Abstract
Transmembrane signaling proteins play a crucial role in the transduction of information across cell membranes. One function of regulated intramembrane proteolysis (RIP) is the release of signaling factors from transmembrane proteins. To study the role of transmembrane domains (TMDs) in modulating structure and activity of released signaling factors, we purified heterologously expressed human transmembrane proteins and their proteolytic processing products from Escherichia coli. Here we show that CD74 and TNFα are heme binding proteins. Heme coordination depends on both a cysteine residue proximal to the membrane and on the oligomerization of the TMD. Furthermore, we show that the various processing products have different modes of heme coordination. We suggest that RIP changes the mode of heme binding of these proteins and generates heme binding peptides with yet unexplored functions. The identification of a RIP modulated cofactor binding of transmembrane signaling proteins sheds new light on the regulation of cell signaling pathways.
Highlights
Transmembrane signaling proteins play a crucial role in the transduction of information across cell membranes
To exclude artificial heme binding via the His-tag fused to CD74-transmembrane domains (TMDs)[25–64], we replaced the 10xHis affinity tag in further experiments with maltose-binding protein (MBP)
We suggest to classify CD74, TNFα, and transferrin receptor-1 (Tfr1) as a new family of heme-binding proteins, called THOMAS proteins (Thiolate-Heme Oligomeric type II trans-Membrane proteins with heme binding mode Adjusted by SPPL2a/b, Fig. 7)
Summary
Transmembrane signaling proteins play a crucial role in the transduction of information across cell membranes. One function of regulated intramembrane proteolysis (RIP) is the release of signaling factors from transmembrane proteins. To study the role of transmembrane domains (TMDs) in modulating structure and activity of released signaling factors, we purified heterologously expressed human transmembrane proteins and their proteolytic processing products from Escherichia coli. The remaining membrane-bound fragment is processed by intramembrane-cleaving proteases such as signal peptide peptidase-like (SPPL) proteases. Subsequent RIP of CD74-NTF by the SPPL protease SPPL2a generates the intracellular domain (ICD) involved in inward signaling. Biological functions of CD74 and TNFα have been extensively studied, little is known about the role of their transmembrane domains in modulating structure and activity of the RIP-released fragments. We observed that the type II transmembrane proteins CD74, TNFα, and other substrates of SPPL2a/b proteases such as Bri, FasL, and Tfr are heme-binding proteins. We suggest that these short ICDs may function as intracellular heme sensor peptides participating in cellular signaling and speculate that full-length type II transmembrane proteins and their NTFs have so far unknown heme-dependent (enzymatic) functions
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