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 [article]

Sung-Mi Kim, Yi Liu, YongQiang Wang, Shay Karkashon, Ariel Lewis-Ballester, Syun-Ru Yeh, Maria Almira Correia
2019 bioRxiv   pre-print
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)
more » ... ystal structure has identified a high-affinity (Kd ≈ 0.5 μM) Trp-binding exosite in each of its 4 monomeric subunits. Mutation of the Glu105, Trp208 and Arg211 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 t1/2. Additionally, although previous studies reported that upon hepatic heme-depletion, the heme-free apoTDO turns over with a t1/2 ≈ 2.2 h relative to the t1/2 of 7.7 h of holoTDO, mutating the axial heme-ligating His328 to Ala has the opposite effect of prolonging hTDO t1/2. 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.
doi:10.1101/793380 fatcat:45thbz25gfapjhao3midkbqhhe