No abstract

Mammalian histone demethylases of the KDM5 family are mediators of gene expression dynamics during developmental, cellular differentiation, and other nuclear processes. They belong to the large group of JmjC domain containing, 2-oxoglutarate (2-OG) dependent oxygenases and target methylated lysine 4 of histone H3 (H3K4me1/2/3), an epigenetic mark associated with active transcription. In recent years, KDM5 demethylases have gained increasing attention due to their misregulation in many cancer entities and are intensively explored as therapeutic targets. Despite these implications, the molecular basis of KDM5 function has so far remained only poorly understood. Little is known about mechanisms of nucleosome recognition, the recruitment to genomic targets, as well as the local regulation of demethylase activity. Experimental evidence suggests close physical and functional interactions with epigenetic regulators such as histone deacetylase (HDAC) containing complexes, as well as the retinoblastoma protein (RB). To understand the regulation of KDM5 proteins in the context of chromatin, these interactions have to be taken into account. Here, we review the current state of knowledge on KDM5 function, with a particular emphasis on molecular interactions and their potential implications. We will discuss and outline open questions that need to be addressed to better understand histone demethylation and potential demethylation-independent functions of KDM5s. Addressing these questions will increase our understanding of histone demethylation and allow us to develop strategies to target individual KDM5 enzymes in specific biological and disease contexts.

A disintegrin and metalloproteinase (ADAM) 17 has been implicated in many shedding processes. Major substrates of ADAM17 are TNF-α, IL-6R, and ligands of the epidermal growth factor receptor. The essential role of the protease is emphasized by the fact that ADAM17 deficiency is lethal in mice. To study ADAM17 function in vivo, we generated viable hypomorphic ADAM17 mice called ADAM17ex/ex mice. Recent studies indicated regulation of proteolytic ADAM17 activity by cellular processes such as cytoplasmic phosphorylation and removal of the prodomain by furin cleavage. Maturation and thus activation of ADAM17 is not fully understood. So far, studies of ADAM17 maturation have been mainly limited to mouse embryonic fibroblasts or transfected cell lines relying on nonphysiologic stimuli such as phorbol esters, thus making interpretation of the results difficult in a physiologic context. In this article, we present a robust cell system to study ADAM17 maturation and function in primary cells of the immune system. To this end, HoxB8 conditionally immortalized macrophage precursor cell lines were derived from bone marrow of wild-type and hypomorphic ADAM17ex/ex mice, which are devoid of measurable ADAM17 activity. ADAM17 mutants were stably expressed in macrophage precursor cells, differentiated to macrophages under different growth factor conditions (M-CSF versus GM-CSF), and analyzed for cellular localization, proteolytic activity, and podosome disassembly. Our study reveals maturation and activity of ADAM17 in a more physiological-immune cell system. We show that this cell system can be further exploited for genetic modifications of ADAM17 and for studying its function in immune cells.

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on primitive hematopoietic stem cells, activated platelets, CD4+ and CD8+ T cells, and keratinocytes. In recent years, CD109 was also associated with different tumor entities and identified as a possible future diagnostic marker linked to reduced patient survival. Also, different cell signaling pathways were proposed as targets for CD109 interference including the TGFβ, JAK-STAT3, YAP/TAZ, and EGFR/AKT/mTOR pathways. Here, we identify the metalloproteinase meprin β to cleave CD109 at the cell surface and thereby induce the release of cleavage fragments of different size. Major cleavage was identified within the bait region of CD109 residing in the middle of the protein. To identify the structural localization of the bait region, homology modeling and single-particle analysis were applied, resulting in a molecular model of membrane-associated CD109, which allows for the localization of the newly identified cleavage sites for meprin β and the previously published cleavage sites for the metalloproteinase bone morphogenetic protein-1 (BMP-1). Full-length CD109 localized on extracellular vesicles (EVs) was also identified as a release mechanism, and we can show that proteolytic cleavage of CD109 at the cell surface reduces the amount of CD109 sorted to EVs. In summary, we identified meprin β as the first membrane-bound protease to cleave CD109 within the bait region, provide a first structural model for CD109, and show that cell surface proteolysis correlates negatively with CD109 released on EVs.