SAP与PIX在免疫细胞信号传导中作用机制的研究

发布时间：2018-08-09 10:32

【摘要】： X染色体连锁的淋巴增生综合症是一种免疫缺陷的遗传性疾病。XLP的患者在受EB病毒感染后,出现致死的爆发性单核细胞增生。有些患者出现渐进性的丙种球蛋白异常和严重的淋巴细胞增生异常,如恶性淋巴癌。有些情况下,患者也会出现自身免疫疾病,如脉管炎,肠炎或再生障碍性贫血。甚至在没有EB病毒的感染下,患者也会出现类似的异常。XLP患者的多种缺陷表明X染色体连锁的淋巴增生综合症是一种根本性的免疫缺陷疾病。在1998年,同时有三个实验室发现,SAP/SH2D1A/DSHP这个基因的异常引起了XLP疾病。SAP基因在自然杀伤细胞(NK)和T细胞中表达。人的SAP蛋白是一个由128个氨基酸组成的小分子蛋白,其中氨基端的6-102个氨基酸组成了一个SH2功能域,而羧基端的26个氨基酸与任何已知的功能域都没有同源性。许多的研究表明,SAP能与SLAM家族的受体结合,包括SLAM, 2B4(CD244),CD84, Ly9(CD229), NTB-A(Ly108)和CRACC。SAP通过其SH2功能域与这些受体胞质部分的TxYxxV/I模块结合。因此,这些受体可能通过招募SAP传导下游信号。而在X染色体连锁的淋巴增生综合症患者中缺乏行使正常功能的SAP蛋白,由此人们推断,XLP患者表现出的免疫缺陷症状可能是由于一个或多个SLAM家族受体的信号无法通过SAP传递,最终导致了免疫反应的严重异常。在XLP患者中,有多种免疫细胞出现功能异常,包括CD4阳性T细胞, CD8阳性T细胞, NK细胞和B细胞。此外,SAP敲除小鼠的免疫缺陷也表明了SAP在免疫调节中起关键作用。这些小鼠极易受啮齿类淋巴细胞脉络丛脑膜炎病毒和伽玛疱疹病毒-68的感染,其CD8阳性T细胞的抗病毒反应也存在异常。从SAP敲除小鼠中分离到的CD4阳性T细胞在CD3抗体刺激下,无法正常分泌白细胞介素-(4IL-4),说明其T细胞表面抗原受体(TCR)介导的信号通路也存在异常。许多实验室对SAP在T细胞和NK细胞中的信号传导机制进行了研究。已有报道证明,SAP的SH2功能域能与Src家族激酶Fyn的SH3功能域结合,将Fyn招募到SLAM家族受体上,并由此介导SLAM受体下游的信号通路,最终导致T细胞活化。然而SLAM缺陷小鼠和Fyn缺陷小鼠表现出的免疫缺陷症状不完全相同,提示除Fyn之外,还可能有其它的信号分子参与了SLAM-SAP的信号通路。XLP患者表现出的多种症状也支持了这种提示。通过酵母双杂交筛选系统发现PIX家族成员与SAP结合。PIX家族是一类小分子鸟苷酸酶Rac/Cdc42的鸟苷转换因子,目前发现有两个成员,αPIX和βPIX。PIX蛋白由N端的SH3功能域, DH功能域, PH功能域和C端的一个卷曲螺旋功能域组成。PIX通过SH3功能域与PAK中的脯氨酸富集区结合。通过一系列的免疫共沉淀实验,我们证明了在T细胞中,SAP与PIX有直接的相互作用,SAP介导了PIX被招募到SLAM家族受体上,传导SLAM受体下游的信号。而与SAP类似的另一个仅含SH2功能域的蛋白EAT-2,却并不能与PIX结合,也不能将PIX招募到SLAM家族受体上。通过SAP中关键点氨基酸突变,我们发现PIX和SAP的结合是通过SAP-SH2功能域中的第二个界面,所以不影响SAP与SLAM的结合。通过构建截断体和关键点氨基酸突变,我们发现,PIX的SH3功能域对于SAP的结合是充分而且必要的。在哺乳动物细胞中,SAP与PAK竞争结合PIX。此外,SAP与PIX,活化状态的Cdc42形成了复合体,提示SAP与PIX可能将SLAM家族受体的信号通路与Cdc42介导的下游信号通路相连。为了揭示PIX与SAP在相关信号传导过程中的作用,我们利用已有的报告基因系统检测了SAP在NFkB,NFAT,AP-1及p53信号通路中的作用。SAP的过量表达在NFkB,AP-1及p53的信号通路中没有明显的作用。而在T细胞中表达SAP能显著激活PMA和离子酶素诱导的NFAT(活化T细胞核因子)活性。SAP的这个效果是通过与钙离子信号协同作用实现的。目前为止,只有活化形式的Ras有如此强的激活作用,提示SAP可能在T细胞活化中有特殊作用。而SAP的同源类似物EAT-2对NFAT的激活没有作用。此外,SAP的关键点氨基酸突变体SAP-R32Q和SAP-R78A都没有这个效果,证明SLAM受体和SAP下游的信号对于SAP的激活作用是必需的。失活状态的Ras完全地阻断了SAP对NFAT的激活,提示Ras的信号通路对SAP激活NFAT是必需的,SAP可能通过直接参与Ras信号通路起作用,也可能通过其它的信号通路影响Ras的信号传导起作用。为了验证SAP-PIX的结合对SAP在NFAT的激活作用中是有意义的,我们构建了功能性阻断体PIX-SH3功能域。通过NFAT报告基因检测,发现PIX-SH3功能域的表达完全抑制了SAP对NFAT的激活效果,而表达不能与SAP结合的PIX-SH3-W43P/W44G功能域并不影响SAP对NFAT信号的激活,说明SAP-PIX的结合在T细胞的活化中可能有特殊作用。此外,Fyn-SH3功能域的表达也不影响SAP对NFAT信号的激活,说明在T细胞中信号通路中,PIX可能是与SAP结合的另一个主要目标。在此,我们的研究揭示了X染色体连锁的淋巴增生综合症基因产物SAP与Rac/Cdc42的鸟苷转换因子PIX存在相互作用并参与T细胞信号通路,能帮助阐述X染色体连锁的淋巴增生综合症的病因学,为研究XLP的治疗方法打下基础。
[Abstract]:X chromosome linked lymphoproliferative syndrome is a kind of immune deficient hereditary disease.XLP that causes fatal mononuclear cell proliferation after EB virus infection. Some patients appear progressive gamma globulin and severe lymphocytic dysplasia, such as malignant lymphoid cancer. In some cases, patients will also come out. Autoimmune diseases, such as vasculitis, enteritis, or aplastic anemia. Even without EB virus infection, patients also have a variety of defects in patients with similar abnormal.XLP indicating that X chromosome linked lymphoproliferative syndrome is a fundamental immunodeficiency disease.
In 1998, three laboratories found that the abnormality of the SAP/SH2D1A/DSHP gene caused the XLP disease.SAP gene to be expressed in natural killer cells (NK) and T cells. The human SAP protein is a small molecular protein composed of 128 amino acids, of which 6-102 amino acids in the amino terminal form a SH2 functional domain and 26 of the carboxyl end. The amino acids are not homologous with any known functional domain. Many studies have shown that SAP can bind to the receptors of the SLAM family, including SLAM, 2B4 (CD244), CD84, Ly9 (CD229), NTB-A (Ly108), and CRACC.SAP by its SH2 functional domain, which are combined with the cytoplasmic parts of these receptors. Downstream signals, and the lack of normal function SAP protein in patients with X chromosome linked lymphoproliferative syndrome, it is inferred that the immune deficiency symptoms in XLP patients may be due to the inability of one or more SLAM family receptors to pass through SAP, and ultimately lead to a severe immune response.
In XLP patients, many immune cells have abnormal function, including CD4 positive T cells, CD8 positive T cells, NK cells and B cells. In addition, the immunodeficiency of SAP knockout mice also indicates that SAP plays a key role in immune regulation. These mice are highly susceptible to the sense of rodent lymphocytic choroidal meningitis virus and gamma herpes virus -68. The antiviral response of the CD8 positive T cells is also abnormal. The CD4 positive T cells isolated from the SAP knockout mice can not normally secrete the interleukin (4IL-4) under the stimulation of CD3 antibody, indicating that the signal pathway mediated by the T cell surface antigen receptor (TCR) is also abnormal.
Many laboratories have studied the signal transduction mechanism of SAP in T and NK cells. It has been reported that the SH2 functional domain of SAP can be combined with the SH3 functional domain of the Src family kinase Fyn and recruits Fyn to the SLAM family receptor, and thus mediates the signal pathway downstream of SLAM receptor and ultimately leads to the activation of the T cells. The immune deficiency symptoms in YN deficient mice are not exactly the same, suggesting that other signal molecules may be involved in the signal pathway of the SLAM-SAP in addition to Fyn, and a variety of symptoms also support the hint.
The yeast two hybrid screening system found that PIX family members and SAP.PIX family are a guanosine conversion factor of a class of small molecular guanosine enzyme Rac/Cdc42, and there are two members. The alpha and beta PIX.PIX proteins are composed of SH3 functional domains of N end, DH functional domain, PH functional domain and C end. The domain is combined with the proline enrichment area in PAK.
Through a series of immunoprecipitation experiments, we have shown that SAP and PIX have direct interaction in T cells. SAP mediates PIX being recruited to the SLAM family receptor and conducting the downstream signal of the SLAM receptor. While another protein EAT-2, similar to SAP, only contains SH2 functional domains, can not be combined with PIX and can not recruit PIX to the family. By mutation of the key point amino acid in SAP, we found that the combination of PIX and SAP is through the second interfaces in the SAP-SH2 functional domain, so it does not affect the binding of SAP to SLAM.
By constructing the truncate and key point amino acid mutations, we found that the SH3 functional domain of PIX is sufficient and necessary for the binding of SAP. In mammalian cells, SAP and PAK are competing with PIX. in addition to SAP and PIX, and Cdc42 in the activation state forms a complex, suggesting that SAP and PIX may mediate the signaling pathway of the SLAM family receptor. The downstream signal paths are connected.
In order to reveal the role of PIX and SAP in the related signal transduction, we have detected the effect of SAP in the signaling pathways of NFkB, NFAT, AP-1, and p53 in the signaling pathway of NFkB, NFAT, AP-1 and p53, and we have detected no significant effect in the NFkB, AP-1 and p53 signaling pathways. The effect of NFAT (activated T nuclear factor) active.SAP is achieved by synergistic action with the calcium ion signal. So far, only the active form of Ras has such strong activation, suggesting that SAP may have a special role in the activation of T cells. And the SAP homologous analogue EAT-2 has no effect on the activation of NFAT. In addition, SAP is closed. The key point amino acid mutants SAP-R32Q and SAP-R78A do not have this effect, proving that the signal of the SLAM receptor and the downstream of the SAP is necessary for the activation of SAP. The Ras in the inactivation state completely blocks the activation of the SAP to NFAT, suggesting that the Ras signaling pathway is necessary for SAP activation NFAT, and SAP may play a role by directly participating in the signaling pathway. It may also affect the signal transduction of Ras through other signaling pathways.
In order to verify the significance of the combination of SAP-PIX in the activation of SAP in NFAT, we constructed the functional domain of the functional blockage PIX-SH3. Through NFAT reporter gene detection, it was found that the expression of the PIX-SH3 functional domain completely inhibited the activation effect of SAP on NFAT, and the PIX-SH3-W43P/W44G functional domain, which was not associated with SAP, did not affect SAP. The activation of the NFAT signal indicates that the binding of SAP-PIX may have special effects on the activation of T cells. In addition, the expression of the Fyn-SH3 functional domain does not affect the activation of SAP to NFAT signals, indicating that PIX may be another major target in the T cell signaling pathway to the combination of SAP.
Here, our study reveals that the X chromosome linked lymphoproliferative syndrome gene product SAP interacts with the guanosine conversion factor PIX of Rac/Cdc42 and participates in the T cell signaling pathway, which can help to elucidate the etiology of X linked lymphoproliferative syndrome and lay the foundation for the study of the treatment of XLP.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【相似文献】