Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits

Livinskaya, Veronika A.; Barlev, Nickolai A.; Aa, Nikiforov

doi:10.1016/j.pep.2014.02.011

Cited by 6 publications

(5 citation statements)

References 26 publications

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“…In order to insure purification of all proteolyti cally active forms of proteasomes in complex with var ious regulators it was necessary to select a subunit of the 20S complex. We did not consider for this role any subunit of the 20S α type proteasome, since first, these proteins could be in the free state within cell and not just in the complex with proteasomes [23,24], and second, it had been demonstrated that tagging of α5 (PSMA5) or α7 (PSMA3) subunits completely pre vented incorporation of the subunits into the 20S pro teasome [25]. Subunit β7 of the 20S proteasome was selected because despite the fact it forms proteolytic cavity of the 20S proteasome, this subunit first, is not catalytically active and second, compared to all other subunits of the of β type it has shorter lifetime [26].…”

Section: Resultsmentioning

confidence: 99%

Mass spectrometric analysis of affinity-purified proteasomes from the human myelogenous leukemia K562 cell line

2014

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Proteasomes carry out regulated proteolysis of most proteins and thereby play a crucial role in the regulation of different cellular processes. Dissecting subunit composition and post-translational modifications of proteasome is one of the important milestones in understanding their functions and mechanisms of regulation in the cell. To this end a strategy we followed a strategy for affinity purification of proteasomes from human myeloid leukemia cells with subsequent mass spectrometric analysis. Proteasomes were purified from the stable cell line K562 expressing HTBH tag-labeled 20S proteasome subunit β7 (PSMB4) by non-covalent affinity purification on biotin-avidin beads, followed by elution with TEV protease. We identified all known subunits of the 26S proteasome, as well as PA200 and regulators PA28γ amongst the eluted proteins, using MALDI-ICR mass spectrometry. We have shown that the proteasomes are associated with heat shock proteins, components of the ubiquitin-proteasome system of some cytoskeleton proteins. A number of novel phosphorylation, ubiquitination and N-terminal modification of proteasome subunits were found for 16 proteasome subunits. Our results might be useful for further proteomic studies of proteasomes.

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Section: Resultsmentioning

confidence: 99%

Mass spectrometric analysis of affinity-purified proteasomes from the human myelogenous leukemia K562 cell line

2014

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“…Two subunits of proteasome PSA3and PSA7 were also found to be differentially expressed, are involved in the regulation of several crucial cellular process, including apoptosis, immune responses, DNA repair, protein quality control and cell cycle progression [ 134 ]. PSA3 (also known as PSMA3) [ 15 ], was down-regulated in cis-resistant cell line as compared to its sensitive counterpart when A2780 was used as a reference, this is consistent with finding of Moghanibashi et al in which PSA3 was reported to be down-regulated in ESCC [ 135 ].…”

Section: Discussionmentioning

confidence: 99%

Sequenced Combinations of Cisplatin and Selected Phytochemicals towards Overcoming Drug Resistance in Ovarian Tumour Models

Althurwi

Beale

et al. 2020

IJMS

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In the present study, cisplatin, artemisinin, and oleanolic acid were evaluated alone, and in combination, on human ovarian A2780, A2780ZD0473R, and A2780cisR cancer cell lines, with the aim of overcoming cisplatin resistance and side effects. Cytotoxicity was assessed by MTT reduction assay. Combination index (CI) values were used as a measure of combined drug effect. MALDI TOF/TOF MS/MS and 2-DE gel electrophoresis were used to identify protein biomarkers in ovarian cancer and to evaluate combination effects. Synergism from combinations was dependent on concentration and sequence of administration. Generally, bolus was most synergistic. Moreover, 49 proteins differently expressed by 2 ≥ fold were: CYPA, EIF5A1, Op18, p18, LDHB, P4HB, HSP7C, GRP94, ERp57, mortalin, IMMT, CLIC1, NM23, PSA3,1433Z, and HSP90B were down-regulated, whereas hnRNPA1, hnRNPA2/B1, EF2, GOT1, EF1A1, VIME, BIP, ATP5H, APG2, VINC, KPYM, RAN, PSA7, TPI, PGK1, ACTG and VDAC1 were up-regulated, while TCPA, TCPH, TCPB, PRDX6, EF1G, ATPA, ENOA, PRDX1, MCM7, GBLP, PSAT, Hop, EFTU, PGAM1, SERA and CAH2 were not-expressed in A2780cisR cells. The proteins were found to play critical roles in cell cycle regulation, metabolism, and biosynthetic processes and drug resistance and detoxification. Results indicate that appropriately sequenced combinations of cisplatin with artemisinin (ART) and oleanolic acid (OA) may provide a means to reduce side effects and circumvent platinum resistance.

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“…Both cerebral hemispheres were analyzed in piglets that received AAV. An HEK293 cell lysate was loaded into each gel as an inter-gel standard and proteasome subunit control because the proteasome β5 and α3 subunit identities have been confirmed by transient transfection in HEK293 cells [47]. GFP protein (Biovision, Milpitas, CA, USA) was loaded into gels for the AAV experiments as a positive control.…”

Section: Western Blot Analysismentioning

confidence: 99%

Oleuropein Activates Neonatal Neocortical Proteasomes, but Proteasome Gene Targeting by AAV9 Is Variable in a Clinically Relevant Piglet Model of Brain Hypoxia-Ischemia and Hypothermia

Demerdash

Chen

O’Brien

et al. 2021

Cells

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Cerebral hypoxia-ischemia (HI) compromises the proteasome in a clinically relevant neonatal piglet model. Protecting and activating proteasomes could be an adjunct therapy to hypothermia. We investigated whether chymotrypsin-like proteasome activity differs regionally and developmentally in the neonatal brain. We also tested whether neonatal brain proteasomes can be modulated by oleuropein, an experimental pleiotropic neuroprotective drug, or by targeting a proteasome subunit gene using recombinant adeno-associated virus-9 (AAV). During post-HI hypothermia, we treated piglets with oleuropein, used AAV-short hairpin RNA (shRNA) to knock down proteasome activator 28γ (PA28γ), or enforced PA28γ using AAV-PA28γ with green fluorescent protein (GFP). Neonatal neocortex and subcortical white matter had greater proteasome activity than did liver and kidney. Neonatal white matter had higher proteasome activity than did juvenile white matter. Lower arterial pH 1 h after HI correlated with greater subsequent cortical proteasome activity. With increasing brain homogenate protein input into the assay, the initial proteasome activity increased only among shams, whereas HI increased total kinetic proteasome activity. OLE increased the initial neocortical proteasome activity after hypothermia. AAV drove GFP expression, and white matter PA28γ levels correlated with proteasome activity and subunit levels. However, AAV proteasome modulation varied. Thus, neonatal neocortical proteasomes can be pharmacologically activated. HI slows the initial proteasome performance, but then augments ongoing catalytic activity. AAV-mediated genetic manipulation in the piglet brain holds promise, though proteasome gene targeting requires further development.

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Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits

Cited by 6 publications

References 26 publications

Mass spectrometric analysis of affinity-purified proteasomes from the human myelogenous leukemia K562 cell line

Mass spectrometric analysis of affinity-purified proteasomes from the human myelogenous leukemia K562 cell line

Sequenced Combinations of Cisplatin and Selected Phytochemicals towards Overcoming Drug Resistance in Ovarian Tumour Models

Oleuropein Activates Neonatal Neocortical Proteasomes, but Proteasome Gene Targeting by AAV9 Is Variable in a Clinically Relevant Piglet Model of Brain Hypoxia-Ischemia and Hypothermia

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