Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

整合素/MFG-E8穿梭车将HIV-1病毒样颗粒装载到滤泡树突状细胞上。在人类免疫缺陷病毒-1(HIV-1)感染期间，淋巴器官滤泡树突状细胞(FDCs)是感染性病毒的储存库，也是治疗的障碍。在这项研究中，我们确定了淋巴器官窦衬里巨噬细胞(SMS)的一个子集，它提供了一个细胞-细胞接触门户，以及一个穿梭系统，它促进FDCs和B细胞摄取HIV-1病毒样颗粒(VLP)。门静脉功能的中心是桥联糖蛋白MFG-E8。利用磷脂酰丝氨酸(PS)结合区和RGD基序，MFG-E8帮助靶向HIV-1 VLP到v整合素携带的SM。FDC和HIV-1gp120特异性B细胞均从Sms上的MFG-E8富含部位收集HIV-1VLP。缺乏MFG-E8或整合素封锁严重限制了艾滋病毒-1 VLP在FDC网络上的传播。我们的结果确定了一种通过SMS感染HIV-1的机制，该机制促进了HIV-1在细胞间传播到FDCs和B细胞。 在B细胞和巨噬细胞中确定CD38-LRRK2-TFEB通路。CD38是一种细胞表面受体，在B细胞和巨噬细胞中高度表达，负责产生几种不同的第二信使。富亮氨酸重复蛋白2(LRRK2)是一种在B细胞和巨噬细胞中表达的多功能蛋白，但与CD38无关。LRRK2具有临床意义，因为它的突变与帕金森氏病有关。我们发现CD38结扎引起B细胞和巨噬细胞中钙依赖的转录因子EB(TFEB)的核易位。CD38参与触发依赖于细胞外和溶酶体钙离子的双相钙信号。CD38和LRRK2共定位于细胞膜，两种蛋白通过免疫共沉淀相互作用。LRRK2缺失小鼠的细胞在CD38刺激后钙反应减弱，而KI小鼠的细胞则表现出相反的表型。与这些发现一致的是，LRRK2缺失细胞在CD38结扎后TFEB激活方面存在缺陷。已知TFEB的激活有助于介导巨噬细胞从氧化磷酸化到糖酵解的转换。相应地，LRRK2缺失的巨噬细胞在内毒素刺激后糖酵解活性降低，而LRRK2Ki巨噬细胞的活性增加。有趣的是，我们还发现CD38刺激抑制巨噬细胞炎症体诱导的IL-1的释放，这种抑制依赖于LRRK2的存在。总之，我们已经确定了一个先前未知的CD38-LRRK2-TFEB信号轴，它与B细胞和巨噬细胞的功能有关。 BCL-2通过靶向Gasdermin D和MLKL中的BH-3样结构域来调节下垂和坏死性下垂。细胞凋亡是多细胞生物体中细胞程序性死亡的一种形式。BCL-2通过中和含有BH3结构域的蛋白，直接激活成孔蛋白Bax和BAK，从而防止细胞凋亡和促进细胞存活。然而，目前尚不清楚Bcl2对其他细胞死亡效应因子的调节作用，如Gasdermin D(GSDMD)或混合谱系激酶域样蛋白(MLKL)，它们的激活分别导致下垂和坏死性下垂。在这项研究中，我们在GSDMD和MLKL中都发现了一个BH3样结构域，它介导了与Bcl-2的相互作用。Bcl2的存在降低了caspase-1、4或5对D275的GSDMD的切割作用，增强了对D87的GSDMD的切割作用。GSDMD D87裂解使焦链执行程序失活。Bcl2的存在也限制了RIP3介导的MLKL的磷酸化，从而减少了MLKL的寡聚并缓和了对坏死性下垂的诱导。我们的观察表明，Bcl-2的存在限制了三种形式的细胞死亡的诱导：细胞凋亡、下垂和坏死性下垂。 胞质模式识别受体NLRP3在人类和啮齿动物中都能感知宿主衍生的危险信号和某些微生物衍生的产物。NLRP3激活组装一个炎症体复合体，该复合体包含适配蛋白ASC和caspase-1，其激活触发促炎症细胞因子IL-1和IL-18的成熟和释放。NLRP3的丝氨酸5(S5)磷酸化阻止其寡聚和激活，而磷酸酶PP2A对该残基的去磷酸化允许NLRP3激活。然而，介导NLRP3 S5磷酸化的蛋白激酶尚不清楚。我们已经证明AKT与NLRP3结合并在S5上将其磷酸化，限制了NLRP3的寡聚。这种磷酸化事件还通过减少NLRP3在赖氨酸496上的泛素化来稳定NLRP3，赖氨酸496抑制了蛋白酶体通过E3连接酶Trim31介导的降解。AKT激酶活性的药理调控可相互调节NLRP3炎症小体介导的IL-1β的产生。抑制AKT可减少小鼠腹腔注射脂多糖后IL-1β的产生。我们认为AKT、Trim31和PP2A共同调节NLRP3蛋白水平和寡聚倾向，从而为NLRP3激活设定一个严格调控的阈值。

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.