Mistargeting of Elastase in Bone Marrow Failure

描述(由申请人提供)： 中性粒细胞是具有吞噬功能的白细胞，其数量不足(“中性粒细胞减少症”)容易引起细菌和真菌感染。人类中性粒细胞减少性骨髓衰竭的两种主要遗传性形式是循环性中性粒细胞减少症(CN)和易患白血病的严重先天性中性粒细胞减少症(SCN)。“良性中性粒细胞减少症”在非洲人后裔中很常见，也可能对健康造成影响。编码中性粒细胞弹性蛋白酶(NE)的基因ELA2突变引起CN，是SCN最常见的原因。最近的研究发现，狗的CN是由AP3转运蛋白的突变引起的，一些SCN可能是由ELA2启动子变异或转录抑制因子Gfi1的突变引起的，这两种情况都会导致NE的过度表达，这表明在这些和其他骨髓衰竭综合征中存在中性粒细胞减少的假说：NE是AP3的跨膜“货物”蛋白。NE跨膜区的突变导致过多的NE聚集在颗粒中，导致CN。NE的AP3识别信号的突变或AP3本身的突变使NE错误地定位于质膜。类似地，由于ELA2启动子变异或Gfi1突变导致的NE的过度表达压倒了正常的AP3介导的转运途径，并将NE转移到质膜上。有三个特定的目的旨在验证这一假说，确定非裔美国人群体中常见的ELA2启动子变异是否与良性的民族中性粒细胞减少症有关，并识别其他中性粒细胞减少症基因：1表征NE的膜关系：1.1开发针对预测的NE细胞质和管腔表面的抗体；1.2测试膜结合的NE在模型和生物底物上的变化催化；1.3检查AP3相互作用在其他物种和其他蛋白酶中的进化保守性。2评价NE错配是中性粒细胞减少症的一般机制：2.1确定NE错位定位的潜在底物和辅助因子；2.2检查其他中性粒细胞减少症中NE的转运。3发现可能通过NE过度表达而导致中性粒细胞减少的其他突变：3.1确定ELA2启动子变异是否有助于中性粒细胞减少症；3.2测量ELA2 C-199A等位基因在良性民族性中性粒细胞减少症患者中的频率；3.3确定新的中性粒细胞减少症基因。

DESCRIPTION (provided by applicant): Neutrophils are phagocytic white blood cells, and a deficiency of their numbers ("neutropenia") predisposes to bacterial and fungal infection. The two principal inherited forms of human neutropenic bone marrow failure are cyclic neutropenia (CN) and leukemia-predisposing, severe congenital neutropenia (SCN). "Benign ethnic neutropenia" is common to individuals of African descent and may also have health consequences. Mutations of the gene ELA2, encoding neutrophil elastase (NE), cause CN and are the most frequent cause of SCN. Recent findings that canine CN is caused by mutations of the AP3 transporter and that some SCN may result from ELA2 promoter variants or mutations of the transcriptional repressor Gfi1, both of which lead to over-expression of NE, suggest a hypothesis for neutropenia in these and other bone marrow failure syndromes: NE is a transmembrane "cargo" protein for AP3. Mutation of NE's transmembrane domains causes too much NE to accumulate in granules, resulting in CN. Mutation of NE's AP3-recognition signal, or of AP3 itself, mislocalizes NE to the plasma membrane. Similarly, over-expression of NE consequent to ELA2 promoter variants or Gfi1 mutations overwhelms normal AP3-mediated trafficking pathways and diverts NE to the plasma membrane. Three Specific Aims are directed toward testing this hypothesis, determining if a common ELA2 promoter variant in the African-American population associates with benign ethnic neutropenia, and identifying additional neutropenia genes: 1 Characterize NE's membrane relationship: 1.1 Develop antibodies to predicted cytoplasmic and luminal surfaces of NE; 1.2 Test membrane-bound NE for altered catalysis on model and biological substrates; 1.3 Examine the evolutionary conservation of AP3 interactions in other species and for other proteases. 2 Evaluate mistrafficking of NE as a general mechanism for neutropenia: 2.1 Identify potential substrates and cofactors of mislocalized NE; 2.2 Inspect NE trafficking in other neutropenic disorders. 3 Discover other mutations that may cause neutropenia through over-expression of NE: 3.1 Determine if ELA2 promoter variation contributes to neutropenia; 3.2 Measure the frequency of the ELA2 C-199A allele in individuals of African descent with benign ethnic neutropenia; 3.3 Identify new neutropenia genes.