Characterization of the structural determinants of the ubiquitin-dependent proteasomal degradation of human hepatic tryptophan 2,3-dioxygenase.

Human hepatic tryptophan 2,3-dioxygenase (hTDO) is a homotetrameric hemoprotein. It is one of the most rapidly degraded liver proteins with a half-life (t1/2) of ∼2.3 h, relative to an average t1/2 of ∼2-3 days for total liver protein. The molecular mechanism underlying the poor longevity of hTDO remains elusive. Previously, we showed that hTDO could be recognized and ubiquitinated by two E3 ubiquitin (Ub) ligases, gp78/AMFR and CHIP, and subsequently degraded via Ub-dependent proteasomal degradation pathway. Additionally, we identified 15 ubiquitination K-sites and demonstrated that Trp-binding to an exosite impeded its proteolytic degradation. Here, we further established autophagic-lysosomal degradation as an alternative back-up pathway for cellular hTDO degradation. In addition, with protein kinases A and C, we identified 13 phosphorylated Ser/Thr (pS/pT) sites. Mapping these pS/pT sites on the hTDO surface revealed their propinquity to acidic Asp/Glu (D/E) residues engendering negatively charged DEpSpT clusters vicinal to the ubiquitination K-sites over the entire protein surface. Through site-directed mutagenesis of positively charged patches of gp78, previously documented to interact with the DEpSpT clusters in other target proteins, we uncovered the likely role of the DEpSpT clusters in the molecular recognition of hTDO by gp78 and plausibly other E3 Ub-ligases. Furthermore, cycloheximide-chase analyses revealed the critical structural relevance of the disordered N- and C-termini not only in the Ub-ligase recognition, but also in the proteasome engagement. Together, the surface DEpSpT clusters and the N- and C-termini constitute an intrinsic bipartite degron for hTDO physiological turnover.

Inhibition Mechanisms of Human Indoleamine 2,3 Dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) catalyze the same dioxygenation reaction of Trp to generate N-formyl kynurenine (NFK). They share high structural similarity, especially in the active site. However, hIDO1 possesses a unique inhibitory substrate binding site (Si) that is absent in hTDO. In addition, in hIDO1, the indoleamine group of the substrate Trp is H-bonded to S167 through a bridging water, while that in hTDO is directly H-bonded to H76. Here we show that Trp binding to the Si site or the mutation of S167 to histidine in hIDO1 retards its turnover activity and that the inhibited activity can be rescued by an effector, 3-indole ethanol (IDE). Kinetic studies reveal that the inhibited activity introduced by Trp binding to the Si site is a result of retarded recombination of the ferryl moiety with Trp epoxide to form NFK and that IDE reverses the effect by preventing Trp from binding to the Si site. In contrast, the abolished activity induced by the S167H mutation is primarily a result of ∼5000-fold reduction in the O2 binding rate constant, possibly due to the blockage of a ligand delivery tunnel, and that IDE binding to the Si site reverses the effect by reopening the tunnel. The data offer new insights into structure-based design of hIDO1-selective inhibitors.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) play a central role in tryptophan metabolism and are involved in many cellular and disease processes. Here we report the crystal structure of human TDO (hTDO) in a ternary complex with the substrates L-Trp and O2 and in a binary complex with the product N-formylkynurenine (NFK), defining for the first time the binding modes of both substrates and the product of this enzyme. The structure indicates that the dioxygenation reaction is initiated by a direct attack of O2 on the C2 atom of the L-Trp indole ring. The structure also reveals an exo binding site for L-Trp, located ~42 Å from the active site and formed by residues conserved among tryptophan-auxotrophic TDOs. Biochemical and cellular studies indicate that Trp binding at this exo site does not affect enzyme catalysis but instead it retards the degradation of hTDO through the ubiquitin-dependent proteasomal pathway. This exo site may therefore provide a novel L-Trp-mediated regulation mechanism for cellular degradation of hTDO, which may have important implications in human diseases.

Intermediaries of branched chain amino acid metabolism induce fetal hemoglobin, and repress SOX6 and BCL11A, in definitive erythroid cells.

High levels of fetal hemoglobin (HbF) can ameliorate human ß-globin gene disorders. The short chain fatty acid butyrate is the paradigmatic metabolic intermediary that induces HbF. Inherited disorders of branched-chain amino acid (BCAA) metabolism have been associated with supranormal HbF levels beyond infancy, e.g., propionic acidemia (PA) and methylmalonic acidemia (MMA). We tested intermediaries of BCAA metabolism for their effects on definitive erythropoiesis. Like butyrate, the elevated BCAA intermediaries isovalerate, isobutyrate, and propionate, induce fetal globin gene expression in murine EryD in vitro, are associated with bulk histone H3 hyperacylation, and repress the transcription of key gamma globin regulatory factors, notably BCL11A and SOX6. Metabolic intermediaries that are elevated in Maple Syrup Urine Disease (MSUD) affect none of these processes. Percent HbF and gamma (γ) chain isoforms were also measured in non-anemic, therapeutically optimized subjects with MSUD (Group I, n=6) or with Isovaleric Acidemia (IVA), MMA, or PA (Group II, n=5). Mean HbF was 0.24 ± 0.15% in Group I and 0.87 ± 0.13% in Group II (p=.01); only the Gγ isoform was detected. We conclude that a family of biochemically related intermediaries of branched chain amino acid metabolism induces fetal hemoglobin during definitive erythropoiesis, with mechanisms that mirror those so far identified for butyrate.

Induction of fetal/embryonic globin gene expression depends on intact cell signaling in definitive erythroid cells.

OBJECTIVE: The induction of fetal hemoglobin during definitive erythropoiesis is a major therapeutic goal in ß-globin gene disorders. Butyrate induces fetal hemoglobin, and p38 phosphorylation has been implicated in this process. We studied p38 and the effect of its inhibitors in a physiologic primary cell model of fetal/embryonic globin gene induction during definitive erythropoiesis. METHODS: p38 phosphorylation was evaluated in a short-term culture of definitive erythroid precursor (EryD) cells following butyrate induction, absent prolonged exposure to cytokines. The impact of p38 inhibitors on embryonic/fetal globin gene induction by butyrate and on normal erythroid processes, including proliferation, differentiation, cell cycle occupancy, and RNA transcription, was also examined. RESULTS: p38 phosphorylation, maximal at harvest of murine fetal liver-derived EryD (FL EryD), when minimal embryonic/fetal globin gene expression is seen, is suppressed by EPO (as reported by others). Butyrate initially delays EPO-mediated suppression of p38 phosphorylation, but p38 phosphorylation thereafter, at 30 minutes to 48 hours, is equivalent and at low levels in EPO-treated FL EryD, with or without butyrate. Inhibitors of p38, at 10-50 µM, prevent butyrate-mediated induction of embryonic/fetal globin gene expression. We found that p38 inhibitors, which also disrupt non-p38 signaling pathways, perturb cell division, erythroid differentiation, transit through the cell cycle, and RNA transcription in primary EryD. CONCLUSION: p38 inhibitors interrupt normal erythropoiesis and the capacity for embryonic/fetal globin gene induction. However, p38 signaling is maximal in primary EryD at harvest, when embryonic globin genes are minimally expressed, and diminishes thereafter. We conclude that p38 inhibitors disrupt cellular pathways that are essential to butyrate-induced embryonic/fetal globin gene expression. However, levels of p38 phosphorylation are not coordinate with embryonic/fetal globin gene expression in EryD, and increased signaling through p38 may not be the sine qua non for embryonic/fetal globin gene induction.

Short-chain fatty acid-mediated effects on erythropoiesis in primary definitive erythroid cells.

Short-chain fatty acids (SCFAs; butyrate and propionate) up-regulate embryonic/fetal globin gene expression through unclear mechanisms. In a murine model of definitive erythropoiesis, SCFAs increased embryonic beta-type globin gene expression in primary erythroid fetal liver cells (eFLCs) after 72 hours in culture, from 1.7% (+/- 1.2%) of total beta-globin gene expression at day 0 to 4.9% (+/- 2.2%) in propionate and 5.4% (+/- 3.4%) in butyrate; this effect was greater in butyrate plus insulin/erythropoietin (BIE), at 19.5% (+/- 8.3%) compared with 0.1% (+/- 0.1%) in ins/EPO alone (P < .05). Fetal gamma-globin gene expression was increased in human transgene-containing eFLCs, to 35.9% (+/- 7.0%) in BIE compared with 4.4% (+/- 4.2%) in ins/EPO only (P < .05). Embryonic globin gene expression was detectable in 11 of 15 single eFLCs treated with BIE, but in0 of 15 ins/EPO-only treated cells. Butyrate-treated [65.5% (+/- 9.9%)] and 77.5% (+/- 4.0%) propionate-treated eFLCs were highly differentiated in culture, compared with 21.5% (+/- 3.5%) in ins/EPO (P < .005). Importantly, signaling intermediaries, previously implicated in induced embryonic/fetal globin gene expression (STAT5, p42/44, and p38), were not differentially activated by SCFAs in eFLCs; but increased bulk histone (H3) acetylation was seen in SCFA-treated eFLCs. SCFAs induce embryonic globin gene expression in eFLCS, which are a useful short-term and physiologic primary cell model of embryonic/fetal globin gene induction during definitive erythropoiesis.

tRNase Z catalysis and conserved residues on the carboxy side of the His cluster.

tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3'-end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the beta-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for the coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein, we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops, and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V, and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reduces efficiency by approximately 1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases.