used for the treatment of epilepsy, seizures, migraines, and bipolar disorders. Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase activated...     

Plasma gelsolin facilitates interaction between β2 glycoprotein I and α5β1 integrin

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma β₂‐glycoprotein I (β₂GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen‐activated protein kinase (MAPK) pathway plays an important role in aPL‐induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with β₂GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous β₂GPI interacts with plasma gelsolin, which binds to integrin a₅β₁ through fibronectin. The tethering of β₂GPI to monoclonal anti‐β₂GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti‐β₂GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti‐integrin a₅β₁ antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin‐integrin signalling pathway, was phosphorylated by anti‐β₂GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti‐β₂GPI antibody‐induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS.     

Comparison of ecophysiological responses to heavy snow in two varieties ofAucuba japonica with different areas of distribution

Aucuba japonica varieties are common evergreen understory shrubs in Japan.Aucuba japonica var.borealis is distributed on the Sea of Japan side of Honshu and Hokkaido where heavy snow cover lasts for more than 3 months in winter.Aucuba japonica var.japonica is distributed in areas with shallow or no snow on the Pacific Ocean side of Honshu and Shikoku. The ecophysiological characteristics of var.borealis were compared with those of var.japonica to examine the effects of heavy and long-term snow cover on the life cycle of var.borealis. Shoots of both varieties were shaded in crushed ice for 110 days, but their photosynthetic activities, chlorophyll contents and the chlorophylla/b ratio was not affected. The leaves of var.borealis were no less frost tolerant than those of var.japonica. In spite of the difference in environmental factors, both varieties had similar characteristics in seasonal changes of photosynthesis, respiration and chlorophylla/b ratio. These results suggest that var.japonica could survive in areas with heavy snow where it does not normally occur. Leaf net production (LNP) was estimated based on the microclimatic data and seasonal photosynthetic and respiration rates. The difference in the annual LNP between the two varieties was equivalent to the difference in the LNP during the snow season. One of the major effects of snow cover is to interrupt and reduce the production period of var.borealis.     

Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.     

Crystal structure of the RNA 2'-phosphotransferase from Aeropyrum pernix K1

In the final step of tRNA splicing, the 2'-phosphotransferase catalyzes the transfer of the extra 2'-phosphate from the precursor-ligated tRNA to NAD. We have determined the crystal structure of the 2'-phosphotransferase protein from Aeropyrum pernix K1 at 2.8 Angstroms resolution. The structure of the 2'-phosphotransferase contains two globular domains (N and C-domains), which form a cleft in the center. The N-domain has the winged helix motif, a subfamily of the helix-turn-helix family, which is shared by many DNA-binding proteins. The C-domain of the 2'-phosphotransferase superimposes well on the NAD-binding fold of bacterial (diphtheria) toxins, which catalyze the transfer of ADP ribose from NAD to target proteins, indicating that the mode of NAD binding by the 2'-phosphotransferase could be similar to that of the bacterial toxins. The conserved basic residues are assembled at the periphery of the cleft and could participate in the enzyme contact with the sugar-phosphate backbones of tRNA. The modes by which the two functional domains recognize the two different substrates are clarified by the present crystal structure of the 2'-phosphotransferase.     

Benzimidazole derivatives as novel nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 1: Benzimidazole-5-sulfonamides

A new class of benzimidazole-5-sulfonamides has been identified as nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Initial structure-activity relationships are presented resulting in compounds 19 and 28 with submicromolar dual functional activity on human and rat receptors.