Woods, N. T. | Search bioRxiv

bioRxiv2019

E3 Ligase UBR5 HECT domain mutations in lymphoma control maturation of B cells via alternative splicing

Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have mutations in UBR5 with the majority of mutations within the HECT domain of the protein which can accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of UBR5 in B cell maturation and activation we generated a conditional mutant disrupting UBR5s C-terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in the spleen and up-regulation of proteins associated with mRNA splicing via the spliceosome. Our studies reveal a novel role of UBR5 in B cell maturation by regulating alternative splicing of key transcripts during B cell development and suggests UBR5 mutations may promote mantle cell lymphoma initiation.\n\nKEY POINTSO_LIUtilizing a novel mouse model mimicking MCL patient mutations, the loss of UBR5s HECT domain causes alterations in B cell development.\nC_LIO_LIUBR5 mutations lead to stabilization of UBR5 and aberrant splicing.\nC_LI

immunology↗

bioRxiv2019

Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

As an obligate intracellular pathogenic bacterium, C. trachomatis develops within a membrane-bound vacuole, termed the inclusion. The inclusion membrane is modified by chlamydial inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. An in vivo understanding of Inc-Inc and Inc-eukaryotic protein interactions and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. Previous bacterial two-hybrid studies established that certain Incs have the propensity to bind other Incs while others have limited Inc-Inc interactions. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To test this hypothesis, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, and chose to analyze Inc proteins with varying Inc-binding propensities. We inducibly expressed these Incs fused to APEX2 in Chlamydia trachomatis L2, verified their localization and labeling activities by transmission electron microscopy, and used affinity purification-mass spectrometry to identify biotinylated proteins. To analyze our mass spectrometry results for statistical significance, we used Significance Analysis of INTeractome (SAINT), which demonstrated that our Inc-APEX2 constructs labeled Inc proteins as well as known and previously unreported eukaryotic proteins that localize to the inclusion. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. One eukaryotic protein, LRRFIP1 (LRRF1) was found in all of our Inc-APEX2 datasets, which is consistent with previously published AP-MS datasets. For the first time, we demonstrate by confocal and super-resolution microscopy that endogenous LRRF1 localizes to the chlamydial inclusion. We also used bacterial two-hybrid studies and pulldown assays to determine if LRRF1 was identified as a true interacting protein or was proximal to our Inc-APEX2 constructs. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.\n\nAuthor summaryMany intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound \"bacteria containing vacuole\" (BCV) that, in most cases, prevents association with the lysosome. Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. Here, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. The inclusion membrane is modified by chlamydial type III secreted inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. Our data highlight the utility of APEX2 to capture the complex protein-protein interactions at a membrane site in vivo in the context of infection.

microbiology↗