Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

为了更好地了解MAP4K2和MAP4K5在体内的作用，我们分离了小鼠MAP4K2和MAP4K5基因。已经创建了map4k2-/-和map4k5-/-小鼠，并将其回交到用于产生双基因敲除(KO)小鼠的C57BL/6背景上。我们将重点放在双KO小鼠身上，因为它们表现出滤泡B细胞数量的急剧减少，边缘带B细胞的扩张，血清抗体的升高，对新抗原的体液反应缺陷，以及自身免疫的证据。这些小鼠的B细胞寿命缩短，在体外激活后表现出caspase3的过度激活。随着对MAP4Ks参与河马途径的认识，我们获得了缺乏LATS1的小鼠和具有LATS2等位基因的小鼠。正在对这些小鼠进行分析，以评估Lats激酶在Map4k2/5双KO小鼠中发现的表型中的作用。 富含亮氨酸的重复蛋白2(LRRK2)是一个由2527个氨基酸组成的蛋白质，具有多个功能和蛋白质相互作用结构域。它在B淋巴细胞和巨噬细胞中有很好的表达。LRRK2的常染色体显性突变是帕金森氏病最常见的遗传原因，遗传连锁研究已确定其可能与克罗恩病和麻风有关。鉴于LRRK2突变的患者表现出自噬、溶酶体和免疫激活的变化，我们调查了LRRK2调节转录因子EB(TFEB)是否被认为是自噬和溶酶体生物发生的主要转录调节因子。我们的研究表明，TFEB是巨噬细胞和B细胞的免疫应答基因。在分子机制的剖析中，我们发现LRRK2增加了CREB的活性，从而控制TFEB的转录。此外，LRRK2在其C末端磷酸化TFEB，从而稳定蛋白质。我们还发现LRRK2介导的TFEB核转位依赖于溶酶体钙通道TPC2和钙调神经磷酸酶。除了它在LRRK2信号通路中的作用外，我们还发现TFEB是Wnt信号通路的负调控因子。为了证实这一结果，我们开发了一个缺乏TFEB表达的HEK293T细胞系，方法是使用CRISPR破坏TFEB基因。 最近，我们也开始通过活体显微镜检查各种抗原是如何输送到淋巴结中的B细胞和T细胞的。人类免疫缺陷病毒(HIV)gp120的包膜蛋白正在作为候选疫苗进行测试。利用活体显微镜跟踪gp120的转运，我们发现了一种新的机制，即gp120被捕获并被运送到淋巴细胞和树突状细胞。初步研究还检查了HIV-1病毒样颗粒(VLP)的定位和传递。使用GPF标记的VLP，我们使用活体双光子显微镜和厚淋巴结共聚焦显微镜来研究HIV VLP的摄取和转运到滤泡树突状细胞网络并最终运送到B细胞。在与Paolo Lusso实验室的合作中，我们发现表达α4β7的HIV-1 VLP通过与MAdCAM的相互作用靶向Peyer‘s斑块的高内皮细胞。 严重急性呼吸综合征(SARS)是一种新发现的病毒性传染病。我们研究了SARS病毒的三个开放阅读框架ORF-9b、ORF-8b和ORF-3a的影响。我们对ORF-9b的研究结果已经发表(J免疫193：3080-9,2014)。在最近的研究中，我们发现ORF8b迅速聚集在细胞中，触发NLRP3炎症体的激活。这会导致炎症细胞迅速死亡。当ORF8b瞬时表达时，ORF8b与NLRP3和ASC共定位于胞质点状结构中。对ORF-3a的研究表明，ORF-3a的表达导致溶酶体损伤，触发TFEB向细胞核的移位。SARS-3a的表达也会导致RIP3依赖但MLKL不依赖的细胞死亡。 细胞色素c从线粒体内膜释放出来，并被Caridolipin锚定，触发Apaf-1凋亡体的形成。心磷脂还与NLRP3相互作用，将NLRP3募集到线粒体，促进炎性小体组装。我们研究了胞浆细胞色素c是否影响巨噬细胞中NLRP3炎性小体的激活。我们发现细胞色素c与NLRP3的LRR结构域结合，细胞色素c减少了NLRP3与心磷脂以及NLRP3与NEK7之间的相互作用，NEK7是NLRP3齐聚所需的NLRP3炎症体的一个新成分。细胞色素c的蛋白转导可抑制NLRP3炎症小体的激活，而部分沉默细胞色素c的表达则可增强NLRP3的表达。在体外炎症小体实验中加入细胞色素c严重限制了caspase-1的激活。 细胞凋亡、上睑下垂和下垂都是导致细胞死亡的效应蛋白激活的特征。BCL-2通过与这些蛋白中存在的BH3结构域结合来抑制这些促凋亡蛋白。坏死性下垂使用效应蛋白MLKL，而热下垂使用效应蛋白Gasdermin D。我们发现MLKL和Gasdermin D都含有能够结合Bcl-2的BH3样结构域。高水平的Bcl-2表达抑制了触发坏死性下垂和下垂的信号。相反，Bcl2水平的降低使细胞对坏死性下垂和下垂都敏感。

To better understand the role of MAP4K2 and MAP4K5 in vivo, the murine map4k2 and map4k5 genes have been isolated. Both map4k2-/- and map4k5-/- mice have been created and backcrossed on to a C57Bl/6 background used to generate double knock-out (KO) mice. We have focused on the double KO mice as they exhibit a sharp reduction in the number of follicular B cells, an expansion of marginal zone B cells, elevated serum antibodies, defective humoral responses to neo-antigens, and evidence of autoimmunity. B cells from these mice have a shortened lifespan and exhibit an exaggerated activation of caspase 3 following in vitro activation. With the recognition that MAP4Ks can participate in the Hippo pathway, we have acquired mice lacking LATS1 and mice with a floxed allele of LATS2. These mice are being analyzed to assess the role of LATS kinases in the phenotype found in the map4k2/5 double KO mice. Leucine-rich repeat kinase 2 (LRRK2) is a large, 2527 amino acid protein with multiple functional and protein interaction domains. It is well expressed in B lymphocytes and macrophages. Autosomal dominant mutations in LRRK2 are the most common genetic cause of Parkinsons disease and genetic linkage studies have identified its potential involvement in Crohn's disease and leprosy. Given that patients with LRRK2 mutations show changes in autophagy, lysosomes, and in immune activation, we investigated whether LRRK2 regulated Transcription Factor EB (TFEB),a basic helix-loop-helix protein considered the master transcriptional regulator of autophagy and lysosomal biogenesis. Our studies show that TFEB is an immune response gene in macrophages and B cells. In dissecting the molecular mechanisms, we found that LRRK2 increases CREB activity, which controls TFEB transcription. In addition, LRRK2 phosphorylates TFEB on its C-terminus, which stabilizes the protein. We also showed that Lrrk2 mediated TFEB nuclear translocation is dependent on the lysosomal calcium channel TPC2 and calcineurin. Besides it role in Lrrk2 signaling we have also identified TFEB as a negative regulator of the Wnt signaling pathway. To confirm this result we developed a HEK293T cell line that lacks TFEB expression by disrupting the gene using CRISPR. Recently, we have also begun to examine how various antigens are delivered to B and T cells in the lymph node by intravital microscopy. The envelope protein of the human immunodeficiency virus (HIV) gp120 is being tested as a vaccine candidate. Using intravital microscopy to follow the transit of gp120 we uncovered a novel mechanism by which gp120 is captured and delivered to lymphocytes and dendritic cells. Preliminary studies have also examined the localization and delivery of HIV-1 viral like particles (VLPs). Using GPF labeled VLPs we used intravital 2-photon microscopy and thick lymph node confocal microscopy to study the uptake and delivery of HIV VLPs to the follicular dendritic cell network and eventually to B cells. In a collaboration with Paolo Lusso laboratory we found that HIV-1 VLPs expressing alpha4beta7 target to Peyer's patch high endothelium via an interaction with MAdCAM. Severe acute respiratory syndrome (SARS) is a recently recognized viral infectious disease. We have studied the impact of three open reading frames from the SARS virus, ORF-9b, ORF-8b and ORF-3A. The results of our studies of ORF-9b have been published (J Immunol 193:3080-9, 2014). In more recent studies we have found that ORF8b rapidly aggregates in cells triggering the activation of NLRP3 inflammasomes. This leads to a rapid inflammatory cell death. When transiently expressed ORF8b co-localizes in cytosolic dot-like structures with NLRP3 and ASC. Studies of ORF-3a indicate that the expression of ORF-3a causes lysosomal damage triggering the translocation of TFEB to the nucleus. Expression of SARS-3a also causes a RIP3 dependent, but MLKL independent cell death. The release of cytochrome c from the inner mitochondrial membrane, where it is anchored by caridolipin, triggers the formation of the Apaf-1 apoptosome. Cardiolipin also interacts with NLRP3 recruiting NLRP3 to mitochondria and facilitating inflammasome assembly. We have investigated whether cytosolic cytochrome c impacts NLRP3 inflammasome activation in macrophages. We found that cytochrome c binds to the LRR domain of NLRP3 and that cytochrome c reduces the interactions between NLRP3 and cardiolipin and between NLRP3 and NEK7, a recently recognized component of the NLRP3 inflammasome needed for NLRP3 oligomerization. Protein transduction of cytochrome c impairs NLRP3 inflammasome activation, while partially silencing cytochrome c expression enhances it. The addition of cytochrome c to an in vitro inflammasome assay severely limited caspase-1 activation. Apoptosis, pyroptosis and necroptosis all feature the activation of effector proteins that causes cellular death. Bcl-2 inhibits the pro-apoptotic proteins by binding to the BH3 domain present in those proteins. Necroptosis uses the effector protein, MLKL, and pyroptosis the effector protein gasdermin D. We have found that both MLKL and gasdermin D contain BH3-like domains that are capable of binding Bcl-2. Expression of high levels of Bcl-2 inhibit signals that trigger necroptosis and pyroptosis. Conversely, reduction in Bcl-2 levels sensitizes cells to both necroptosis and pyroptosis.