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 b5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of b5c/X but not b5i, thereby explaining the selectivity of PR-957 for b5i. 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.

Proteolytic degradation is an essential cellular process which is primarily carried out by the 20S proteasome core particle (CP), a protease of 720 kDa and 28 individual subunits. As a result of its central functional role, the proteasome represents an attractive drug target that has been extensively investigated during the last decade and validated by the approval of bortezomib by the US Food and Drug Administration (FDA). Currently, several optimized second-generation proteasome inhibitors are being explored as anticancer drugs in clinical trials, and most of them target both constitutive proteasomes (cCPs) and immunoproteasomes (iCPs). However, selective inhibition of the iCPs, a distinct class of proteasomes predominantly expressed in immune cells, appears to be a promising therapeutic rationale for the treatment of autoimmune disorders. Although a few selective agents have already been identified, the recently determined crystal structure of the iCP will further promote the development and optimization of iCP-selective compounds.

Mammalian genomes encode seven catalytic proteasome subunits, namely, β1c, β2c, β5c (assembled into constitutive 20S proteasome core particles), β1i, β2i, β5i (incorporated into immunoproteasomes), and the thymoproteasome-specific subunit β5t. Extensive research in the past decades has yielded numerous potent proteasome inhibitors including compounds currently used in the clinic to treat multiple myeloma and mantle cell lymphoma. Proteasome inhibitors that selectively target combinations of β1c/β1i, β2c/β2i, or β5c/β5i are available, yet ligands truly selective for a single proteasome activity are scarce. In this work we report the development of cell-permeable β1i and β5i selective inhibitors that outperform existing leads in terms of selectivity and/or potency. These compounds are the result of a rational design strategy using known inhibitors as starting points and introducing structural features according to the X-ray structures of the murine constitutive and immunoproteasome 20S core particles.

Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.

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Quantification of 25-hydroxyvitamin D 3 (25-hydroxycholecalciferol) in serum is the best-established indicator of vitamin D status (1 ). Vitamin D 3 (cholecalciferol) is absorbed from the diet, and, given sufficient ultraviolet irradiation, nutritionally adequate amounts of vitamin D 3 are formed in the skin from its precursor, 7-dehydrocholesterol. In the liver, vitamin D 3 undergoes hydroxylation to 25-hydroxyvitamin D 3 , which is further metabolized in the kidney to form the active metabolite 1,25-dihydroxyvitamin D 3 . Vitamin D 2 (ergocalciferol) is derived solely from plant sources; relevant serum concentrations of 25-hydroxyvitamin D 2 are observed only after ingestion of vitamin D 2 drug preparations, and biological equivalence to vitamin D 3 has never been demonstrated conclusively.The prevalence of hypovitaminosis D has been recognized as substantial (2, 3 ), even in regions with high sun exposure (4 ), contributing not only to osteoporosis (5, 6 ) but possibly to a loss of muscle strength in aging as well (7 ).Various assays are used for the quantification of circulating 25-hydroxyvitamin D 3 that incorporate either vitamin D-binding globulin or anti-vitamin D antibodies for analyte recognition (8 -10 ). Fully automated tests have become available during recent years (11 ). Several HPLC methods with ultraviolet detection have been described as well (12,13 ), but their routine use is limited by complex sample preparation requirements.A key problem for the quantification of circulating 25-hydroxyvitamin D 3 is the strong binding of the molecule to vitamin D-binding globulin. Precipitation of serum constituents by use of organic solvents or acids can lead to variable coprecipitation of the analyte. Release of 25-hydroxyvitamin D 3 from its bonds to the binding protein is technically challenging in automated assays in particular. In light of these analytical problems, a reference method for the quantification of 25-hydroxyvitamin D 3 is desired to permit validation of routine immunoassays. Gas chromatography-mass spectrometry methods were developed years ago (14 -17 ), but they are extremely complex and did not gain use for quality-control programs or routine assay validation. Liquid chromatography-tandem mass spectrometry (LC-TMS) requires substantially less time-consuming sample clean-up and offers far shorter analytical run times than gas chromatographymass spectrometry. The feasibility of vitamin D quantification by LC-TMS was demonstrated previously (18 ), but the procedure included derivatization, which is complex and labor-intensive. Reference systems based on LC-MS were accepted in clinical chemistry recently (19,20 ), and we decided to develop a convenient and specific isotopedilution LC-TMS method for the quantification of 25-hydroxyvitamin D 3 in serum as a candidate reference method.For use as an internal standard, stable-isotope-labeled 25-hydroxyvitamin D 3 was synthesized as described previously (14, 17 ); the molecule contained three deuterium atoms and one 13 C atom. 25-Hydroxyvitamin D...

The CCAAT box is a frequent element of eukaryotic promoters, and its specific recognition by the conserved heterotrimeric CCAAT-binding complex (CBC) constitutes a key step in promoter organization and regulation of transcription. Here, we report the crystal structures of the CBC from Aspergillus nidulans in the absence and in complex with double-stranded DNA at 1.8 Å resolution. The histone-like subunits HapC and HapE induce nucleosome-like DNA bending by interacting with the sugar-phosphate backbone. Minor groove sensing and widening by subunit HapB tightly anchor the CBC to the CCAAT box, as shown by structural and biochemical data. Furthermore, crucial interactions of the DNA duplex with subunit HapB provide an explanation for the sequence specificity of the CBC. The herein-described mode of transcription factor binding answers the question of how histone proteins gained sequence specificity for the CCAAT box.

Inhibition of the 20S proteasome by bortezomib (Velcade) constitutes a successfully applied therapy for blood cancer. However, emerging resistance restricts its medicinal use. For example, mutations in the proteolytically active β5-subunit of the proteasome, the main target of inhibitors, were reported to impair drug binding and thus to reduce therapeutic efficacy. Using yeast as a model system, we describe here a systematic evaluation of these mutations by cell growth analysis, proteasome inhibition assays, and X-ray crystallography. The 11 mutants examined display decreased proliferation rates, impaired proteolytic activity, and marked resistance to bortezomib as well as the α',β'-epoxyketone inhibitors carfilzomib (Kyprolis) and ONX 0914, while the second-generation compound carfilzomib was the least affected. In total, 49 proteasome X-ray structures, including structural data on proteasome-carfilzomib complexes, reveal three distinct molecular mechanisms that hamper both drug binding and natural substrate turnover to an extent that is still compatible with cell survival.