Crystal Structure of Epoxomicin:20S Proteasome Reveals a Molecular Basis for Selectivity of α‘,β‘-Epoxyketone Proteasome Inhibitors

Groll, M.; Kim, Kyung Bo; Kairies, Norman; Huber, Robert; Crews, Craig M.

doi:10.1021/ja993588m

Cited by 301 publications

(328 citation statements)

References 23 publications

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“…Using the crystal structure of the proteasome in complex with epoxomicin, which has structural similarity to the backbone of the suc-LLVY-AMC substrate, 23,34 in silico structural prediction of the b5 subunit in complex with suc-LLVY-AMC was performed, suggesting decreased binding affinity of this substrate in the mutant b5 in BTZ-resistant cells. These in silico results were confirmed using b5-activity assay after native gel electrophoresis, which showed a profound decreased conversion of suc-LLVY-AMC in 8226/ BTZ100 protein extract, although the b5 subunit was heavily overexpressed.…”

Section: Discussionmentioning

confidence: 99%

“…23) and the bovine proteasomal crystal structure (MMDB ID: 19465, PDB ID: 1IRU) 24 were used as templates. BTZ was manually docked into the bovine b5 subunit using Molecular Operating Environment (MOE) v2009 (Chemical Computing Group, Montreal, Quebec, Canada) and energy minimized using the Assisted Model Building with Energy Refinement (Amber99) force field.…”

Section: Computational Modeling Of B5 Mutationsmentioning

confidence: 99%

“…We performed in silico 3D modeling analysis of the b5-substrate tetrapeptide suc-LLVY-AMC. For our in silico 3D structural studies, we used a crystal structure of the yeast proteasome in complex with epoxomicin 23 and we used epoxomicin as a scaffold to generate atomic coordinates for bound suc-LLVY-AMC substrate. In addition, the substrate was docked into the WT bovine crystal structure of the proteasome, after which we introduced the different b5-subunit mutations.…”

Section: Mutations In B5 Subunit Hinder Suc-llvy-amc Bindingmentioning

confidence: 99%

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Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells

et al. 2011

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Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the b5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZresistant cells revealed upregulation of mutant b5 subunit of the proteasome. These newly identified b5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the b5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant b5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the a7 subunit of the proteasome can overcome this drug resistance modality.

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Section: Discussionmentioning

confidence: 99%

Section: Computational Modeling Of B5 Mutationsmentioning

confidence: 99%

Section: Mutations In B5 Subunit Hinder Suc-llvy-amc Bindingmentioning

confidence: 99%

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Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells

et al. 2011

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“…For example, bortezomib is a reversible PI, whereas carfilzomib binds selectively and irreversibly to the proteasome. 14,33,34 In addition, there may be differences in the synergistic effects between bortezomib and other agents relative to carfilzomib. The specific mechanisms that cause bortezomib resistance 35 will need to be more fully elucidated to determine the underlying basis for carfilzomib's ability to overcome bortezomib resistance.…”

Section: Discussionmentioning

confidence: 99%

Replacement of bortezomib with carfilzomib for multiple myeloma patients progressing from bortezomib combination therapy

et al. 2014

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In this open-label, intra-patient phase I/II trial, bortezomib was replaced with carfilzomib (escalated from 20 to 45 mg/m 2 on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle) for multiple myeloma (MM) patients who progressed while on or within 12 weeks of receiving a bortezomib-containing combination regimen. Study objectives included determination of the maximum tolerated dose (MTD), overall response rate (ORR), clinical benefit rate (CBR), time to progression, time to response, duration of response, progression-free survival and overall survival (OS). Of 38 registered patients, 37 were treated and evaluable for efficacy and safety. Thirty-one carfilzomib-based regimens using 14 different drug combinations were tested. One regimen (carfilzomib (45 mg/m 2 ), ascorbic acid (1000 mg) and cyclophosphamide (2.2 mg/kg)) reached MTD. ORR and CBR were 43.2 and 62.2%, respectively. Median progressionfree survival, time to progression and OS were 8.3, 9.9 and 15.8 months, respectively. Hematologic adverse events (AEs; Xgrade 3) included lymphopenia (35.1%), thrombocytopenia (24.3%), anemia (10.8%) and neutropenia (10.8%). Nonhematologic AEs (Xgrade 3) included fever (5.4%) and hypokalemia (5.4%). These results demonstrate that replacing bortezomib with carfilzomib is safe and can be effective for MM patients failing bortezomib-containing combination regimens. This trial was registered at

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“…Epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome with concomitant modification of the amino-terminal catalytic Thr residue of the 20S proteasome (Groll et al 2000;Meng et al 1999). Gliotoxin, a noncompetitive inhibitor of the chymotrypsin-like activity of the 20S proteasome, acts by reversible covalent modification involving mixed disulfide bonds at or near the active site of the chymotrypsin-like activity (Kroll et al 1999).…”

Section: Inhibition Assaysmentioning

confidence: 99%

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

Götze

Saborowski

Martínez‐Cruz

et al. 2017

Cell Stress and Chaperones

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Crustaceans are intensively farmed in aquaculture facilities where they are vulnerable to parasites, bacteria, or viruses, often severely compromising the rearing success. The ubiquitin-proteasome system (UPS) is crucial for the maintenance of cellular integrity. Analogous to higher vertebrates, the UPS of crustaceans may also play an important role in stress resistance and pathogen defense. We studied the general properties of the proteasome system in the hemocytes of the whiteleg shrimp, Penaeus vannamei, and the European brown shrimp Crangon crangon. The 20S proteasome was the predominant proteasome population in the hemocytes of both species. The specific activities of the trypsin-like (Try-like), chymotrypsinlike (Chy-like), and caspase-like (Cas-like) enzymes of the shrimp proteasome differed between species. P. vannamei exhibited a higher ratio of Try-like to Chy-like activities and Caslike to Chy-like activities than C. crangon. Notably, the Chy-like activity of P. vannamei showed substrate or product inhibition at concentrations of more than 25 mmol L −1 . The K M values ranged from 0.072 mmol L −1 for the Try-like activity of P. vannamei to 0.309 mmol L −1 for the Cas-like activity of C. crangon. Inhibition of the proteasome of P. vannamei by proteasome inhibitors was stronger than in C. crangon. The pH profiles were similar in both species. The Try-like, Chy-like, and Cas-like sites showed the highest activities between pH 7.5 and 8.5. The proteasomes of both species were sensitive against repeated freezing and thawing losing~80-90% of activity. This study forms the basis for future investigations on the shrimp response against infectious diseases, and the role of the UPS therein.

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Crystal Structure of Epoxomicin:20S Proteasome Reveals a Molecular Basis for Selectivity of α‘,β‘-Epoxyketone Proteasome Inhibitors

Cited by 301 publications

References 23 publications

Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells

Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells

Replacement of bortezomib with carfilzomib for multiple myeloma patients progressing from bortezomib combination therapy

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

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