High yield bacterial expression and purification of active recombinant PA28αβ complex

Feuvre, Aurélie Y. Le; Dantas‐Barbosa, Carmela; Baldin, Véronique; Coux, Olivier

doi:10.1016/j.pep.2008.10.014

Cited by 10 publications

(11 citation statements)

References 32 publications

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“…The identity of the antigen recognized by the 011H12 DAb was confirmed by ELISA. Soluble purified 011H12 DAb showed reactivity with the active PA28αβ complex (30) and to a lesser extent with individual PA28 subunits (Fig. 3B).…”

Section: Resultsmentioning

confidence: 96%

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Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

Sánchez-Martín

Martı́nez-Torrecuadrada

Teesalu

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.phage library | phage display | single domain antibody | physiological selection | tumor-associated antigen

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Section: Resultsmentioning

confidence: 96%

“…The PA28 complex has been observed in the nucleus (mainly PA28γ) and the cytoplasm [mainly PA28αβ (30,53)]. How PA28 subunits might be targeted to the cell surface is unclear; however, several other intracellular proteins are aberrantly expressed at the cell surface in malignant tumors of various types (4,42).…”

Section: Discussionmentioning

confidence: 99%

Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

Sánchez-Martín

Martı́nez-Torrecuadrada

Teesalu

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…at an apparent molecular weight larger than twice what was expected. Human recombinant PA28αβ was produced and purified from E. coli, as previously described (27). Human PA28γ was expressed in E. coli BL21 DE3 CodonPlus.…”

Section: Methodsmentioning

confidence: 99%

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

Jonik-Nowak

Menneteau

Fesquet

et al. 2017

Preprint

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PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However its exact mechanisms of action are unclear and likely to involve additional partners that remain to be identified. Here we report the identification of the first cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal Bodies by inhibition of its association with the key Cajal body component coilin. Altogether, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.Significance StatementThe 20S proteasome is a key actor of the control of protein levels and integrity in cells. To perform its multiple functions, it works with a series of regulators, among which a nuclear complex called PA28γ. In particular, PA28γ participates in the regulation of cell proliferation and nuclear dynamics. We describe here the characterization of a novel protein, PIP30/FAM192A, which binds tightly to PA28γ and favors its interaction with the 20S proteasome while inhibiting its association with coilin, a central component of nuclear Cajal bodies. Thus PIP30/FAM192A critically controls the interactome and consequently the functions of PA28γ, and appears to be a new player in the fine regulation of intracellular proteostasis in the cell nucleus.

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“…After expression in Escherichia coli, the protein complex was purified essentially as previously described for PA28␣␤ (43).…”

Section: Methodsmentioning

confidence: 99%

“…In Vitro Reconstitution Assay-Purified recombinant PA28␥ was mixed with 1 g of human 20S proteasome (purified from HeLa cells (43) and treated with 25 M epoxomicin or solvent) in activity buffer (20 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 10% glycerol) (final volume 20 l). After incubation at 37°C for 5 min, the samples were loaded onto a native gel as described (44).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

Welk

Coux

Kleene

et al. 2016

Journal of Biological Chemistry

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The proteasome is an intracellular protease complex consisting of the 20S catalytic core and its associated regulators, including the 19S complex, PA28␣␤, PA28␥, PA200, and PI31. Inhibition of the proteasome induces autoregulatory de novo formation of 20S and 26S proteasome complexes. Formation of alternative proteasome complexes, however, has not been investigated so far. We here show that catalytic proteasome inhibition results in fast recruitment of PA28␥ and PA200 to 20S and 26S proteasomes within 2-6 h. Rapid formation of alternative proteasome complexes did not involve transcriptional activation of PA28␥ and PA200 but rather recruitment of preexisting activators to 20S and 26S proteasome complexes. Recruitment of proteasomal activators depended on the extent of active site inhibition of the proteasome with inhibition of ␤5 active sites being sufficient for inducing recruitment. Moreover, specific inhibition of 26S proteasome activity via siRNA-mediated knockdown of the 19S subunit RPN6 induced recruitment of only PA200 to 20S proteasomes, whereas PA28␥ was not mobilized. Here, formation of alternative PA200 complexes involved transcriptional activation of the activator. Alternative proteasome complexes persisted when cells had regained proteasome activity after pulse exposure to proteasome inhibitors. Knockdown of PA28␥ sensitized cells to proteasome inhibitor-mediated growth arrest. Thus, formation of alternative proteasome complexes appears to be a formerly unrecognized but integral part of the cellular response to impaired proteasome function and altered proteostasis.The proteasome, one of the main proteolytic systems of the cell, is essential for maintenance of protein homeostasis and thus of cellular functions. The proteolytic activity of this multicatalytic protease resides inside the 20S core particle, built of four stacked rings of seven ␣ and ␤ subunits with ␣ 7

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High yield bacterial expression and purification of active recombinant PA28αβ complex

Cited by 10 publications

References 32 publications

Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

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