Human hepatic tryptophan 2,3-dioxygenase ubiquitin-dependent protein degradation: The critical role of its exosite as the molecular lynchpin of its substrate-mediated protein stabilization

Hepatic tryptophan 2,3-dioxygenase (TDO) is a cytoplasmic homotetrameric hemoprotein and the rate-limiting enzyme in the irreversible degradation of the essential amino acid L -tryptophan ( L-Trp ) to N-formylkynurenine, thus controlling the flux of L -Trp into its serotonergic and kynureninic/NAD pathways. TDO has long been recognized to be substrate-inducible via protein stabilization, but the molecular mechanism of this stabilization has remained elusive. Recent elucidation of human TDO (hTDO) crystal structure has identified a high-affinity (Kd ≈ 0.5 μM) Trp-binding exosite in each of its 4 monomeric subunits. Mutation of the Glu 105 , Trp 208 and Arg 211 comprising this exosite not only abolished the high-affinity L -Trp binding, but also accelerated the ubiquitin-dependent proteasomal degradation of hTDO. We have further characterized this hTDO degradation by documenting that its ubiquitination by gp78/AMFR and CHIP E2/E3 ligase complexes occurs on external Lys-residues within or vicinal to acidic Asp/Glu and phosphorylated pSer/pThr (DEpSpT)-clusters. Furthermore, we have identified the unstructured hTDO N- and C-termini as imparting relatively high proteolytic instability, as their deletion (ΔNC) markedly prolonged hTDO t 1/2 . Additionally, although previous studies reported that upon hepatic heme-depletion, the heme-free apoTDO turns over with a t 1/2 ≈ 2.2 h relative to the t 1/2 of 7.7 h of holoTDO, mutating the axial heme-ligating His 328 to Ala has the opposite effect of prolonging hTDO t 1/2 . Most importantly, introducing the exosite mutation into the ΔNC-deleted or H328A-mutant completely abolished their prolonged half-lives irrespective of L -Trp presence or absence, thereby revealing that the exosite is the molecular lynchpin that defines L -Trp-mediated TDO induction via protein stabilization.