Structural mechanism for statin inhibition of HMG-CoA reductase

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. Statins are HMGR inhibitors with inhibition constant values in the nanomolar range that effectively lower serum cholesterol levels and are widely prescribed in the treatment of hypercholesterolemia. We have determined structures of the catalytic portion of human HMGR complexed with six different statins. The statins occupy a portion of the binding site of HMG-CoA, thus blocking access of this substrate to the active site. Near the carboxyl terminus of HMGR, several catalytically relevant residues are disordered in the enzyme-statin complexes. If these residues were not flexible, they would sterically hinder statin binding.     

体外Src蛋白酪氨酸激酶抑制剂筛选模型的建立

在体外构建一个以Src蛋白为靶位点的蛋白酪氨酸激酶抑制剂快速筛选模型,为筛选Src蛋白酪氨酸激酶抑制剂奠定基础.基因工程表达GST-v-Src蛋白,收集包涵体蛋白,并经变性复性处理.以生物素化的聚Glu∶Tyr(4∶1)为激酶反应底物、以辣根过氧化物酶(horseradish peroxidase,HRP)标记的磷酸酪氨酸特异性单克隆抗体(HRP-PY20)检测磷酸酪氨酸残基的酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)测定获得蛋白的激酶活性,并进行抑制剂筛选.该模型具有靶向性强、快速、简便可行、高通量的特点.用该方法对6种治疗肿瘤配方中常用的中草药进行筛选,筛选出3种中草药水煎剂富含Src蛋白酪氨酸激酶抑制剂成分.     

EGF receptor and erbB-2 tyrosine kinase domains confer cell specificity for mitogenic signaling

The epidermal growth factor (EGF) receptor (EGFR) can efficiently couple with mitogenic signaling pathways when it is transfected into interleukin-3 (IL-3)-dependent 32D hematopoietic cells. When expression vectors for erbB-2, which is structurally related to EGFR, or its truncated counterpart, delta NerbB-2, were introduced into 32D cells, neither was capable of inducing proliferation. This was despite overexpression and constitutive tyrosine kinase activity of their products at levels associated with potent transformation of fibroblast target cells. Thus, EGFR and erbB-2 couple with distinct mitogenic signaling pathways. The region responsible for the specificity of intracellular signal transduction was localized to a 270-amino acid stretch encompassing their respective tyrosine kinase domains. Thus, tissue- or cell-specific regulation of growth factor receptor signaling can occur at a point after the initial interaction of growth factor with receptor. Such specificity in signal transduction may account for the selection of certain oncogenes in some malignancies.     

A steric mechanism for inhibition of CO binding to heme proteins

The crystal structures of myoglobin in the deoxy- and carbon monoxide-ligated states at a resolution of 1.15 angstroms show that carbon monoxide binding at ambient temperatures requires concerted motions of the heme, the iron, and helices E and F for relief of steric inhibition. These steps constitute the main mechanism by which heme proteins lower the affinity of the heme group for the toxic ligand carbon monoxide.     

Maturation of a stress-activated mechanism inhibiting induction of tyrosine transaminase

Rats of various ages were subjected to the stress of 30 minutes on a noisy reciprocating shaker 4 hours before their liver tyrosine transaminase and tryptophan pyrrolase activities were measured. Adrenalectomized infants and adults and hypophysectomized adults were also stressed. Intact, stressed infants exhibited an increase in tyrosine transaminase activity, while intact, stressed adults showed no change. In the stressed adrenalectomized adult, tyrosine transaminase activity markedly decreased, while adrenalectomized infants showed no change. Hypophysectomy largely, but not completely, abolished inhibition in the adults. Tryptophan pyrrolase activity, when present, was increased by stress in all age groups, but the increase was abolished by adrenalectomy and hypophysectomy. The results suggest stress-activation of a pituitary mechanism that inhibits or represses activation of tyrosine transaminase and that may not function during early postnatal life.     

Structural evidence for a two-metal-ion mechanism of group I intron splicing

We report the 3.4 angstrom crystal structure of a catalytically active group I intron splicing intermediate containing the complete intron, both exons, the scissile phosphate, and all of the functional groups implicated in catalytic metal ion coordination, including the 2'-OH of the terminal guanosine. This structure suggests that, like protein phosphoryltransferases, an RNA phosphoryltransferase can use a two-metal-ion mechanism. Two Mg2+ ions are positioned 3.9 angstroms apart and are directly coordinated by all six of the biochemically predicted ligands. The evolutionary convergence of RNA and protein active sites on the same inorganic architecture highlights the intrinsic chemical capacity of the two-metal-ion catalytic mechanism for phosphoryl transfer.     

Protein tyrosine kinase Wee1B is essential for metaphase II exit in mouse oocytes

Waves of cyclin synthesis and degradation regulate the activity of Cdc2 protein kinase during the cell cycle. Cdc2 inactivation by Wee1B-mediated phosphorylation is necessary for arrest of the oocyte at G2-prophase, but it is unclear whether this regulation functions later during the metaphase-to-anaphase transition. We show that reactivation of a Wee1B pathway triggers the decrease in Cdc2 activity during egg activation. When Wee1B is down-regulated, oocytes fail to form a pronucleus in response to Ca(2+) signals. Calcium-calmodulin-dependent kinase II (CaMKII) activates Wee1B, and CaMKII-driven exit from metaphase II is inhibited by Wee1B down-regulation, demonstrating that exit from metaphase requires not only a proteolytic degradation of cyclin B but also the inhibitory phosphorylation of Cdc2 by Wee1B.     

Structural basis for the inhibition of protein biosynthesis: mode of action of tubulosine

A structural model for the inhibition of protein biosynthesis was previously formulated on the basis of a topochemical analogy between the ipecac alkaloids and the glutarimide antibiotics. The structure of tubulosine satisfies the requirements of this model. The prediction that such a compound would exhibit amebicidal activity and act by selectively inhibiting the transfer reaction in protein biosynthesis is confirmed.     

Ionic mechanism of cholinergic inhibition in molluscan neurons

Acetylcholine, the inhibitory transmitter to the so-called H-neurons of molluscs, produces its effect by increasing the permeability of the subsynaptic membrane to chloride ions. The change in permeability gives rise to a net influx of this anion, which hyperpolarizes the neuron. The presence of an outward pump of chloride ions is postulated to account for the required electrochemical gradient. The participation of potassium ions in this inhibitory phenomenon was not detected.     

Structural bioinformatics-based design of selective, irreversible kinase inhibitors

The active sites of 491 human protein kinase domains are highly conserved, which makes the design of selective inhibitors a formidable challenge. We used a structural bioinformatics approach to identify two selectivity filters, a threonine and a cysteine, at defined positions in the active site of p90 ribosomal protein S6 kinase (RSK). A fluoromethylketone inhibitor, designed to exploit both selectivity filters, potently and selectively inactivated RSK1 and RSK2 in mammalian cells. Kinases with only one selectivity filter were resistant to the inhibitor, yet they became sensitized after genetic introduction of the second selectivity filter. Thus, two amino acids that distinguish RSK from other protein kinases are sufficient to confer inhibitor sensitivity.     

Structure of a tyrosine phosphatase adhesive interaction reveals a spacer-clamp mechanism

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. Human RPTPmu is a type IIB receptor protein tyrosine phosphatase that both forms an adhesive contact itself and is involved in regulating adhesion by dephosphorylating components of cadherin-catenin complexes. Here we describe a 3.1 angstrom crystal structure of the RPTPmu ectodomain that forms a homophilic trans (antiparallel) dimer with an extended and rigid architecture, matching the dimensions of adherens junctions. Cell surface expression of deletion constructs induces intercellular spacings that correlate with the ectodomain length. These data suggest that the RPTPmu ectodomain acts as a distance gauge and plays a key regulatory function, locking the phosphatase to its appropriate functional location.     

Turnover of rat liver tyrosine transaminase: stabilization after inhibition of protein synthesis

Turnover of the rat liver tyrosine transaminase in vivo was measured by a label and chase procedure under conditions where the amount of enzyme undergoes no change. Half-life of the (14)C-labeled enzyme in this basal condition was found to be 1.5 +/- 0.3 hours. Inhibitors of protein synthesis (cycloheximide or puromycin) do not appreciably influence the basal enzyme level over a 5-hour period, although these drugs will block hormonal induction of this enzyme. In pulse-labeling experiments, cycloheximide blocked transaminase synthesis almost completely. The conclusion that enzyme degradation, as well as synthesis, must be blocked when protein synthesis is stopped was confirmed in experiments showing that labeled enzyme is stable in the liver of rats treated with cycloheximide The participation of a continuously synthesized polypeptide in the degradative phase of transaminase turnover is suggested.     

DDT-induced inhibition of avian shell gland carbonic anhydrase: a mechanism for thin eggshells

The shell-forming glands of Japanese quail fed p,p'-DDT or p,p'-DDE had carbonic anhydrase activity 16 to 19 percent lower than shell glands of quail on a diet free of pesticides.     

Structural mechanism of endosome docking by the FYVE domain

The recruitment of trafficking and signaling proteins to membranes containing phosphatidylinositol 3-phosphate [PtdIns(3)P] is mediated by FYVE domains. Here, the solution structure of the FYVE domain of the early endosome antigen 1 protein (EEA1) in the free state was compared with the structures of the domain complexed with PtdIns(3)P and mixed micelles. The multistep binding mechanism involved nonspecific insertion of a hydrophobic loop into the lipid bilayer, positioning and activating the binding pocket. Ligation of PtdIns(3)P then induced a global structural change, drawing the protein termini over the bound phosphoinositide by extension of a hinge. Specific recognition of the 3-phosphate was determined indirectly and directly by two clusters of conserved arginines.     

The product of the c-fms proto-oncogene: a glycoprotein with associated tyrosine kinase activity

The c-fms proto-oncogene is a member of a gene family that has been implicated in tumorigenesis. Glycoproteins encoded by c-fms were identified in cat spleen cells by means of an immune-complex kinase assay performed with monoclonal antibodies to v-fms-coded epitopes. The major form of the normal cellular glycoprotein has an apparent molecular weight of 170,000 and, like the product of the viral oncogene, serves as a substrate for an associated tyrosine-specific protein kinase activity in vitro. The results suggest that the transforming glycoprotein specified by v-fms is a truncated form of a c-fms-coded growth factor receptor.     

Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells

Several pathways of transmembrane signaling in lymphocytes involve protein-tyrosine phosphorylation. With the exception of p56lck, a tyrosine kinase specific to T lymphoid cells that associates with the T cell transmembrane proteins CD4 and CD8, the kinases that function in these pathways are unknown. A murine lymphocyte complementary DNA that represents a new member of the src family has now been isolated and characterized. This complementary DNA, termed blk (for B lymphoid kinase), specifies a polypeptide of 55 kilodaltons that is related to, but distinct from, previously identified retroviral or cellular tyrosine kinases. The protein encoded by blk exhibits tyrosine kinase activity when expressed in bacterial cells. In the mouse and among cell lines, blk is specifically expressed in the B cell lineage. The tyrosine kinase encoded by blk may function in a signal transduction pathway that is restricted to B lymphoid cells.     

抑制杨木APMP纸浆返黄技术的研究(Ⅲ)——助剂用量及抑制杨木APMP纸浆返黄机理的研究

优选助剂组合ZW-1/抗坏血酸/次亚磷酸钠处理黑杨APMP纸浆取得了令人满意的光稳定效果.用紫外光辐照、干/湿热老化处理,作者对组合中各助剂优化用量以及助剂组合处理改善黑杨APMP纸浆光稳定性能的机理进行了研究.结果表明,组合中各助剂优化用量分别为0.25%,0.5%,1.0%;助剂组合处理能够有效改善黑杨APMP纸浆光稳定性能,是因为减少了模拟光老化过程中芳核羧基的含量.     

香菇多糖抗氧化及其抗真菌机制初步研究

香菇子实体干粉经乙醇除杂、水提、除蛋白、乙醇分级沉淀等不同方法后,得到Len-40、Len-60、Len-80等3种不同多糖组分,采用邻苯三酚自氧化体系和Fenton体系考察其清除自由基的能力,滤纸片法考察其对真菌的抑菌效果.结果表明,香菇多糖不同组分均具有抗氧化活性,抗氧化能力由强到弱依次为Len-40＞Len-60＞Len-80;该多糖对温度不敏感,通过导致菌丝断裂、相互缠绕、死亡等方式达到抑菌的效果.     

Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor

The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the proband's lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.