Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity.

Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit ß5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of ß5c/X but not ß5i, thereby explaining the selectivity of PR-957 for ß5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.

7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver.

The cytochrome P450 subfamily CYP3A belongs to the most important detoxification enzymes. Because the main CYP3A isoforms are not polymorphic and therefore detract themselves from genetic screening as a potent prediction marker for drug metabolism or induction effects, effective in vitro testing of a putative drug-CYP3A interaction is indicated. We used mouse liver microsomes treated with the model drug phenytoin to set up an effective and reliable in vitro test system. A metabolic assay analyzing 7-alkoxyresorufin-O-dealkylation showed specific CYP3A-dependent 7-benzyloxyresorufin oxidation (BROD). This was confirmed by testing other alkoxyresorufins (7-ethoxy-, 7-methoxy-, and 7-pentoxyresorufin) in mice and correlation of the data with testosterone 6beta-hydroxylation and a plethora of isoform-specific chemical inhibitors (orphenadrine, chloramphenicol, nifedipine, ketoconazole, and sulfaphenazole). Isoform-specific expression and induction of CYP3A11 in mouse liver was tested by RNase protection assay, reverse transcription polymerase chain reaction (RT-PCR), and immunoblot. With the BROD assay, we could clearly dissect CYP3A11 from other P450s induced by phenytoin-like CYP2C29, CYP2B9, CYP1A1, and CYP4A. We conclude that the BROD assay is a specific tool to assign CYP3A induction by drugs or other chemicals, at least in a mouse model system.

Concordant up-regulation of cytochrome P450 Cyp3a11, testosterone oxidation and androgen receptor expression in mouse brain after xenobiotic treatment.

Inactivation of testosterone by specific hydroxylations is a main function of cytochrome P450 (P450, CYP) in the brain. Recent data imply that induction of brain P450s by neuroactive drugs alters steroid hormone levels and endocrine signalling, giving rise to endocrine disorders. In this study, we investigated this drug-hormone crosstalk in mouse brain. Phenytoin led to a significant increase of 2alpha-, 2beta-, 6beta-, 16alpha- and 16beta-hydroxytestosterones, while 6alpha- and 15alpha-hydroxytestosterones showed no significant alteration of their metabolism compared with untreated controls. Inhibition of testosterone hydroxylation using the chemical inhibitors orphenadrine, chloramphenicol, ketoconazole and nifedipine as well as antibodies against CYP3A- and 2B-isoforms pointed to major role of Cyp3a11 and an only minor function of Cyp2b9/10 in mouse brain. Cyp3a11 revealed to be the major isoform affected by phenytoin. There was considerable overlap of Cyp3a11 and AR expression in neuronal structures of the limbic system, namely the hippocampus, amygdala, hypothalamus and thalamus. Phenytoin treatment led to an increase of both, Cyp3a11 and AR expression in the limbic system. Additionally, the coherence between CYP3A and AR expression was analysed in PC-12 cells. Inhibition of phenytoin-induced endogenous CYP3A2 and AR by ketoconazole led a reduction of their expression to basal levels. We conclude that Cyp3a11 plays a crucial role in directing drug action to hormonal response within the limbic system of mouse brain in a so-called drug-hormone crosstalk.

Author Correction: The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation.

The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 3. The HTML version of the Article has been updated to include a corrected version of the Supplementary Information.

The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation.

FAT10 is a ubiquitin-like modifier that directly targets proteins for proteasomal degradation. Here, we report the high-resolution structures of the two individual ubiquitin-like domains (UBD) of FAT10 that are joined by a flexible linker. While the UBDs of FAT10 show the typical ubiquitin-fold, their surfaces are entirely different from each other and from ubiquitin explaining their unique binding specificities. Deletion of the linker abrogates FAT10-conjugation while its mutation blocks auto-FAT10ylation of the FAT10-conjugating enzyme USE1 but not bulk conjugate formation. FAT10- but not ubiquitin-mediated degradation is independent of the segregase VCP/p97 in the presence but not the absence of FAT10's unstructured N-terminal heptapeptide. Stabilization of the FAT10 UBDs strongly decelerates degradation suggesting that the intrinsic instability of FAT10 together with its disordered N-terminus enables the rapid, joint degradation of FAT10 and its substrates without the need for FAT10 de-conjugation and partial substrate unfolding.

Xenobiotics in the limbic system--affecting brain's network function.

Xenobiotic compounds enter the brain through nutrition, environmentals, and drugs. In order to maintain intrinsic homeostasis, the brain has to adapt to xenobiotic influx. Among others, steroid hormones appear as crucial mediators in this process. However, especially in the therapy of neurological diseases or brain tumors, long-term application of neuroactive drugs is advised. Several clinically important malignancies based on hormonal dysbalance rise up after treatment with neuroactive drugs, for example, sexual and mental disorders or severe cognitive changes. A drug-hormone cross talk proceeding over drug-mediated cytochrome P450 induction predominantly in the limbic system and the blood-brain barrier, consequently altered steroid hormone metabolism, and P450-mediated change of steroid hormone receptor expression and signaling may serve as an explanation for such disorders. Especially, the interplay between the expression of AR and P450 at the blood-brain barrier and in structures of the limbic system is of considerable interest in understanding brain's reaction on xenobiotic treatment. This chapter summarizes present models and concepts on brain's reaction after xenobiotics crossing the blood-brain barrier and invading the limbic system.