Pyrrole derivatives as potent inhibitors of lymphocyte-specific kinase: Structure,synthesis, and SAR

We have described the synthesis, enzyme inhibitory activity, structure–activity relationships, and proposed binding mode of a novel series of pyrrole derivatives as lymphocyte-specific kinase (Lck) inhibitors. The most potent analogs exhibited good enzyme inhibitory activity (IC₅₀s <10 nM) for Lck kinase inhibition.     

Discovery of novel N-acylsulfonamide analogs as potent and selective EP3 receptor antagonists

A series of novel N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated for their inhibitory effect on PGE₂-induced uterine contraction in pregnant rats. Among those tested, a series of N-acylbenzenesulfonamide analogs were found to be more potent than the corresponding carboxylic acid analogs in both the in vitro and in vivo evaluations. The structure activity relationships (SAR) are also discussed.     

The carbohydrate-binding module (CBM)-like sequence is crucial for rice CWA1/BC1 function in proper assembly of secondary cell wall materials

We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials.Key words: carbohydrate-binding module, COBRA-LIKE, CWA1/BC1, glycosylphosphatidylinositol-anchored protein, secondary cell wall formationThe main function of carbohydrate-binding modules (CBMs) of microbes and plants is to attach the enzyme to a variety of cell surface glycans and thereby increase the local concentration of substrate, leading to more efficient catalysis.^1–4 Almost all CBMs studied to date contain surface-exposed aromatic rings, which have been shown to be the main sites of interaction with polysaccharides. These residues form face-to-face hydrophobic stacking interactions in which a Trp residue or ring of a Tyr residue interacts with the non-polar face of a sugar ring.^5–9 CBMs have been classified into families based on amino acid sequence similarity. Currently, there are 59 defined families of CBMs and these CBMs display substantial variation in ligand specificity (http://www.cazy.org/Carbohydrate-Binding-Modules.html). Among these CBM families, the large family of CBM2 has been further classified into two subgroups, CBM2a and 2b, which have shown to bind cellulose and xylan, respectively.^10–12 CBM2a characteristically possess three exposed Trp residues,¹³ whereas CBM2b have two Trp residues,¹⁴ which are conserved among the CBM2 members (Fig. 1A).Open in a separate windowFigure 1Sequence alignment of the CBM-like sequence of CWA1/BC1, the BC1L proteins and bacterial CBM2 members. (A) Sequence alignment between bacterial CBM2a, 2b and CWA1/BC1. The three surface-exposed Trp residues of CBM2a members are indicated by asterisks and W. The CBM sequences of CBM2a are: CfiCenA, Cellulomonas fimi endo-1,4-glucanase; CfiCex, C. fimi exo-beta-1,4-glucanase. Those of CBM2b are: CfiXylD1, C. fimi endo-1,4-beta-xylanase D; CfiXylD2, C. fimi endo-1,4-beta-xylanase. CWA1/BC1 shows weak similarity to CBM2, and some Trp residues are conserved with bacterial CBM2 members. (B) Sequence alignment of CWA1/BC1, the BC1L proteins and CWA1/BC1 orthologs, Zea maiz Brittle Stalk 2 (ZmBk2) and Arabidopsis thaliana COBRA-LIKE 4 (AtCOBL4). The CBM-like sequence of CWA1/BC1, especially the Trp residues, is highly conserved among the analyzed sequences. Substituted Trp (W) residues to Val (V) in CWA1/BC1 are indicated by closed triangles. Numbers at the left are the positions of the amino acids in each protein, with gaps (dashes) included to maximize alignments. Identical and similar amino acids are shaded and gray, respectively.Our recent study showed that the defect of the rice CWA1/BC1 (CELL WALL ARCHITECTURE 1/BRITTLE CULM 1) gene induced abnormal secondary cell wall formation with amorphous and bulky structures at the cytoplasm side and CWA1/BC1 encodes one of COBRA-like glycosylphosphatidylinositol (GPI)-anchored proteins, which are specifically found in plants, suggesting that CWA1/BC1 regulates assembly of secondary cell wall materials in rice sclerenchyma. Furthermore, several reports have shown that the N-terminus of rice CWA1/BC1 and other COBRA-like GPI-anchored proteins in Arabidopsis (12 members) and maize Brittle Stalk 2 (Bk2) share weak similarity to a CBM2 in several bacterial cellulases.^15,16 However, the importance of CBM-like sequence in COBRA family members has not been clarified. To investigate the nature of CWA1/BC1, we compared the CBM-like sequence in rice CWA1/BC1 with bacterial CBM2, 10 members of the BC1-like (BC1L) protein in rice and CWA1/BC1 orthologs, Arabidopsis COBL4 and maize Bk2. Furthermore, we constructed three-point mutated CWA1/BC1, in which three conserved Trp residues in CBM-like sequence were substituted for Val residues (CWA1/BC1^W→V), and introduced it into the cwa1 mutant to evaluate the necessity of the CBM-like sequence for proper function of CWA1/BC1. We discuss a putative explanation, based on our results, of the properties and possible functions of CWA1/BC1.     

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. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation,catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.     

Purification,Biochemical Characterization,and Cloning of Phospholipase D from <Emphasis Type="Italic">Streptomyces racemochromogenes</Emphasis> Strain 10-3

We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71–76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, l-serine, and 2-aminoethanol hydrochloride with a conversion rate of 82–97% in a simple one-phase system, which was comparable to the rate of other Streptomyces PLDs in a complicated biphasic system.     

Molecular Functions of Oxygen-Evolving Complex Family Proteins in Photosynthetic Electron Flow

Oxygen-evolving complex (OEC) protein is the original name for membrane-peripheral subunits of photosystem (PS) Ⅱ. Recently,multiple isoforms and homologs for OEC proteins have been identified in the chloroplast thylakoid lumen, indicating that functional diversification has occurred in the OEC family. Gene expression profiles suggest that the Arabidopsis OEC proteins are roughly categorized into three groups: the authentic OEC group, the stressresponsive group, and the group including proteins related to the chloroplast NAD(P)H dehydrogenase (NDH) complex involved in cyclic electron transport around PSI. Based on the above gene expression profiles, molecular functions of the OEC family proteins are discussed together with our current knowledge about their functions.     

Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.