Lysine 188 substitutions convert the pattern of proteasome activation by REGgamma to that of REGs alpha and beta.
Lysine 188 substitutions convert the pattern of proteasome activation by REGgamma to that of REGs alpha and beta.
复制标题
DOI：
--
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
J. Li;X. Gao;J. Ortega;T. Nazif;L. Joss;M. Bogyo;A. Steven;M. Rechsteiner
中科院分区：
文献类型：
--
作者：
J. Li;X. Gao;J. Ortega;T. Nazif;L. Joss;M. Bogyo;A. Steven;M. Rechsteiner
11S REG (PA28) 是结合蛋白酶体并刺激肽水解的多聚环。 REGalpha 激活 P1 位上具有疏水性、酸性或碱性残基的肽的蛋白酶体水解，而 REGgamma 仅激活碱性残基后的裂解。我们分离出 REGgamma 突变体，能够激活诊断所有三个活性蛋白酶体 β 亚基的荧光肽的水解。最强大的 REGgamma 特异性突变体涉及用 Glu 或 Asp 替代 Lys188。 REGgamma(K188E/D) 变体在蛋白酶体激活方面与 REGalpha 几乎相同，但组装成稳定性较差的七聚体/六聚体。基于REGalpha晶体结构，REGgamma的Lys188通过七聚体面向水通道，增加了REG通道作为底物选择性门的可能性。然而，125I-YL3-VS 对蛋白酶体胰凝乳蛋白酶样亚基的共价修饰表明，REGgamma(K188E) 对所有三个蛋白酶体活性位点的激活并非由于门控松弛所致。我们认为 REGgamma(K188E) 七聚体的稳定性降低使得它们能够在蛋白酶体结合时改变构象，从而减轻通常由野生型 REGgamma 分子施加的 CT 和 PGPH 位点的抑制。
11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGalpha activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGgamma only activates cleavage after basic residues. We have isolated REGgamma mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome beta subunits. The most robust REGgamma specificity mutants involve substitution of Glu or Asp for Lys188. REGgamma(K188E/D) variants are virtually identical to REGalpha in proteasome activation but assemble into less stable heptamers/hexamers. Based on the REGalpha crystal structure, Lys188 of REGgamma faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin-like subunits by 125I-YL3-VS demonstrates that REGgamma(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGgamma(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGgamma molecule.