Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state

Abo, Yohichi; Hagiya, Akiko; Naganuma, Takao; Tohkairin, Yukiko; Shiomi, Kunihiro; Kajiura, Zenta; Hachimori, Akira; Uchiumi, Toshio; Nakagaki, Masayuki

doi:10.1016/j.bbrc.2004.07.196

Cited by 2 publications

(1 citation statement)

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“…Ribosomal P proteins are proposed to exist as a pentamer of 3 proteins, P0 (38 kD), P1 (19 kD) and P2 (17 kD) with 2 P1/P2 heterodimers binding one molecule of P0 through the P1 molecule [20–22]. The pentamer binds the GTPase‐associated domain of 28S rRNA of the 60S subunit and interacts with elongation factors 1 α and 2 [23,45]. It has been suggested that this pentamer is responsible for autoantibody production [24], that autoantibodies recognize both linear and conformational epitope(s) [25,26], and that CD4 T lymphocytes help antibody production [27].…”

Section: Introductionmentioning

confidence: 99%

Major immunoreactive domains of human ribosomal P proteins lie N-terminal to a homologous C-22 sequence: application to a novel ELISA for systemic lupus erythematosus

Lin

Dubljevic

Fritzler

et al. 2005

Clinical and Experimental Immunology

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SummaryThe aim of this study was to identify immunoreactive domains on human ribosomal P0, P1 and P2 proteins, other than the C-22 peptide, to develop a novel ELISA using a combination of these proteins and to compare this ELISA with one using the C-22 peptide. Human recombinant P0, P1, P2 and mutant P0 lacking the homologous C-22 peptide (N-P0) were produced in bacteria and tested by ELISA and immunoblotting using sera from 48 patients with systemic lupus erythematosus (SLE), 48 with an unrelated inflammatory disorder (Crohn's disease) and 47 healthy controls. ELISA with P0, P1 and P2, premixed at equimolar concentrations, gave higher OD readings than each protein tested individually. Eighteen SLE sera tested positive by ELISA with premixed P0, P1, P2 but only 3 tested positive with the C-22 peptide. Twentytwo SLE sera reacted positively, as determined by immunoblotting, with 5 different P protein combinations: P1P2, P0P1P2, P1, P0P1, P0 and P1. Only sera reactive with all three P proteins reacted with the C-22 peptide, with absent or minimal reactivity with N-P0. Native antigens yielded sensitivity (6/48, 13%) similar to the C-22 peptide assay. An ELISA with premixed P1 and P2 gave higher OD values than the arithmetic means with P1 or P2. Fifteen SLE patients had antibodies to double stranded (ds)-DNA, of which 6 also had antibodies to P0P1P2 by ELISA but 12 reactive with P0P1P2 did not have discernable ds-DNA antibodies. Ribosomal P autoantibodies react mainly with epitopes N-terminal to a homologous C-22 peptide. An ELISA with premixed P0, P1 and P2 has 5-fold greater sensitivity (38%) for SLE than an assay with the conventional C-22 peptide (7%). The combined sensitivity for SLE for antibodies to P0P1P2 and ds-DNA is 56%, higher than C-22 and ds-DNA, 38%. Only one of the SLE patients had neuropsychiatric lupus.

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Section: Introductionmentioning

confidence: 99%

Major immunoreactive domains of human ribosomal P proteins lie N-terminal to a homologous C-22 sequence: application to a novel ELISA for systemic lupus erythematosus

Lin

Dubljevic

Fritzler

et al. 2005

Clinical and Experimental Immunology

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Towards a model of GCN2 activation

Masson

2019

Biochemical Society Transactions

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Cells must be able to sense and adapt to their surroundings to thrive in a dynamic environment. Key to adapting to a low nutrient environment is the Integrated Stress Response (ISR), a short-lived pathway that allows cells to either regain cellular homeostasis or facilitate apoptosis during periods of stress. Central to the ISR is the protein kinase General Control Non-depressible 2 (GCN2), which is responsible for sensing starvation. Upon amino acid deficiency, GCN2 is activated and initiates the ISR by phosphorylating the translation initiation factor eIF2α, stalling protein translation, and activating the transcription factor ATF4, which in turn up-regulates autophagy and biosynthesis pathways. A key outstanding question is how GCN2 is activated from an autoinhibited state. Until recently, a model of activation focussed on the increase of deacylated tRNA associated with amino acid starvation, with deacylated tRNA binding directly to GCN2 and releasing autoinhibition. However, in vivo experiments have pointed towards an alternative, deacylated-tRNA-independent mechanism of activation. Here, we review the various factors that may facilitate GCN2 activation, including recent research showing that the P-stalk complex, a ribosome-associated heteropentameric protein complex, is a potent activator of GCN2.

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Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state

Cited by 2 publications

References 26 publications

Major immunoreactive domains of human ribosomal P proteins lie N-terminal to a homologous C-22 sequence: application to a novel ELISA for systemic lupus erythematosus

Major immunoreactive domains of human ribosomal P proteins lie N-terminal to a homologous C-22 sequence: application to a novel ELISA for systemic lupus erythematosus

Towards a model of GCN2 activation

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