Mechanism of action of STAMP - a new comodulator of glucocorticoid receptors

该项目的两个目的是1）观察Amax，EC 50和PAA与辅因子如STAMP的变化是否可以分离，以及2）寻找STAMP的其他活性。 目标1已经使用GR、共活化剂TIF 2和STAMP的三元体系解决，原因有两个。 首先，我们以前建立了TIF 2和STAMP增加Amax和PAA，并以超过累加的方式降低GR介导的基因表达的EC 50。 第二，潜在的生物活性蛋白质表面的排列的数量比两个因子高得多，从而增加了可能参与选择性基因转录的可能组合。 在全细胞生物测定中使用三种因子（GR，TIF 2和STAMP）中每种因子的三种不同大小的片段，我们发现每种蛋白质的不同区域可以选择性地改变三个参数（Amax，EC 50和PAA）。 这些结果揭示了内分泌治疗的药理学操作的新的潜在目标。 同样重要的是，仅对Amax的常规研究将仅观察到GR调节基因表达所涉及的那些因子的子集，并且将错过那些在不影响Amax的情况下改变EC 50和/或PAA的药剂。 目的2通过在同质遗传背景中制备STAMP KO小鼠来实现。 STAMP是一种转录辅因子，可增强p160辅激活因子SRC-1和TIF 2在糖皮质激素受体调节的基因诱导和抑制中的活性。 然而，STAMP也被其他人描述为具有聚谷氨酰化活性的酪氨酸微管蛋白连接酶样家族成员（TTLL 5）。 此外，我们先前报道了STAMP关于几种细胞类型的生长的类固醇非依赖性活性（He等人，2010，BMC Cancer，10，128）。 为了确定STAMP在整个动物中的功能，制备了含有破坏的Stamp基因的小鼠，导致蛋白质中间的翻译提前终止。 纯合子靶向突变体（Stamptm/tm）雌性动物表现正常。 Stamptm/tm雄性似乎也正常，除了它们几乎总是不育的，这是由于精子成熟和运动缺陷的独特组合，涉及与微管蛋白多聚谷氨酰化减少相关的精子尾部轴丝破坏。 其他结构中的轴丝似乎不受影响。 尽管睾丸中wt STAMP mRNA的水平比检查的任何其他器官高20倍，但wt与Stamptm/tm雄性的精子发育器官没有明显变化。 这种男性生育能力的缺陷与先前确定的对精子功能重要的24个基因中的任何一个都无关。 因此，STAMP似乎参与了精子成熟和运动的独特途径，这可能与男性生育力有关。 总之，我们正在使用我们最近制备的STAMP KO小鼠来寻找KO动物的表型缺陷。 GR/TIF 2/STAMP三元复合物作用的详细研究已经确定GR调节诱导的三个参数（EC 50、PAA和Amax）不协调调节。 相反，这三种蛋白质的不同蛋白质表面可以选择性地调节各个参数，这表明可能存在可以改变一个、两个或所有三个参数的活性的药物。 这些综合研究结果有助于我们在分子水平上定义类固醇激素的作用并了解其在人体生理学中的作用的长期目标。

The two aims of this project are 1) to see if changes in Amax, EC50, and PAA with cofactors such as STAMP can be dissociated, and 2) to search for other activities of STAMP. Aim 1 has been addressed using the ternary system of GR, the coactivator TIF2, and STAMP for two reasons. First, we previously established that TIF2 and STAMP augment the Amax and PAA, and decrease the EC50, of GR-mediated gene expression in a more than additive manner. Second, the number of permutations of potential biologically active protein surfaces is much higher with three than two factors, thereby increasing the possible combinations that could be involved in selective gene transcription. Using three differently sized fragments of each of three factors (GR, TIF2, and STAMP) in whole cell bioassays, we found that different regions of each protein can selectively alter none, some, or all three parameters (Amax, EC50, and PAA). These results reveal new potential targets for pharmacological manipulation of endocrine therapies. Equally important is the demonstration that conventional studies of just the Amax will observe only a subset of those factors involved in GR-regulated gene expression and will miss those agents that modify the EC50 and/or PAA without affecting the Amax. Aim 2 has been pursued by preparing the STAMP KO mice in a homogeneous genetic background. STAMP is a transcriptional cofactor that augments the activity of the p160 coactivators SRC-1 and TIF2 in glucocorticoid receptor-regulated gene induction and repression. However, STAMP has also been described by others as a tyrosine tubulin ligase-like family member (TTLL5) with polyglutamylation activity. Furthermore, we previously reported steroid-independent activity of STAMP regarding the growth of several cell types (He et al., 2010, BMC Cancer, 10, 128). To determine STAMP functions in whole animals, mice containing a disrupted Stamp gene, resulting in premature termination of translation in the middle of the protein, were prepared. Homozygous targeted mutant (Stamptm/tm) females appear normal. Stamptm/tm males also seem normal except that they are almost always sterile due to a unique combination of sperm maturation and motility defects involving disruption of sperm tail axonemes that is associated with reduced tubulin polyglutamylation. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of wt vs. Stamptm/tm males despite the levels of wt STAMP mRNA in the testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to any of the 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique pathway of sperm maturation and motility that may be relevant for male fertility. In summary, we are using our recently prepared STAMP KO mice to look for phenotypic defects of KO animals. Detailed studies of the action of the ternary complex of GR/TIF2/STAMP have established that the three parameters of GR-regulated induction (EC50, PAA, and Amax) are not coordinately regulated. Instead, different protein surfaces of the three proteins can selectively modulate individual parameters, which indicates that pharmaceuticals may exist that can alter the activities of one, two, or all three parameters. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.