Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

Cytokinesis and cancer:Polo loves ROCK'n' Rho(A)

Cytokinesis is the last step of the M (mitosis) phase,yet it is crucial for the faithful division of one cell into two.Cytokinesis failure is often associated with cancer.Cytokinesis can be morphologically divided into four steps:cleavage furrow initiation,cleavage furrow ingression,midbody formation and abscission.Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis.At the same time,Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis,including cytokinesis.Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks.More specifically,Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1,thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains.Ect2 itself can be phosphorylated by Plk1 in vitro.Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity.Once activated,RhoA-GTP will activate downstream effectors,including ROCK1 and ROCK2.ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen,and Plk1 can phosphorylate ROCK2 in vitro.We review current understandings of the interplay between Plk1,RhoA proteins and other proteins (e.g.,NudC,MKLP2,PRC1,CEP55) involved in cytokinesis,with partitular emphasis of its clinical implications in cancer.     

川芎及两种主要药效成分对废用性肌萎缩的影响

目的：探讨川芎及川芎中起活血作用的两种主要药效成分（阿魏酸钠和川芎嗪）对后肢去负荷大鼠比目鱼肌萎缩的影响与作用。方法：尾部悬吊法建立大鼠废用性肌萎缩模型,用免疫组化技术及血液流变学方法观察药物对比目鱼肌各项指标的影响。结果：与后肢去负荷大鼠相比①高剂量的阿魏酸钠和川芎嗪使比目鱼肌I型肌纤维横截面积分别增加了37.3%和39.4%（P〈0.05）;②三种药物均能明显抑制梭外肌纤维MHCII表达水平的升高（P〈0.01）;③使肌梭内核袋2纤维MHCII的表达由阳性转变为阴性;④并能明显降低低切变率下的全血粘度。结论：川芎及两种主要药效成分阿魏酸钠与川芎嗪均能不同程度地对抗废用性肌萎缩的发生,以高剂量川芎嗪与阿魏酸钠的药效最为明显。     

Myosin VI and Optineurin Are Required for Polarized EGFR Delivery and Directed Migration

The polarized trafficking of membrane proteins into the leading edge of the cell is an integral requirement for cell migration. Myosin VI and its interacting protein optineurin have previously been shown to operate in anterograde trafficking pathways, especially for the polarized delivery of cargo to the basolateral domain in epithelial cells. Here we show that in migratory cells ablation of myosin VI or optineurin inhibits the polarized delivery of the epidermal growth factor receptor (EGFR) into the leading edge and leads to profound defects in lamellipodia formation. Depletion of either myosin VI or optineurin, however, does not impair the overall ability of cells to migrate in a random migration assay, but it dramatically reduces directed migration towards a growth factor stimulus. In summary, we identified a specific role for myosin VI and optineurin in directionally persistent cell migration, which involves the polarized delivery of vesicles containing EGFR into the leading edge of the cell.     

Axial dispositions and conformations of myosin crossbridges along thick filaments in relaxed and contracting states of vertebrate striated muscles by X-ray fiber diffraction

X-ray diffraction patterns from live vertebrate striated muscles were analyzed to elucidate the detailed structural models of the myosin crown arrangement and the axial disposition of two-headed myosin crossbridges along the thick filaments in the relaxed and contracting states. The modeling studies were based upon the previous notion that individual myosin filaments had a mixed structure with two regions, a "regular" and a "perturbed". In the relaxed state the distributions and sizes of the regular and perturbed regions on myosin filaments, each having its own axial periodicity for the arrangement of crossbridge crowns within the basic period, were similar to those reported previously. A new finding was that in the contracting state, this mixed structure was maintained but the length of each region, the periodicities of the crowns and the axial disposition of two heads of a crossbridge were altered. The perturbed regions of the crossbridge repeat shifted towards the Z-bands in the sarcomere without changing the lengths found in the relaxed state, but in which the intervals between three successive crowns within the basic period became closer to the regular 14.5-nm repeat in the contracting state. In high resolution modeling for a myosin head, the two heads of a crossbridge were axially tilted in opposite directions along the three-fold helical tracks of myosin filaments and their axial orientations were different from each other in perturbed and regular regions in both states. Under relaxing conditions, one head of a double-headed crossbridge pair appeared to be in close proximity to another head in a pair at the adjacent crown level in the axial direction in the regular region. In the perturbed region this contact between heads occurred only on the narrower inter-crown levels. During contraction, one head of a crossbridge oriented more perpendicular to the fiber axis and the partner head flared axially. Several factors that significantly influence the intensities of the myosin based-meridional reflections and their relative contributions are discussed.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.     

Inhibition of the myosin light chain kinase prevents hypoxia-induced blood-brain barrier disruption

Increased mortality after stroke is associated with development of brain edema. The aim of the present study was to examine the contribution of endothelial myosin light chain (MLC) phosphorylation to hypoxia-induced blood-brain barrier (BBB) opening. Measurements of trans-endothelial electrical resistance (TEER) were performed to analyse BBB integrity in an in vitro co-culture model (bovine brain microvascular endothelial cells (BEC) and rat astrocytes). Brain fluid content was analysed in rats after stroke induction using a two-vein occlusion model. Dihydroethidium was used to monitor intracellular generation of reactive oxygen species (ROS) in BEC. MLC phosphorylation was detected using immunohistochemistry and immunoblot analysis. Hypoxia caused a decrease of TEER values by more than 40%, which was prevented by inhibition of the MLC-kinase (ML-7, 10 micromol/L). In addition, ML-7 significantly reduced the brain fluid content in vivo after stroke. The NAD(P)H-oxidase inhibitor apocynin (500 micromol/L) prevented the hypoxia-induced TEER decrease. Hypoxia-dependent ROS generation was completely abolished by apocynin. Furthermore, ML-7 and apocynin blocked hypoxia-dependent phosphorylation of MLC. Our data demonstrate that hypoxia causes a breakdown of the BBB in vitro and in vivo involving ROS and the contractile machinery.     

unc-94 encodes a tropomodulin in Caenorhabditis elegans

unc-94 is one of about 40 genes in Caenorhabditis elegans that, when mutant, displays an abnormal muscle phenotype. Two mutant alleles of unc-94, su177 and sf20, show reduced motility and brood size and disorganization of muscle structure. In unc-94 mutants, immunofluorescence microscopy shows that a number of known sarcomeric proteins are abnormal, but the most dramatic effect is in the localization of F-actin, with some abnormally accumulated near muscle cell-to-cell boundaries. Electron microscopy shows that unc-94(sf20) mutants have large accumulations of thin filaments near the boundaries of adjacent muscle cells. Multiple lines of evidence prove that unc-94 encodes a tropomodulin, a conserved protein known from other systems to bind to both actin and tropomyosin at the pointed ends of actin thin filaments. su177 is a splice site mutation in intron 1, which is specific to one of the two unc-94 isoforms, isoform a; sf20 has a stop codon in exon 5, which is shared by both isoform a and isoform b. The use of promoter-green fluorescent protein constructs in transgenic animals revealed that unc-94a is expressed in body wall, vulval and uterine muscles, whereas unc-94b is expressed in pharyngeal, anal depressor, vulval and uterine muscles and in spermatheca and intestinal epithelial cells. By Western blot, anti-UNC-94 antibodies detect polypeptides of expected size from wild type, wild-type-sized proteins of reduced abundance from unc-94(su177), and no detectable unc-94 products from unc-94(sf20). Using these same antibodies, UNC-94 localizes as two closely spaced parallel lines flanking the M-lines, consistent with localization to the pointed ends of thin filaments. In addition, UNC-94 is localized near muscle cell-to-cell boundaries.     

非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。     

平滑肌肌球蛋白轻链激酶的非激酶作用及对ATP 酶活性的调节

肌球蛋白轻链激酶（myosin light chain kinase, MLCK）具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响,以进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制.采用PCR技术构建MLCK部分氨基酸缺失的重组表达载体pGEX-F6-5/D,经大肠杆菌表达得到可溶性GST融合蛋白,利用SDS-PAGE及Western 印迹鉴定表达的MLCK在细胞中的分布,结果还显示,提取液的上清和沉淀中均有MLCK片段的表达.运用亲和层析技术分离并纯化删除前、后表达的MLCK片段（F6.5和F6-5/D）,经谷胱甘肽琼脂糖凝胶 4B 纯化,SDS-PAGE鉴定显示为单一表达条带.应用EnzChek磷分析试剂盒和孔雀绿两种方法分别测定不同浓度的MLCK对非磷酸化肌球蛋白Mg²⁺-ATP酶活性的影响.两种MLCK的片段均具有激活ATP酶活性的作用,并随MLCK浓度的增加,酶的活性增加.比较删除前后不同MLCK片段对ATP酶活性的影响结果显示,删除MLCK片段1002位丙氨酸至1019位亮氨酸后,对ATP酶的激活作用较删除前明显降低,表明删除的部分氨基酸序列为MLCK非激酶活性所必需的区域.利用电镜技术观察到MLCK片段（F6.5）使非磷酸化肌球蛋白构象发生明显的变化.加入MLCK片段后肌球蛋白的构象由非活性型转化为活性型,并且MLCK片段还具有促进肌球蛋白单体形成肌丝的作用.