Abstract
The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignant-transforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease-resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44°C for the full-length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58°C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein-protein interaction.
| Original language | English |
|---|---|
| Pages (from-to) | 522-529 |
| Number of pages | 8 |
| Journal | Cell Biochemistry and Biophysics |
| Volume | 44 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2006 |
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Keywords
- Circular dichroism
- elF3
- INT6
- Limited proteolysis
- PCI domain
ASJC Scopus subject areas
- Cell Biology
- Clinical Biochemistry
- Biophysics
- Biochemistry
Cite this
Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor. / Murai, Marcelo J.; Carneiro, Flávia R G; Gozzo, Fabio C.; Ierardi, Daniela F.; Pertinhez, Thelma A.; Zanchin, Nilson I T.
In: Cell Biochemistry and Biophysics, Vol. 44, No. 3, 2006, p. 522-529.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor
AU - Murai, Marcelo J.
AU - Carneiro, Flávia R G
AU - Gozzo, Fabio C.
AU - Ierardi, Daniela F.
AU - Pertinhez, Thelma A.
AU - Zanchin, Nilson I T
PY - 2006
Y1 - 2006
N2 - The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignant-transforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease-resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44°C for the full-length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58°C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein-protein interaction.
AB - The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignant-transforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease-resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44°C for the full-length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58°C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein-protein interaction.
KW - Circular dichroism
KW - elF3
KW - INT6
KW - Limited proteolysis
KW - PCI domain
UR - http://www.scopus.com/inward/record.url?scp=33646871009&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33646871009&partnerID=8YFLogxK
U2 - 10.1385/CBB:44:3:522
DO - 10.1385/CBB:44:3:522
M3 - Article
C2 - 16679540
AN - SCOPUS:33646871009
VL - 44
SP - 522
EP - 529
JO - Cell Biochemistry and Biophysics
JF - Cell Biochemistry and Biophysics
SN - 1085-9195
IS - 3
ER -