Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

B 淋巴细胞中的信号转导：关键信号分子的鉴定

• 批准号: 10272061
• 负责人: JOHN H KEHRL
• 金额: $ 121.56万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272061
• 关键词: AKT inhibition; Adaptor Signaling Protein; Antibody-Producing Cells; Antigens; Apoptosis; Autoimmune Diseases; Autophagocytosis; B-Lymphocytes; BCL2 gene; BH3 Domain; Bax protein; Binding; CASP1 gene; Calcium; Calcium Signaling; Cell Death; Cell Surface Receptors; Cell membrane; Cell physiology; Cells; Clinical; Co-Immunoprecipitations; Complex; Crohn' s disease; Cyclic AMP-Dependent Protein Kinases; Defect; Event; Follicular Dendritic Cells; Glycolysis; Glycoproteins; HIV Envelope Protein gp120; HIV-1; Human; Imaging Techniques; Immune response; Immunity; Infection; Inflammasome; Innate Immune System; Integrins; Interleukin-1; Interleukin-18; Intraperitoneal Injections; Knock-in; Knock-in Mouse; Knockout Mice; LATS1 gene; LATS2 gene; LRRK2 gene; Leprosy; Ligation; Link; Lysine; Mediating; Microbe; Mus; Mutation; Nuclear Translocation; Null Lymphocytes; Organism; Oxidative Phosphorylation; Parkinson Disease; Pathway interactions; Pattern recognition receptor; Pharmacology; Phase; Phenotype; Phosphatidylserines; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Production; Protein Dephosphorylation; Protein Kinase; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Proto-Oncogene Proteins c-akt; RGD (sequence); RIPK3 gene; Receptor Signaling; Regulation; Rodent; Second Messenger Systems; Serine; Signal Pathway; Signal Transduction; Signaling Molecule; Sinus; Site; Sum; System; Tissues; Ubiquitination; VDAC1 gene; Viral; Virus; cell type; curative treatments; cytokine; extracellular; germinal center kinases; interest; intravital microscopy; lymph nodes; lymphoid organ; macrophage; member; multicatalytic endopeptidase complex; particle; pathogen; prevent; programs; protein function; response; transcription factor; ubiquitin-protein ligase; uptake

An Integrin/MFG-E8 shuttle loads HIV-1 viral like particles onto follicular dendritic cells. During human immunodeficiency virus-1 (HIV-1) infection lymphoid organ follicular dendritic cells (FDCs) serve as a reservoir for infectious virus and an obstacle to curative therapies. In this study we identified a subset of lymphoid organ sinus lining macrophage (SMs) that provide a cell-cell contact portal, and a shuttling system, which facilitates the uptake of HIV-1 viral like particles (VLPs) by FDCs and B cells. Central for portal function was the bridging glycoprotein MFG-E8. Using a phosphatidylserine (PS) binding domain and an RGD motif, MFG-E8 helped target HIV-1 VLPs to v integrin bearing SMs. Both FDCs and HIV-1 gp120 specific B cells collected HIV-1 VLPs from MFG-E8 rich sites on SMs. Lack of MFG-E8 or integrin blockade severely limited the spread of HIV-1 VLPs onto FDC networks. Our results identify a mechanism for HIV-1 uptake by SMs that facilitates the cell to cell spread of HIV-1 to FDCs and B cells. Defining a CD38-LRRK2-TFEB pathway in B cells and macrophages. CD38 is a cell surface receptor highly expressed in B cells and macrophages responsible for generating several different second messengers. Leucine rich repeat kinase 2 (LRRK2) is a large multiple function protein expressed in B cells and macrophages, but not previously connected to CD38. LRRK2 is of clinical interest because mutations in it are linked to Parkinsons disease. We have found that CD38 ligation caused a calcium dependent nuclear translocation of transcription factor EB (TFEB) in B cells and macrophages. CD38 engagement triggers a bi-phasic calcium signal that depended upon extracellular and lysosomal calcium. CD38 and LRRK2 co-localized at the plasma membrane, and the two proteins robustly interacted by co-immunoprecipitation. Cells from LRRK2 null mice showed decreased calcium responses after CD38 stimulation, while cells from kinase overactive LRRK2 knock-in (KI) mice had the opposite phenotype. Consistent with these findings, LRRK2 null cells showed defects in TFEB activation following CD38 ligation. TFEB activation is known to help mediate the switch from oxidative phosphorylation to glycolysis in macrophages. Accordingly, LRRK2 null macrophages had decreased glycolytic activity after LPS stimulation, while LRRK2 KI macrophages had increased activity. Interestingly we also found that CD38 stimulation limits macrophage inflammasome induced IL-1 release, and this inhibition depended upon the presence of LRRK2. In sum, we have identified a previously unknown CD38-LRRK2-TFEB signaling axis with functional implications for both B cells and macrophages. Bcl-2 regulates pyroptosis and necroptosis by targeting BH-3 like domains in gasdermin D and MLKL. Apoptosis is a form of programmed cell death in multicellular organisms. Bcl-2 prevents apoptosis and promotes cellular survival by neutralizing BH3 domain containing proteins, which directly activate the pore-forming proteins BAX and BAK. However, Bcl-2 is not known to regulate other cell death effectors such as gasdermin D (GSDMD) or mixed lineage kinase domain-like (MLKL), whose activation causes pyroptosis and necroptosis, respectively. In this study, we identified a BH3-like domain in both GSDMD and MLKL that mediates an interaction with Bcl-2. The presence of Bcl-2 reduced GSDMD cleavage at D275 by caspase-1, 4 or 5, and enhanced the GSDMD cleavage at D87. The GSDMD D87 cleavage inactivates the pyroptotic execution program. The presence of Bcl-2 also limited RIP3 mediated phosphorylation of MLKL, which reduced MLKL oligomerization and tempered the induction of necroptosis. Our observations suggest that the presence of Bcl-2 limits the induction of three forms of cell death apoptosis, pyroptosis, and necroptosis. The cytosolic pattern recognition receptor NLRP3 senses host-derived danger signals and certain microbe-derived products in both humans and rodents. NLRP3 activation assembles an inflammasome complex, which contains the adapter protein ASC and caspase-1, whose activation triggers the maturation and release of the proinflammatory cytokines IL-1 and IL-18. Serine 5 (S5) phosphorylation of NLRP3 prevents its oligomerization and activation, while dephosphorylation of this residue by the phosphatase PP2A allows NLRP3 activation. However, the protein kinase that mediates NLRP3 S5 phosphorylation is unknown. We have shown that AKT associates with NLRP3 and phosphorylates it on S5, limiting NLRP3 oligomerization. This phosphorylation event also stabilizes NLRP3 by reducing its ubiquitination on lysine 496, which inhibits its proteasome mediated degradation by the E3 ligase Trim31. Pharmacologic manipulation of AKT kinase activity reciprocally modulates NLRP3 inflammasome mediated IL-1beta production. Inhibition of AKT reduced IL-1beta production following the intraperitoneal injection of LPS into mice. We propose that AKT, Trim31 and PP2A together modulate NLRP3 protein levels and tendency to oligomerize, thereby setting a tightly regulated threshold for NLRP3 activation.

整合素/MFG-E8穿梭体将HIV-1病毒样颗粒装载到滤泡树突细胞上。 在人类免疫缺陷病毒-1（HIV-1）感染过程中，淋巴器官滤泡树突状细胞（FDC）作为感染性病毒的储存库和治疗性治疗的障碍。在这项研究中，我们确定了一个淋巴器官窦内衬巨噬细胞（SM）的子集，提供了一个细胞-细胞接触门户，和一个穿梭系统，这有利于吸收HIV-1病毒样颗粒（VLP）的FDC和B细胞。门脉功能的中心是桥接糖蛋白MFG-E8。使用磷脂酰丝氨酸（PS）结合结构域和RGD基序，MFG-E8帮助将HIV-1 VLP靶向至携带整合素的SM。FDCs和HIV-1 gp 120特异性B细胞都从SM上富含MGF-E8的位点收集HIV-1 VLP。MFG-E8或整联蛋白阻断剂的缺乏严重限制了HIV-1 VLP在FDC网络中的传播。我们的研究结果确定了一种机制，为艾滋病毒-1的吸收SM，促进细胞间传播的艾滋病毒-1的FDC和B细胞。 在B细胞和巨噬细胞中定义CD 38-LRRK 2-TFE B途径。 CD 38是在B细胞和巨噬细胞中高度表达的细胞表面受体，负责产生几种不同的第二信使。富含亮氨酸重复序列激酶2（LRRK 2）是在B细胞和巨噬细胞中表达的大的多功能蛋白质，但先前未连接至CD 38。LRRK 2具有临床意义，因为它的突变与帕金森病有关。我们已经发现，CD 38连接引起B细胞和巨噬细胞中转录因子EB（TFE B）的钙依赖性核转位。 CD 38结合触发依赖于细胞外和溶酶体钙的双相钙信号。CD 38和LRRK 2共定位于质膜，并且这两种蛋白质通过免疫共沉淀强烈相互作用。来自LRRK 2敲除小鼠的细胞在CD 38刺激后显示出降低的钙应答，而来自激酶过度活化的LRRK 2敲入（KI）小鼠的细胞具有相反的表型。与这些发现一致，LRRK 2缺失细胞在CD 38连接后显示TFEB活化缺陷。已知TFEB活化有助于介导巨噬细胞中氧化磷酸化向糖酵解的转变。 因此，LRRK 2无效巨噬细胞在LPS刺激后具有降低的糖酵解活性，而LRRK 2 KI巨噬细胞具有增加的活性。有趣的是，我们还发现CD 38刺激限制了巨噬细胞炎性小体诱导的IL-1释放，并且这种抑制依赖于LRRK 2的存在。总之，我们已经确定了一个以前未知的CD 38-LRRK 2-TFE B信号传导轴，其对B细胞和巨噬细胞都具有功能意义。 Bcl-2通过靶向gasdermin D和MLKL中的BH-3样结构域调节细胞凋亡和坏死性凋亡。 细胞凋亡是多细胞生物中程序性细胞死亡的一种形式。Bcl-2通过中和含有BH 3结构域的蛋白质（直接激活成孔蛋白BAX和巴克）来防止细胞凋亡并促进细胞存活。然而，还不知道Bcl-2调节其他细胞死亡效应物，如gasdermin D（GSDMD）或混合谱系激酶结构域样（MLKL），其活化分别引起细胞凋亡和细胞坏死。 在这项研究中，我们确定了一个BH 3样结构域在GSDMD和MLKL介导的Bcl-2的相互作用。Bcl-2的存在减少了GSDMD在D275处被半胱天冬酶-1，4或5切割，并增强了GSDMD在D87处的切割。GSDMD D87裂解使自燃执行程序失活。Bcl-2的存在也限制了RIP 3介导的MLKL磷酸化，这减少了MLKL寡聚化并缓和了坏死性凋亡的诱导。我们的观察结果表明，Bcl-2的存在限制了诱导三种形式的细胞死亡凋亡，pyroptosis和necroptosis。 胞质模式识别受体NLRP 3在人类和啮齿动物中感知宿主衍生的危险信号和某些微生物衍生的产物。NLRP 3活化组装炎性体复合物，其含有衔接蛋白ASC和半胱天冬酶-1，其活化触发促炎细胞因子IL-1和IL-18的成熟和释放。NLRP 3的丝氨酸5（S5）磷酸化防止其寡聚化和活化，而磷酸酶PP 2A对该残基的去磷酸化允许NLRP 3活化。然而，介导NLRP 3 S5磷酸化的蛋白激酶是未知的。我们已经表明，AKT与NLRP 3相关联，并在S5上磷酸化它，限制NLRP 3寡聚化。该磷酸化事件还通过减少NLRP 3在赖氨酸496上的泛素化来稳定NLRP 3，这抑制了其由E3连接酶Trim 31介导的蛋白酶体降解。AKT激酶活性的药理学操作可调节NLRP 3炎性体介导的IL-1 β产生。腹腔注射LPS后，抑制AKT可减少IL-1 β的产生。我们提出，AKT，Trim 31和PP 2A一起调节NLRP 3蛋白水平和寡聚化趋势，从而为NLRP 3激活设定严格调节的阈值。