Distinct roles of immunoreceptor tyrosine‐based motifs in immunosuppressive indoleamine 2,3‐dioxygenase 1

Abstract

The enzyme indoleamine 2,3‐dioxygenase 1 (IDO1) catalyses the initial, rate‐limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1's catalytic function has long been considered as the one mechanism responsible for IDO1‐dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine‐based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP‐1 and SHP‐2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine‐phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half‐life. To dissect any differential roles of the two IDO1's ITIMs, we generated protein mutants by replacing one or both ITIM‐associated tyrosines with phospho‐mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 – but not ITIM2 mutant – did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen‐specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1's degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1‐dependent events will occur in a local microenvironment.

Tracking Hidden Binding Pockets Along the Molecular Recognition Path of l‐Trp to Indoleamine 2,3‐Dioxygenase 1

“…PCR products were digested with appropriate restriction enzymes and cloned into the pEF‐BOS plasmid . For both WT and mutated Ido1 constructs, 20 μg of plasmid DNA were transfected by electroporation in 1×10 7 cells of the murine mastocytoma P1.HTR . Stable transfectants were obtained by puromycin (Sigma‐Aldrich) selection.…”

“…[56] For both WT and mutated Ido1 constructs, 20 μg of plasmid DNA were transfected by electroporation in 1 × 10 7 cells of the murine mastocytoma P1.HTR. [57] Stable transfectants were obtained by puromycin (Sigma-Aldrich) selection. P1.HTR cells, stably transfected with the empty plasmid, were used as control.…”

Deficiency of immunoregulatory indoleamine 2,3-dioxygenase 1in juvenile diabetes

“…Relative protein expression was quantified by using an ImageQuant TL LAS4000 mini densitometer and the Analysis Toolbox software (GE Healthcare). Densitometric analysis of the specific signals was performed within a linear range of exposure of the blots, selecting in each experiment the 2 lowest exposure times useful to detect the signals, as described (64). Human IL-6 and TGF-β1 concentrations were determined in PBMC supernatants by using specific ELISA kits (eBioscience Inc. and Promega Corporation, respectively).…”

New Insights from Crystallographic Data: Diversity of Structural Motifs and Molecular Recognition Properties between Groups of IDO1 Structures

“…The structure of IDO1 is folded in two domains: 1) a small domain placed at the N‐terminal region containing two immunoreceptor tyrosine‐based inhibitory motifs (ITIMs); 2) a large domain located at the C‐terminal region hosting the heme cofactor (Figure ). The small domain is implicated in the signaling activity of the enzyme upon phosphorylation of the ITIMs, whereas the large domain accounts for the catalytic activity of IDO1 …”

“…The small domain is implicated in the signaling activity of the enzyme; this suggests that diverse inhibitors bound to the catalytic domain may induce different conformations of the small domain, eventually affecting both the catalytic activity and the signaling function of IDO1.…”