Clawson G A, Norbeck L L, Wise J P, Patierno S R
Department of Pathology, Pennsylvania State University, Hershey 17033.
Cell Growth Differ. 1993 Jul;4(7):589-94.
A nuclear scaffold (NS) protease has previously been implicated in production of the M(r) 46,000 ATP-binding protein in NS (which may acquire nucleoside triphosphatase activity and participate in nucleocytoplasmic transport) by cleavage of a subset of lamins A/C. In a preceding paper (G. Clawson, L. Norbeck, C. Hatem, C. Rhodes, P. Amiri, J. McKerrow, S. Patierno, and G. Fiskum, Cell Growth & Differ., 3: 827-838), this NS protease was identified as a novel, Ca(2+)-regulated serine protease, which was found only in the NS and which appears to represent a unique multicatalytic protease complex. Based upon its predominantly chymotrypsin-like substrate preference, a peptide-chloromethylketone inhibitor (succinyl-AAPF-chloromethylketone, AAPFcmk) was identified. AAPFcmk showed a KI = 56 nM for the NS protease versus 1.4 microM for the endoplasmic reticulum activity. Treatment of C3H/10T1/2 mouse embryo fibroblast cells with 1 microM AAPFcmk produced effects which were confined to the nuclear (and to a lesser extent the endoplasmic reticulum) compartment. In this report, we examine the effects of the AAPFcmk inhibitor on cellular transformation and growth. Growth of C3H/10T1/2 cells was decreased by 34% and 56% at 25 microM and 50 microM AAPFcmk, respectively. Growth inhibition occurred without any major change in DNA content distribution, suggesting effects throughout the cell cycle. Growth inhibition was not observed at lower (< or = 10 microM) concentrations, which decreased transformation of C3H/10T1/2 fibroblasts in a dose-dependent manner by up to 90%, even at femtomolar concentrations of AAPFcmk (in the absence of growth inhibition). Inclusion of irrelevant inhibitors was without affect.(ABSTRACT TRUNCATED AT 250 WORDS)
先前已表明，一种核支架（NS）蛋白酶通过切割核纤层蛋白A/C的一个亚群，参与了NS中分子量为46,000的ATP结合蛋白的产生（该蛋白可能获得核苷三磷酸酶活性并参与核质运输）。在前一篇论文（G. Clawson、L. Norbeck、C. Hatem、C. Rhodes、P. Amiri、J. McKerrow、S. Patierno和G. Fiskum，《细胞生长与分化》，3: 827 - 838）中，这种NS蛋白酶被鉴定为一种新型的、受Ca(2+)调节的丝氨酸蛋白酶，它仅存在于NS中，似乎代表一种独特的多催化蛋白酶复合物。基于其主要类似胰凝乳蛋白酶的底物偏好，鉴定出一种肽氯甲基酮抑制剂（琥珀酰 - AAPF - 氯甲基酮，AAPFcmk）。AAPFcmk对NS蛋白酶的抑制常数KI = 56 nM，而对内质网活性的抑制常数为1.4 μM。用1 μM AAPFcmk处理C3H/10T1/2小鼠胚胎成纤维细胞产生的效应局限于细胞核（以及程度较轻的内质网）区室。在本报告中，我们研究了AAPFcmk抑制剂对细胞转化和生长的影响。在25 μM和50 μM AAPFcmk时，C3H/10T1/2细胞的生长分别下降了34%和56%。生长抑制发生时DNA含量分布没有任何重大变化，表明在整个细胞周期均有影响。在较低（≤10 μM）浓度下未观察到生长抑制，这些浓度甚至在飞摩尔浓度的AAPFcmk（在无生长抑制的情况下）时，也能以剂量依赖方式使C3H/10T1/2成纤维细胞的转化降低高达90%。加入无关抑制剂没有影响。（摘要截短于250字）
