Mellgren R L
Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
J Biol Chem. 1997 Nov 21;272(47):29899-903. doi: 10.1074/jbc.272.47.29899.
Cell-permeant peptidyl aldehydes and diazomethylketones are frequently utilized as inhibitors of regulatory intracellular proteases. In the present study the specificities of several peptidyl inhibitors for purified human mu-calpain and 20 S proteasome were investigated. Acetyl-LLnL aldehyde, acetyl-LLM aldehyde, carbobenzyloxy-LLnV aldehyde (ZLLnVal), and carbobenzyloxy-LLY-diazomethyl ketone produced half-maximum inhibition of the caseinolytic activity of mu-calpain at concentrations of 1-5 x 10(-7) M. In contrast, only ZLLnVal was a reasonably potent inhibitor of the caseinolytic activity of 20 S proteasome, producing 50% inhibition at 10(-5) M. The other inhibitors were at least 10-fold less potent, producing substantial inhibition only at near saturating concentrations in the assay buffer. Further studies with ZLLnVal demonstrated that its inhibition of the proteasome was independent of casein concentration over a 25-fold range. Proteolysis of calpastatin or lysozyme by the proteasome was half-maximally inhibited by 4 and 22 microM ZLLnVal, respectively. Thus, while other studies have shown that ZLLnVal is a potent inhibitor of the hydrophobic peptidase activity of the proteasome, it appears to be a much weaker inhibitor of its proteinase activity. The ability of the cell permeant peptidyl inhibitors to inhibit growth of the yeast Saccharomyces cerevisiae was studied because this organism expresses proteasome but not calpains. Concentrations of ZLLnVal as high as 200 microM had no detectable effect on growth rates of overnight cultures. However, yeast cell lysates prepared from these cultures contained 2 microM ZLLnVal, an amount which should have been sufficient to fully inhibit hydrophobic peptidase activity of yeast proteasome. Degradation of ubiquitinylated proteins in yeast extracts by endogenous proteasome was likewise sensitive only to high concentrations of ZLLnVal. The higher sensitivity of the proteinase activity of calpains to inhibition by the cell permeant inhibitors suggests that calpain-like activities may be targets of these inhibitors in animal cells.
细胞渗透性肽基醛和重氮甲基酮常被用作细胞内调节蛋白酶的抑制剂。在本研究中，研究了几种肽基抑制剂对纯化的人μ-钙蛋白酶和20S蛋白酶体的特异性。乙酰-LLnL醛、乙酰-LLM醛、苄氧羰基-LLnV醛（ZLLnVal）和苄氧羰基-LLY-重氮甲基酮在浓度为1-5×10^(-7)M时对μ-钙蛋白酶的酪蛋白水解活性产生半数最大抑制。相比之下，只有ZLLnVal是20S蛋白酶体酪蛋白水解活性的有效抑制剂，在10^(-5)M时产生50%的抑制。其他抑制剂的效力至少低10倍，仅在测定缓冲液中接近饱和浓度时才产生显著抑制。对ZLLnVal的进一步研究表明，其对蛋白酶体的抑制在25倍的范围内与酪蛋白浓度无关。蛋白酶体对钙蛋白酶抑制蛋白或溶菌酶的蛋白水解分别被4和22μM的ZLLnVal半数最大抑制。因此，虽然其他研究表明ZLLnVal是蛋白酶体疏水肽酶活性的有效抑制剂，但它似乎是其蛋白酶活性的弱得多的抑制剂。研究了细胞渗透性肽基抑制剂抑制酿酒酵母生长的能力，因为这种生物体表达蛋白酶体但不表达钙蛋白酶。高达200μM的ZLLnVal浓度对过夜培养物的生长速率没有可检测到的影响。然而，从这些培养物中制备的酵母细胞裂解物含有2μM的ZLLnVal，这一量应该足以完全抑制酵母蛋白酶体的疏水肽酶活性。内源性蛋白酶体对酵母提取物中泛素化蛋白质的降解同样仅对高浓度的ZLLnVal敏感。钙蛋白酶的蛋白酶活性对细胞渗透性抑制剂抑制的更高敏感性表明，类似钙蛋白酶的活性可能是动物细胞中这些抑制剂的作用靶点。
