Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

National Institute Of Allergy And Infectious Diseases

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Abstract

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 B cell function. 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 these results 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. 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 anti-apoptotic protein Bcl-2 belongs to Bcl-2 family of proteins. Bcl-2 prevents apoptosis by neutralizing the BH3-only proteins. Whether Bcl-2 involves the regulation of GSDMD mediated-pyroptosis or MLKL executed-necroptosis is unknown. We have identified a BH3-like domain in GSDMD and MLKL, which is targeted by Bcl-2. Bcl-2 not only reduces the GSDMD cleavage at D275 by caspase-1, 4 or 5, but also induces the GSDMD cleavage at D87. The D87 cleavage of GSDMD inactivates the pyroptotic execution. Bcl-2 expression reduces inflammasome induced pyroptosis. Bcl-2 also diminishes MLKL phosphorylation by Rip3 and inhibits the oligomerization of MLKL to reduce necroptosis. Our observation suggests that Bcl-2 regulates not only apoptosis, but also pyroptosis and necroptosis.