Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314-28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was approximately 200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.     

GPR120的研究进展

游离脂肪酸作为组织能量来源以及介导各种细胞进程的信号分子,其生理功能长期以来受到广泛关注。外周游离脂肪酸水平的升高与肥胖、脂代谢紊乱以及糖尿病紧密相关。GPR120作为一新的长链脂肪酸受体,参与调节体内一系列的代谢过程,如激素分泌、细胞增殖及脂质生成等。作为肥胖、糖尿病的潜在治疗靶标,值得更深入的研究。     

P120链蛋白与肿瘤

研究参与肿瘤发生的重要信号分子,对揭示肿瘤发生发展有重要意义。链蛋白(catenin)家族成员与肿瘤的发生密切相关,在保持细胞黏附中起着重要作用,其表达缺失或下降将导致细胞分化丧失和具有侵袭性,容易发生浸润和转移。链蛋白家族最早被分为α、β、γ三类,它们都能与E-钙黏着蛋白(E-cadherin)形成复合体。P120cat作为新发现的链蛋白家族成员之一,位于细胞连接处和细胞核内,与肿瘤的发生有着更密切和特殊的关系,在不同的条件下,它既可以作为肿瘤的促进物,又可成为肿瘤的抑制物。     

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases.

The structures of two catalytically modified semisynthetic RNases obtained by replacing phenylalanine 120 with leucine and tyrosine have been determined and refined at a resolution of 2.0 A (R = 0.161 and 0.184, respectively). These structures have been compared with the refined 1.8-A structure (R = 0.204) of the fully active phenylalanine-containing enzyme (Martin PD, Doscher MS, Edwards BFP, 1987, J Biol Chem 262:15930-15938) and with the catalytically defective D121A (2.0 A, R = 0.172) and D121N (2.0 A, R = 0.186) analogs (deMel VSJ, Martin PD, Doscher MS, Edwards BFP, 1992, J Biol Chem 267:247-256). The movement away from the active site of the loop containing residues 65-72 is seen in all three catalytically defective analogs--F120L, D121A, and D121N--but not in the fully active (or hyperactive) F120Y. The insertion of the phenolic hydroxyl of Tyr 120 into a hydrogen-bonding network involving the hydroxyl group of Ser 123 and a water molecule in F120Y is the likely basis for the hyperactivity toward uridine 2',3'-cyclic phosphate previously found for this analog (Hodges RS, Merrifield RB, 1974, Int J Pept Protein Res 6:397-405) as well as the threefold increase in KM for cytidine 2',3'-cyclic phosphate found for this analog by ourselves.     

Correlated mutations at gp120 positions 322 and 440: implications for gp120 structure

Analysis of V3 and C4 sequences of HIV-1 reveals correlated mutations at gp120 positions 322 and 440, and a very strong preference for a positively charged residue at position 440 when position 322 is negatively charged. This observation suggests that these two residues are close to each other and interact electrostatically in R5 viruses. This interaction was used to model V3 in the context of gp120 using NMR data for the V3 loop and the crystal structure of the gp120-core. The interaction between residues 322 and 440 may serve as part of the molecular switch for HIV-1 phenotype conversion.     

连环蛋白家族成员P120ctn的作用及相关机制

连环蛋白P120可在细胞连接处与E-钙黏蛋白结合形成连环蛋白-钙黏蛋白复合体,调控钙黏蛋白介导的细胞黏附作用;在胞质内可与Rho家族GTP酶相互作用调节细胞骨架的运动;在细胞核内可与核转录因子NF-κB和转录抑制因子Kaiso结合,影响炎性反应和细胞增殖.P120对细胞黏附、细胞动力、炎性反应和细胞增殖的影响使其与损伤修复和肿瘤的发生、发展密切相关.深入研究P120的作用及其相关机制对于进一步研究损伤后修复及肿瘤预防和治疗具有深远的意义.