Synthesis and characterization of the regulatory domains of protein kinase C

蛋白激酶 C 调控域的合成和表征

基本信息

批准号：
11660109
负责人：
IRIE Kazuhiro
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660109/
关键词：
cysteine-rich domain phorbol protein kinase C tumor promoter zinc finger エレクトロスプレー質量分析法 HATU

项目摘要

Protein kinase C (PKC) isozymes are major receptors of tumor-promoting phorbol esters. Conventional and novel PKC isozymes (α, β, γ, δ, ε, η, θ) contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12,13-dibutyrate (PDBu) binding sites. To determine the phorbol ester binding sites of these isozymes, the C1 domains consisting of about 50 amino acids of all PKC isozymes have been synthesized by a solid phase Fmoc strategy. All C1B peptides except for α-C1B were successfully folded by zinc treatment as monitored by CD and ESI-MS spectroscopy, and showed potent PDBu binding affinities with the dissociation constants (K_d) of nanomolar range (0.45-1.5 nM), comparable to those of the native PKC isozymes. However, the K_d values of PDBu for many of the C1A peptides could not be determined. We found that some of the C1A peptides experience the temperature dependent inactivation and that elongation of the 50-mer C1 peptides at both N-and C-termini increases their folding efficiency. These findings enabled us to determine the K_d's of PDBu for all PKC C1 peptides except for θ-C1A.The major PDBu binding sites of novel PKC isozymes (δ, ε, η, θ) were C1B domains with K_d values of 0.45-0.81 nM.In contrast, all C1A peptides of conventional PKC isozymes (α, β, γ) exhibited nanomolar K_d values (0.97-1.3 nM). It is noteworthy that both C1 peptides of PKCβ and PKCγ showed strong PDBu binding affinity of nanomolar range. The above results provide a structural blueprint for the rational design of PKCγ-selective modulators for the treatment of neuropathic pain. To extend this approach, the 116-mer peptide containing the double cysteine-rich motifs of PKCγ (γ-C1A-C1B) has been synthesized for the first time.

蛋白激酶C（PKC）同工酶是促进肿瘤的佛比尔酯的主要受体。常规和新型的PKC同工酶（α，β，γ，δ，ε，η，θ）包含两个富含半胱氨酸的C1域（C1A和C1B），它们都是候选的phorbol-12,13-13-二仅候选 - 二倍酸酯（PDBU）（PDBU）。为了确定这些同工酶的佛波酯结合位点，已通过固相FMOC策略合成了由所有PKC同工酶的约50个氨基酸组成的C1结构域。通过CD和ESI-MS光谱法监测，除α-C1b以外的所有C1b肽通过锌处理成功折叠，并显示出与纳米极范围（0.45-1.5 nm）的解离常数（K_D）的潜在PDBU结合亲和力，与天然PKC同素异生物的所有C1B。但是，无法确定许多C1a肽的PDBU的K_D值。我们发现，某些C1a Pepperides经历了温度依赖性的失活，并且在N和C-termini处的50-MER C1肽的伸长会提高其折叠效率。 These findings enabled us to determine the K_d's of PDBu for all PKC C1 peptides except for θ-C1A.The major PDBu binding sites of novel PKC isozymes (δ, ε, η, θ) were C1B domains with K_d values of 0.45-0.81 nM.In contrast, all C1A peptides of conventional PKC isozymes (α, β, γ）暴露于纳摩尔k_d值（0.97-1.3 nm）。值得注意的是，PKCβ和PKCγ的C1肽均表现出强烈的PDBU结合亲和力。上述结果为PKCγ选择性调节剂的合理设计提供了结构性蓝图，用于治疗神经性疼痛。为了扩展这种方法，首次合成了含有PKCγ（γ-C1A-C1B）双半胱氨酸基序（γ-C1A-C1B）的116-Mer肽。