Rapid characterization of spike variants via mammalian cell surface display

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Cascade of autophosphorylation in the beta-subunit of the insulin receptor

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.     

大豆下胚轴可溶性蛋白中钙激活的蛋白激酶

大豆（Ｇｌｙｃｉｎｅ ｍ ａｘ Ｌ．） 下胚轴可溶性蛋白提取液进行自磷酸化，以ＳＤＳ－ＰＡＧＥ电泳分析其标记产物时发现，当有较高浓度的Ｃａ２＋ 存在于反应液中时，有一条１８ ｋＤ蛋白带被高强度标记，同时也可观察到另一条标记强度不高的６７ ｋＤ蛋白带． 当反应时间延长到１５ 或３０ｍ ｉｎ 时，它们的标记强度都逐渐减弱，最终从放射自显影底片上消失；在反应液中加入钙螯合剂ＥＧＴＡ 时，则只有６７ ｋＤ 被高强度标记；在磷酸化反应过程中加入非标记ＡＴＰ，蛋白中的３２Ｐ逐渐被非标记磷取代，表明反应体系处于磷酸化－脱磷酸化的平衡过程中，并有结果显示这一过程是钙依赖性的． 组蛋白Ｈ１ 可以使反应进程加快，表明提取液中的蛋白激酶可以利用它作为底物． 综合结果表明，１８ ｋＤ和６７ ｋＤ蛋白可能是具有自磷酸化能力且对Ｃａ２＋ 敏感的蛋白激酶，它们对Ｃａ２＋ 的不同反应，使得钙信号的传递更具可控性     

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K_m of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K_i of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

The N-terminal part of Binder of SPerm 5 (BSP5), which promotes sperm capacitation in bovine species is intrinsically disordered

Bovine BSP5 belongs to the Binder of SPerm (BSP) family. BSP5 plays a role in the bovine sperm capacitation by promoting cholesterol and phospholipid efflux. The variable N-terminal part in the BSP proteins is the uncharacterized region with no known function. Full-length, N-terminal part, and individual fibronectin type II domains of bovine BSP5 were cloned, expressed and purified from Escherichia coli. His-S tagged N-terminal part showed large variation in migration on SDS-PAGE in comparison to other constructs. Using mass spectrometry it was demonstrated that the His-S-N-terminal part has the expected molecular mass (13 kDa). The recombinant N-terminal part was sensitive to E. coli endogenous proteases during purification. Denaturing purification involving boiling lysis of cells was carried out, as the protein was thermostable. The His-S-N-terminal part lacked structure as determined by CD analysis. Bioinformatics analyses confirmed that the N-terminal part of bovine BSP5 is intrinsically disordered. In addition, bioinformatics analysis indicated that rabbit BSP and multiple forms of BSP proteins of bovine and equine species possess partially or completely disordered N-terminus. The conservation of disorder at the N-terminus in BSP members belonging to different species suggests a role in biological process such as sperm capacitation and/or sperm-egg interactions.     

Drosophila female germline stem cells undergo mitosis without nuclear breakdown

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Discovery of dual ZAP70 and Syk kinases inhibitors by docking into a rare C-helix-out conformation of Syk

The non-receptor tyrosine kinase Syk (spleen tyrosine kinase) is a pharmaceutical relevant target because its over-activation is observed in several autoimmune diseases, allergy, and asthma. Here we report the identification of two novel inhibitors of Syk by high-throughput docking into a rare C-helix-out conformation published recently. Interestingly, both compounds are slightly more active on ZAP70 (Zeta-chain-associated protein kinase 70), which is the kinase closest to Syk in the phylogenetic tree of human kinases. Taken together, the docking pose and experimental results suggest that the higher affinity of the inhibitors for ZAP70 than Syk originates from a more populated C-helix-out conformation in ZAP70. The latter observation is congruent with the 100-fold lower intrinsic activity of ZAP70 than Syk, as the C-helix-out conformation is inactive. The pharmacophore features of DFG-in, C-helix-out compounds are analyzed in relation to DFG-out inhibitors.