CAAX Processing Enzymes as Anticancer Targets

描述(由申请人提供)：许多细胞内信号蛋白(例如，Ras和Rho蛋白)和几个核层蛋白以羧基末端CAAX基序终止。CAAX蛋白经历了三次连续的翻译后修饰。首先，半胱氨酸(即CAAX基序的C)被一对胞浆酶--法尼基转移酶(FTase)和香叶基香叶基转移酶I(GGTase I)法尼化或香叶化。其次，最后三个氨基酸(即-AAX)被RAS和内质网(ER)的完整膜蛋白-α-因子转换酶(RCEL)切割。第三，新暴露的羧基末端的异丙基半胱氨酸被另一种ER蛋白--异丙基半胱氨酸羧甲基转移酶(ICMT)甲基化。这些翻译后修饰使CAAX蛋白的C末端更加疏水，增强了蛋白质与膜表面的附着，并促进了某些蛋白质之间的相互作用。 激活的RAS突变在30%的人类癌症中被检测到，并且在白血病和骨髓增生性疾病中很常见。FTase抑制剂已被用于治疗含有突变激活的RAS蛋白的癌症。不幸的是，K-RAS和N-RAS--人类癌症中最常见的RAS亚型--在设置FTase抑制时很容易被GGTase I香叶化。这种交替的异戊二烯基化途径将注意力集中在该途径中的其他酶上，如GGTase I、rcel和ICMT。令人惊讶的是，没有数据表明抑制这些其他酶对小鼠癌症发展的影响。在这个项目中，这一空白将得到解决。 在初步研究中，已经产生了同时携带Cre诱导的潜在致癌Kras2等位基因(KrasLsL)和可诱导的Mx1-Cre转基因的小鼠。在这些小鼠中诱导CRE会激活潜在的RAS等位基因，并导致一种成熟的、致命的骨髓增生性疾病，这让人想起人类的慢性粒细胞白血病或幼年粒单核细胞白血病。最近，翻译后加工酶(FTase、GGTase I、rcel和ICMT)的条件性等位基因已经产生。因此，现在有可能培育出Cre表达可以同时激活潜在致癌K-RAS等位基因和灭活CAAX处理酶的小鼠。利用这些小鼠，我们将确定有缺陷的CAAX处理对RAS诱导的骨髓增生性疾病的发展、进展和致命性的影响。

DESCRIPTION (provided by applicant): Many intracellular signaling proteins (e.g., the Ras and Rho proteins) and several nuclear lamins terminate with a carboxyl-terminal CAAX motif. CAAX proteins undergo three sequential posttranslational modifications. First, the cysteine (i.e., the C of the CAAX motif) is farnesylated or geranylgeranylated by a pair of cytosolic enzymes--farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I). Second, the last three amino acids (i.e., the -AAX) are cleaved off by Ras and a-factor converting enzyme (Rcel), an integral membrane protease of the endoplasmic reticulum (ER). Third, the newly exposed carboxyl-terminal isoprenylcysteine is methylated by another ER protein, isoprenylcysteine carboxyl methyltransferase (Icmt). These posttranslational modifications render the C-terminus of CAAX proteins more hydrophobic, enhancing the attachment of the proteins to membrane surfaces and facilitating certain protein-protein interactions. Activating Ras mutations have been detected in 30% of all human cancers, and are common in leukemia and myeloproliferative diseases. Inhibitors of FTase have been used to treat cancers that harbor mutationally activated Ras proteins. Unfortunately, K-Ras and N-Ras--the Ras isoforms most often implicated in human cancers--are readily geranylgeranylated by GGTase I in the setting FTase inhibition. This alternate isoprenylation pathway has focused attention on other enzymes in the pathway, such as GGTase I, Rcel, and Icmt. Surprisingly, there are no data on the impact of inhibiting these other enzymes on the development of cancer in mice. In this project, this void will be addressed. In preliminary studies, mice harboring both a Cre-inducible latent oncogenic Kras2 allele (KrasLsL) and the inducible Mx1-Cre transgene have been generated. Induction of Cre in those mice activates the latent Ras allele and results in a full-fledged, lethal, myeloproliferative disease that is reminiscent of chronic myelogenous leukemia or juvenile myelomonocytic leukemia in humans. Recently, conditional "floxed" alleles for the posttranslational processing enzymes (FTase, GGTase I, Rcel, and Icmt) have been generated. Thus, it is now possible to breed mice in which Cre expression can be used to simultaneously activate the latent oncogenic K-Ras allele and inactivate the CAAX processing enzymes. Using these mice, we will define the impact of defective CAAX processing on the development, progression, and lethality of Ras-induced myeloproliferative disease.