Project description:The impact of LPS and LTA stimulation on differentiated bone marrow and Yolk sac Hoxb8 macrophages in comparison to untreated control cells was studied by global protein profiling using a bottom-up approach.
Project description:We recently identified CCDC134 as a novel regulator of TLR responses. To gain a comprehensive understanding of the effects of CCDC134 loss, we conducted total proteome analysis on control sgRen and sgCCDC134 Hoxb8 immortalized murine myeloid progenitors that were differentiated into macrophages. Interestingly, we observed that plasma membrane and endolysosomal TLRs, along with their associated chaperone Gp96, were among the most significantly downregulated proteins. Our findings further demonstrated that CCDC134 is essential for the proper folding and stability of Gp96. As a result, the deletion of CCDC134 in various cell lines and Hoxb8-derived macrophages results in altered TLR folding and trafficking.
Project description:Progenitor cells of yolk sac and bone marrow origin were transduced with an estrogen receptor Hoxb8 fusion protein to generate stable cell lines. Macrophages were differentiated with M-CSF in the absence of estrogen, and then stimulated with IL-4 or LPS. Hoxb8 progenitor cells and differentiated macrophages were analyzed by RNA sequencing.
Project description:Investigation of immune cell differentiation and function is limited by shortcomings of suitable and scalable experimental systems. Although forced expression of certain Hox genes allows immortalization of hematopoietic progenitor cells, their differentiation potential is limited to select myeloid lineages. Here we show that an estrogen-regulated form of Hoxb8 that is retrovirally delivered into bone marrow cells can be used along with FLT3 ligand to conditionally immortalize early hematopoietic progenitor cells (Hoxb8-FL). Hoxb8-FL cells have lost self-renewal capacity and the ability to adopt megakaryocyte/ erythroid lineage fates, but sustain myeloid and lymphoid differentiation potential. Hoxb8-FL cells differentiate in vitro and in vivo into different myeloid and lymphoid cell types, including macrophages, granulocytes, dendritic cells and B- and T-lymphocytes, which are phenotypically and functionally indistinguishable from their primary counterparts. Given the simplicity to generate Hoxb8-FL cells and their unlimited proliferative capacity, this system provides unique opportunities to investigate cell differentiation and immune cell functions. Hoxb8 expressing immortalized cells
Project description:Various pathways can target nascent or fully synthesized precursor polypeptides to the human endoplasmic reticulum (ER). Typically, they involve cytosolic proteins or complexes and their respective receptors on the ER surface. The signal recognition particle (SRP) and the heterodimeric SRP-receptor (SR) represent one such targeting system, others are TRC40 and its heterodimeric TRC-receptor (WRB/CAML) and the components of the SND pathway. Apparently, they all can target precursor polypeptides to the Sec61-channel in the ER membrane. To characterize the substrate specificities of these targeting pathways, we combined siRNA-mediated depletion of membrane receptor subunits in HeLa cells with label-free quantitative proteomics and differential protein abundance analyis.
