Investigations on hyperphosphorylation of paratarg-7, a significant risk factor and frequent antigenic target of paraproteins in MGUS and multiple myeloma.

在以前的研究中，我们已经发现并表征了过度磷酸化的paratarg-7(PP-7)是15%的IgA和IgG型副蛋白以及11%的IgM类副蛋白的抗原靶蛋白。此外，我们已经证明PP-7携带者是迄今为止已知的发生MGUS/MM/WM的最强的危险因素，并且是作为常染色体显性遗传的。在DFG支持该项目的头三年中，我们表明PP-7是&gt；1/3(！)非裔美国人MGUS/MM患者中副蛋白的变化强调了PP-7在这些疾病的发病机制中的重要性。此外，我们成功地确定了额外的副蛋白自身抗原靶向，并证明了与健康对照组相比，所有这些自身抗原靶向都过度磷酸化(也是遗传常染色体显性的)，这表明MGUS/MM/WM患者中自身抗原性副蛋白靶标的过度磷酸化是常见的。研究了副蛋白靶标过度磷酸化的机制，我们证明在健康对照组中，在PKcheeta刺激后，P-7在丝氨酸17处过度磷酸化，并被蛋白磷酸酶2A去磷酸化，与所有其他自身抗原性副蛋白一样。在PP-7携带者和所有其他过度磷酸化的自身抗原性副蛋白靶标的携带者中，PP-7的去磷酸化是由于三聚体PP2A酶复合体中调节亚基B55Delta被B56Gamma3交换而导致的。对与蛋白酪氨酸激酶相关的磷酸化状态和表达的PP2A亚单位进行的序列分析，没有发现与P-7过度磷酸化相关的SNPs或突变。同样，与两个独立合作伙伴合作进行的两次全外显子组测序没有发现导致P-7过度磷酸化的基因变异，全基因组甲基化分析也没有发现。然而，单核苷酸多态和基于微卫星的全基因组连锁分析发现，染色体4q35有一个4.3Mb长的区域，用于定位导致P-7过度磷酸化的遗传变异，LOD评分为6(！)。在建议的DFG支持的第二阶段，我们将使用WTP-7和PP-7 LCL的功能方法缩小DNA区域，并研究从各自感兴趣的遗传变体到副蛋白自身抗原靶的过度磷酸化的途径。我们的研究将为MGUS/MM/WM的发病机制提供重要和全新的信息，这些信息将远远超出它们在paratarg-7过度磷酸化中的作用。

In previous studies we had identified and characterized hyperphosphorylated paratarg-7 (pP-7) as the antigenic target of 15% of all paraproteins of the IgA and IgG type and of 11% of all IgM paraproteins. Moreover, we had shown that pP-7 carriership is the strongest risk factor to develop MGUS/MM/WM known to date and is inherited as an autosomal-dominant trait. During the first three years of DFG support for this project we showed that pP-7 is the target of >1/3 (!) of paraproteins in African-American MGUS/MM patients underlining the importance of pP-7 in the pathogenesis of these diseases. Moreover, we succeeded in identifying additional autoantigenic targets of paraproteins and demonstrated that all these autoantigenic targets were hyperphosphorylated in the respective patients compared to healthy controls (and also inherited autosomal-dominantly), demonstrating that hyperphosphorylation of autoantigenic paraprotein targets is frequent in MGUS/MM/WM patients. Investigating the mechanisms underlying the hyerphosphorylation of the paraprotein targets we demonstrated that in healthy controls P-7 is hyerphosphorylated at serine 17 after stimulation by PKCzeta, and dephosphorylated by protein phosphatase 2A, as are all other autoantigenic paratargs. In pP-7 carriers and carriers of all other hyperphosphorylated autoantigenic paraproteins targets, dephosphorylation of pP-7 is defective due to an exchange of the regulatory subunit B55delta by B56gamma3 in the trimeric PP2A enzyme complex. Sequencing of paratargs and of the for the phosphorylation state responsible PKCzeta as well as the expressed subunits of PP2A, did not reveal SNPs or mutations that were associated with the hyperphosphorylation of P-7. Similarly, whole-exome sequencing which was performed twice in cooperation with two independent cooperating partners did not identify a genetic variant responsible for P-7 hyperphosphorylation, nor did whole-genome methylation analysis. However, a single nucleotide polymorphisms and microsatellite-based genome-wide linkage analysis identified a 4.3 Mb long region of chromosome 4q35 to locate the genetic variant responsible for the hyperphosphorylation of P-7 with a LOD score of 6 (!). In the suggested second period of DFG support, we will narrow-down the DNA region using a functional approach with wtP-7 and pP-7 LCLS and investigate the pathways leading from the respective genetic variant of interest to the hyperphosphorylation of autoantigenic targets of paraproteins. Our investigations will yield important and completely novel information on the pathogenesis of MGUS/MM/WM that will go far beyond their role in paratarg-7 hyperphosphorylation.