Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL(Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit.     

Phosphoinositide 3-kinase delta (PI3Kδ) in leukocyte signaling and function

PI3Kδ is a lipid kinase of the PI3K class IA family involved in early signaling events of leukocytes responding to a wide variety of stimuli. The leukocyte specificity of PI3Kδ is defined by its expression, whereas its signaling function is via the production of phosphoinositide 3,4,5-triphosphates at the proximity of activated receptors for recruiting other signaling molecules. The importance of PI3Kδ in B cell development and function is most apparent, and its role in other leukocyte cell types can be easily demonstrated as well. PI3Kδ participates in the development, activation and migration of T cells and NK cells. The role of PI3Kδ in myeloid cell activities, such as inflammation driven cell infiltration, neutrophil oxidative burst, immune complex mediated macrophage activation, as well as mast cell maturation and degranulation, has been well illustrated in various studies. As a result of the broad effects of PI3Kδ in leukocyte functions, the disruption of PI3Kδ expression or activity leads to decreased inflammatory and immune responses in vivo. The protective role of PI3Kδ inactivation in animal models of arthritis, asthma or obstructive respiratory diseases has been demonstrated. These findings suggest the potential efficacy achievable with PI3Kδ inhibitors in the treatment of autoimmune and respiratory diseases.     

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding V_H CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long V_H CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long V_H CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.     

Homology model and targeted mutagenesis identify critical residues for arachidonic acid inhibition of Kv4 channels

Polyunsaturated fatty acids such as arachidonic acid (AA) exhibit inhibitory modulation of Kv4 potassium channels. Molecular docking approaches using a Kv4.2 homology model predicted a membrane-embedded binding pocket for AA comprised of the S4-S5 linker on one subunit and several hydrophobic residues within S3, S5 and S6 from an adjacent subunit. The pocket is conserved among Kv4 channels. We tested the hypothesis that modulatory effects of AA on Kv4.2/KChIP channels require access to this site. Targeted mutation of a polar residue (K318) and a nonpolar residue (G314) within the S4-S5 linker as well as a nonpolar residue in S3 (V261) significantly impaired the effects of AA on K⁺ currents in Xenopus oocytes. These residues may be important in stabilizing (K318) or regulating access to (V261, G314) the negatively charged carboxylate moiety on the fatty acid. Structural specificity was supported by the lack of disruption of AA effects observed with mutations at residues located near, but not within the predicted binding pocket. Furthermore, we found that the crystal structure of the related Kv1.2/2.1 chimera lacks the structural features present in the proposed AA docking site of Kv4.2 and the Kv1.2/2.1 K⁺ currents were unaffected by AA. We simulated the mutagenic substitutions in our Kv4.2 model to demonstrate how specific mutations may disrupt the putative AA binding pocket. We conclude that AA inhibits Kv4 channel currents and facilitates current decay by binding within a hydrophobic pocket in the channel in which K318 within the S4-S5 linker is a critical residue for AA interaction.     

Homology of prostomial and pharyngeal structures in eunicida (Annelida) based on innervation and morphological similarities

Eunicidan bristle worm families are commonly identified by the shape of their prostomia and pharyngeal structures. However, current hypotheses of homology among these structures are conflicting, making it difficult to assess morphological evolution, reconstruct phylogeny, and produce a stable classification. To generate more consistent hypotheses of homology among eunicidan anterior structures, the author examined the anterior morphology and the nervous system stained with anti‐α‐tubulin and serotonin antibodies in representative species of Eunicidae, Onuphidae, Oenonidae, Dorvilleidae and Lumbrineridae. The shape of the brain varied conspicuously among families; however, it has mostly the same commissures (usually two of the dorsal and five of the ventral roots of the circumoesophageal connective). The stomatogastric system is also conservative in composition, having two main pairs of stomatogastric nerves which vary in their relative position among the different families. Innervation similarities combined with correspondence and topological morphological similarities made it possible to present explicit hypotheses of primary homology of features, such as buccal lips, pharyngeal fold, and dorsolateral fold anterior extension. Buccal lips are present in all families; however, ventral pads on the prostomium of the Dorvillea line of Dorvilleidae are anterior prolongations of the pharyngeal fold and not buccal lips. All examined taxa, except dorvilleid species, have conspicuous dorsolateral fold anterior extension. In Eunicidae, this anterior extension is a transverse band, while in other families it is a pair of folds. Observed similarities also gave insights on the homology of maxillary elements of Dorvilleidae, providing background knowledge for future studies. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

BRCA1蛋白的序列及空间结构的分析

查询人的BRCA1蛋白的氨基酸序列,利用生物信息学的方法进行相似性搜索,获得一系列BRCA1蛋白的氨基酸序列。选择了其中的11条序列,对BRCA1蛋白进行了多重序列分析和进化分析,对BRCA1蛋白的BRCT结构域进行三维同源模型的构建与比较分析。分析结果表明:BRCA1中某些特定部位的氨基酸序列高度保守;确定氨基酸的保守位点并联合进化分析可对基因错义突变的致病性做初步地猜测;相近物种来源的BRCA1具有较近的亲缘关系,而且具有极其相似的三维空间结构。这些为研究BRCA1蛋白的结构与功能关系提供指导意义。     

各地区人类嗜T细胞病毒Ⅰ型的聚类分析

已有相关文献表明人类嗜T细胞病毒Ⅰ型(Human T-lymphotropic virus 1,记为HTLV-Ⅰ)的分布具有区域性,本文旨在提出不同的分析区域性的方法。首先从GenBank中选取来自亚洲、南美洲、非洲的共20条核苷酸序列,用分子生物学软件Vector NTI Suite分析各地区序列样本内部的同源性,然后以各序列的氨基酸含量为对象,定义一个全新的公式进行同源性分析,将该结果与其他研究者采用实验的方法的分析结果比较。结果发现不同的分析方法所得的结论均是一致的。这表明:HTLV-Ⅰ病毒的分布有明显的区域性,文章采用的研究方法对其他流行病学的研究是同样可行的。     

Diversity and three-dimensional structures of the alpha Mcr of the methanogenic Archaea from the anoxic region of Tucuruí Lake, in Eastern Brazilian Amazonia

Methanogenic archaeans are organisms of considerable ecological and biotechnological interest that produce methane through a restricted metabolic pathway, which culminates in the reaction catalyzed by the Methyl-coenzyme M reductase (Mcr) enzyme, and results in the release of methane. Using a metagenomic approach, the gene of the α subunit of mcr (mcrα) was isolated from sediment sample from an anoxic zone, rich in decomposing organic material, obtained from the Tucuruí hydroelectric dam reservoir in eastern Brazilian Amazonia. The partial nucleotide sequences obtained were 83 to 95% similar to those available in databases, indicating a low diversity of archaeans in the reservoir. Two orders were identified - the Methanomicrobiales, and a unique Operational Taxonomic Unit (OTU) forming a clade with the Methanosarcinales according to low bootstrap values. Homology modeling was used to determine the three-dimensional (3D) structures, for this the partial nucleotide sequence of the mcrα were isolated and translated on their partial amino acid sequences. The 3D structures of the archaean Mcrα observed in the present study varied little, and presented approximately 70% identity in comparison with the Mcrα of Methanopyrus klanderi. The results demonstrated that the community of methanogenic archaeans of the anoxic C1 region of the Tucurui reservoir is relatively homogeneous.     

Identification and characterization of splicing variants of PLEKHA5 (Plekha5) during brain development

PLEKHA5 (pleckstrin homology domain-containing protein family A, member 5) belongs to the PLEKHA family (PLEKHA1-6); however, the properties of this protein remain poorly characterized. We have identified and characterized two forms of PLEKHA5 mRNA. The long form of PLEKHA5 (L-PLEKHA5) contains 32 exons, encodes 1282 amino acids, and is specifically expressed in the brain; the short form of PLEKHA5 (S-PLEKHA5) is generated by alternative splicing of L-PLEKHA5, contains 26 exons, encodes 1116 amino acids, and is ubiquitously expressed. Both forms of the protein contain putative Trp-Trp (WW) and pleckstrin homology (PH) domains and are located mainly in the cytosol. Developmental and age-dependent expression studies in the mouse brain have shown that Plekha5 is the most abundantly expressed protein at E13.5 with S-Plekha5 dominancy. L-Plekha5 levels increased gradually with the decrease in total Plekha5 levels; moreover, L-Plekha5 became the dominant protein at E17.5, maintaining its dominance throughout adulthood. Protein-lipid overlay assays have indicated that the PH domain of PLEKHA5 specifically interacts with PI3P, PI4P, PI5P, and PI(3,5)P2. These results suggest that the S- to L-conversion of PLEKHA5 (Plekha5) may play an important role in brain development through association with specific phosphoinositides.     

Mechanical regulation of signaling pathways in bone

A wide range of cell types depend on mechanically induced signals to enable appropriate physiological responses. The skeleton is particularly dependent on mechanical information to guide the resident cell population towards adaptation, maintenance and repair. Research at the organ, tissue, cell and molecular levels has improved our understanding of how the skeleton can recognize the functional environment, and how these challenges are translated into cellular information that can site-specifically alter phenotype. This review first considers those cells within the skeleton that are responsive to mechanical signals, including osteoblasts, osteoclasts, osteocytes and osteoprogenitors. This is discussed in light of a range of experimental approaches that can vary parameters such as strain, fluid shear stress, and pressure. The identity of mechanoreceptor candidates is approached, with consideration of integrins, pericellular tethers, focal adhesions, ion channels, cadherins, connexins, and the plasma membrane including caveolar and non-caveolar lipid rafts and their influence on integral signaling protein interactions. Several mechanically regulated intracellular signaling cascades are detailed including activation of kinases (Akt, MAPK, FAK), β-catenin, GTPases, and calcium signaling events. While the interaction of bone cells with their mechanical environment is complex, an understanding of mechanical regulation of bone signaling is crucial to understanding bone physiology, the etiology of diseases such as osteoporosis, and to the development of interventions to improve bone strength.