Dissecting Intracellular Proteolysis Using Small Molecule Inhibitors and Molecular Probes

Ovaa, Huib; Overkleeft, Herman S.; Kessler, Benedikt M.; Ploegh, Hidde L.

doi:10.1002/9783527619320.ch3a

Cited by 1 publication

(1 citation statement)

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“…In contrast, MG-262 is a peptide boronate. Boronic acids have a high affinity for the Thr hydroxyl group, and the relative strength of the boron-oxygen bond, coupled with much slower off-rates than those of peptide aldehydes, endows them with greater metabolic stability (58). Thus, although they are classified as covalent, competitive, and reversible inhibitors, this property, in addition to their potency and selectivity, makes them not only superior to peptide aldehyde inhibitors but also suitable for clinical use as one prototype (Velcade) has proved.…”

Section: Characterization Of the Physiologic Turnover Of Nativementioning

confidence: 99%

Characterization of the Physiological Turnover of Native and Inactivated Cytochromes P450 3A in Cultured Rat Hepatocytes: A Role for the Cytosolic AAA ATPase p97?

et al. 2007

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Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno-and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in S. cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal and ALD [NH 4 Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase cum immunoprecipitation analyses revealed a basal 52% 35 S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH 4 Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were crosslinked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-crosslinking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic † Supported by NIH grants GM44037 (MAC) and DK26506 (MAC), RR012961 (KFM) and NIH grant P30DK26743 (Liver Core Center Cell and Tissue Biology Core). *Corresponding Author: M. A. Correia Dept. of Cellular and Molecular Pharmacology, Mission Bay Campus, Genentech Hall 600 16th Street, N572F/Box 2280 University of California San Francisco, CA 94158−2280 415−476−3992 (TEL) 415−476−5292 (FAX) e-mail: almira.correia@ucsf.edu. Supporting Information: MS/MS data obtained from the proteomic analyses of CYP3A-protein crosslinked complexes after subjection to SDS-PAGE, sequential gel-slicing of each relevant lane, and in situ tryptic digestion followed by LC-MS/MS analyses are provided. The proteins were identified from more than 1 peptide, but only 1 representative spectrum has been provided for each in the supplementary material. This material is available free of charge via the Internet at http://pubs.acs.org NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 15. Published in final edited form as:Biochemistry. analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.The hepatic hemoproteins cytochromes P450 (P450s) 1 are key enzymes in the oxidative metabolism of various endob...

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Section: Characterization Of the Physiologic Turnover Of Nativementioning

confidence: 99%