Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

为了更好地理解MAP 4K 2和MAP 4K 5在体内的作用，已经分离了鼠map 4k 2和map 4k 5基因。已经创建了map 4k 2-/-和map 4k 5-/-小鼠，并将其与用于产生双敲除（KO）小鼠的C57 B1/6背景进行回交。我们集中在双KO小鼠，因为它们表现出滤泡B细胞数量的急剧减少，边缘区B细胞的扩增，血清抗体升高，对新抗原的体液应答缺陷，以及自身免疫的证据。来自这些小鼠的B细胞具有缩短的寿命，并且在体外活化后表现出过度的半胱天冬酶3活化。由于认识到MAP 4Ks可以参与Hippo通路，我们获得了缺乏LATS 1的小鼠和具有LATS 2的floxed等位基因的小鼠。对这些小鼠进行分析，以评估LATS激酶在map 4k 2/5双KO小鼠中发现的表型中的作用。 富含亮氨酸的重复序列激酶2（Leucine-rich repeat kinase 2，LRRK 2）是一个由2527个氨基酸组成的大蛋白，具有多个功能结构域和蛋白质相互作用结构域。它在B淋巴细胞和巨噬细胞中充分表达。LRRK 2的常染色体显性突变是帕金森病最常见的遗传原因，遗传连锁研究已经确定其与克罗恩病和麻风病的潜在关系。鉴于LRRK 2突变患者显示自噬、溶酶体和免疫活化的变化，我们研究了LRRK 2是否调节转录因子EB（TFEB），一种碱性螺旋-环-螺旋蛋白，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，TFE B是巨噬细胞和B细胞中的免疫应答基因。在剖析分子机制时，我们发现LRRK 2增加CREB活性，其控制TFEB转录。此外，LRRK 2在其C-末端磷酸化TFEB，从而稳定蛋白质。我们还发现Lrrk 2介导的TFEB核转位依赖于溶酶体钙通道TPC 2和钙调神经磷酸酶。除了它在Lrrk 2信号传导中的作用之外，我们还鉴定了TFEB作为Wnt信号传导途径的负调节剂。为了证实这一结果，我们开发了一种HEK 293 T细胞系，通过使用CRISPR破坏基因来缺乏TFEB表达。 最近，我们也开始研究如何通过活体显微镜将各种抗原传递到淋巴结中的B和T细胞。人类免疫缺陷病毒（HIV）gp 120的包膜蛋白正在作为候选疫苗进行测试。使用活体显微镜跟踪gp 120的转运，我们发现了一种新的机制，通过这种机制，gp 120被捕获并传递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）的定位和递送。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向滤泡树突状细胞网络并最终向B细胞的摄取和递送。在与Paolo Lusso实验室的合作中，我们发现表达α 4 β 7的HIV-1 VLP通过与MAdCAM的相互作用靶向派尔集合淋巴结高内皮。 严重急性呼吸综合征（severeacuterespiratorysyndrome，SARS）是一种新近被认识的病毒性传染病。 我们研究了SARS病毒的三个开放阅读框架ORF-9 b、ORF-8b和ORF-3A的影响。我们对ORF-9 b的研究结果已经发表（J Immunol 193：3080-9，2014）。在最近的研究中，我们发现ORF 8b在细胞中迅速聚集，触发NLRP 3炎性小体的激活。这导致炎症细胞迅速死亡。当ORF 8b瞬时表达时，其与NLRP 3和ASC共定位于细胞溶质点样结构中。ORF-3a的研究表明，ORF-3a的表达引起溶酶体损伤，触发TFEB易位到细胞核。SARS-3a的表达还引起RIP 3依赖性但MLKL非依赖性的细胞死亡。 细胞色素c从线粒体内膜释放，在那里它被卡立磷脂锚定，触发Apaf-1核糖体的形成。心磷脂还与NLRP 3相互作用，将NLRP 3募集到线粒体并促进炎性小体组装。我们已经研究了细胞溶质细胞色素c是否影响巨噬细胞中的NLRP 3炎性体活化。我们发现，细胞色素c与NLRP 3的LRR结构域结合，并且细胞色素c减少NLRP 3与心磷脂之间以及NLRP 3与NEK 7之间的相互作用，NEK 7是NLRP 3寡聚化所需的NLRP 3炎性体的最近认识到的组分。细胞色素c的蛋白质转导会削弱NLRP 3炎性体的激活，而部分沉默细胞色素c的表达会增强NLRP 3炎性体的激活。在体外炎性体测定中添加细胞色素c严重限制了半胱天冬酶-1的激活。 细胞凋亡、细胞凋亡和细胞坏死都以引起细胞死亡的效应蛋白的激活为特征。Bcl-2通过结合存在于这些蛋白中的BH 3结构域来抑制促凋亡蛋白。 坏死性凋亡使用效应蛋白MLKL，而焦亡使用效应蛋白gasdermin D。我们已经发现MLKL和gasdermin D都含有能够结合Bcl-2的BH 3样结构域。 高水平的Bcl-2表达抑制触发坏死性凋亡和焦亡的信号。相反，Bcl-2水平的降低使细胞对坏死性凋亡和焦亡两者敏感。

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.