Isolation and Characterization of Bovine Thymus Multicatalytic Proteinase Complex

Eleuteri, Anna Maria; Angeletti, Mauro; Lupidi, Giulio; Tacconi, R; Bini, Luca; Fioretti, Evandro

doi:10.1006/prep.1999.1187

Cited by 37 publications

(32 citation statements)

References 41 publications

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“…Isolation and purification of MPC were carried out following an experimental protocol very similar to that previously utilized for MPC isolation from other bovine organs [14,15] and essentially based on fractionation from 40 to 60% in ammonium sulfate, an ion exchange chromatography and two gel-filtration columns which favor the removal of lower molecular weight contaminants. A higher degree of purification was obtained by adding a hydrophobic interaction chromatography step, which seems to improve the separation of MPC from the copurifying chaperonine Hsp90.…”

Section: Isolation and Purificationmentioning

confidence: 99%

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain

et al. 2002

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The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.

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Section: Isolation and Purificationmentioning

confidence: 99%

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain

et al. 2002

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“…14,15 This variant, which also contains different regulatory subunits known as 11S or PA28, 16 has been found to be highly expressed in cells of hematopoietic origin in several species. [17][18][19] Studies of 20S i function have revealed that it generates peptide fragments with more hydrophobic and basic amino acids at the C-terminus, 20 which are better suited for presentation to major histocompatability class I molecules, providing the rationale for its name. However, the 20S i also participates in many constitutive proteolytic processes, 17,19,[21][22][23][24] and conversely the 20S proteasome may in some cases generate immunogenic epitopes, 25 suggesting that each proteasome variant may provide both housekeeping and specialized functions.…”

Section: Introductionmentioning

confidence: 99%

Targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors

Kuhn

Hunsucker

Chen

et al. 2009

Blood

188

178

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Proteasome inhibition is a validated strategy for therapy of multiple myeloma, but this disease remains challenging as relapses are common, and often associated with increasing chemoresistance. Moreover, nonspecific proteasome inhibitors such as bortezomib can induce peripheral neuropathy and other toxicities that may compromise the ability to deliver therapy at full doses, thereby decreasing efficacy. One novel approach may be to target the immunoproteasome, a proteasomal variant found predominantly in cells of hematopoietic origin that differs from the constitutive proteasome found in most other cell types. Using purified preparations of constitutive and immunoproteasomes, we screened a rationally designed series of peptidyl-aldehydes and identified several with relative specificity for the immunoproteasome. The most potent immunoproteasome-specific inhibitor, IPSI-001, preferentially targeted the ␤1 i subunit of the immunoproteasome in vitro and in cellulo in a dose-dependent manner. This agent induced accumulation of ubiquitin-protein conjugates, proapoptotic proteins, and activated caspasemediated apoptosis. IPSI-001 potently inhibited proliferation in myeloma patient samples and other hematologic malignancies. Importantly, IPSI-001 was able to overcome conventional and novel drug resistance, including resistance to bortezomib. These findings provide a rationale for the translation of IPSIs to the clinic, where they may provide antimyeloma activity with greater specificity and less toxicity than current inhibitors.

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“…Proteasomes were isolated and purified from bovine thymus and brain according to early reported methods (Eleuteri et al, 2000;Amici et al, 2003). Substrates for assaying the ChT-L, T-L, PGPH activities (Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Ser-Thr-Arg-AMC, Z-Leu-Leu-Glu-AMC), recombinant GroES, triethanolamine hydrochloride and aminopeptidase N were purchased from Sigma-Aldrich S.R.L.…”

Section: Methodsmentioning

confidence: 99%

“…ChT-L, T-L, PGPH and BrAAP activities were determined using the peptides Suc-LLVY-AMC, z-LSTR-AMC, z-LLE-AMC and z-GPALG-pAB as substrates. Aminopeptidase N was used for the coupled assays in the determinations of the BrAAP activity (Orlowski and Michaud, 1989;Orlowski et al, 1993;Eleuteri et al, 2000). The reaction mixture was prepared by successive addition of Tris-HCl buffer 50 mM pH 8.0, 5 mg proteasomes and increasing amounts of GroES: finally, the reaction was started by addition of substrate.…”

Section: Effects Of Groes On the Proteolytic Activities Of 20 S Protementioning

confidence: 99%

Co‐chaperonin GroES as a modulator of proteasomal activity

Cuccioloni

Montecchia

Amici

et al. 2008

J of Molecular Recognition

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The proteasome has a crucial part in the degradation of normal, damaged, mutant or misfolded proteins within both the ubiquitin ATP-dependent and the ubiquitin ATP-independent pathways. Proteasome-mediated proteolysis is modulated by diverse factors, and in this regard, chaperonins have been attracting great interest. The investigation on the role of a co-chaperonin, namely GroES, in the modulation of proteasomal activity was the focus of this work. Our study reports on an analytical approach based on combined fluorimetric, chromatographic (applied to the enzymatic activity evaluation), surface plasmon resonance techniques and molecular modelling, addressed to the assessment and characterization of the interaction. Globally, we described a high affinity interaction between GroES and two different 20 S (immuno- and constitutive) proteasomes, uncovering new scenarios on their possible physio-pathological role, specifically on the ability of proteasomes to interact both with unfolding and folding- assisting macromolecules.

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Isolation and Characterization of Bovine Thymus Multicatalytic Proteinase Complex

Cited by 37 publications

References 41 publications

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain

Targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors

Co‐chaperonin GroES as a modulator of proteasomal activity

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