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Gemin5 proteolysis reveals a novel motif to identify L protease targets.

Piñeiro D, Ramajo J, Bradrick SS, Martínez-Salas E - Nucleic Acids Res. (2012)

Bottom Line: Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57.Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells.Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

ABSTRACT
Translation of picornavirus RNA is governed by the internal ribosome entry site (IRES) element, directing the synthesis of a single polyprotein. Processing of the polyprotein is performed by viral proteases that also recognize as substrates host factors. Among these substrates are translation initiation factors and RNA-binding proteins whose cleavage is responsible for inactivation of cellular gene expression. Foot-and-mouth disease virus (FMDV) encodes two proteases, L(pro) and 3C(pro). Widespread definition of L(pro) targets suffers from the lack of a sufficient number of characterized substrates. Here, we report the proteolysis of the IRES-binding protein Gemin5 in FMDV-infected cells, but not in cells infected by other picornaviruses. Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57. In silico search of putative L targets within Gemin5 identified two sequences whose potential recognition was in agreement with proteolysis products observed in infected cells. Mutational analysis revealed a novel L(pro) target sequence that included the RKAR motif. Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells. This protein carries a RRLR motif whose substitution to EELR abrogated L(pro) recognition. Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

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Gemin5 amino acid sequences retrieved in the search in silico of putative substrates of L protease. The Gemin5 open reading frame (1508 amino acids) was subdivided into overlapping subsets of 12 amino acids. Putative targets were selected if they contain K or R amino acids upstream of polar (three out of eight) residues. Sequences meeting these criteria but having proline or E, D residues in the four upstream positions were excluded. Two sequences where the third residue was hydrophobic were selected, 838–854 (A) and 1226–1241 (B). (C) Comparison of the Gemin5 amino acid sequence 838–854 with that of the L-VP4 junction in the viral polyprotein, eIF4GII and eIF4GI. A broken line rectangle depicts the motif of higher resemblance between Gemin5 and L-VP4 junction. Subscript symbols indicate amino acid positions.
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gks172-F4: Gemin5 amino acid sequences retrieved in the search in silico of putative substrates of L protease. The Gemin5 open reading frame (1508 amino acids) was subdivided into overlapping subsets of 12 amino acids. Putative targets were selected if they contain K or R amino acids upstream of polar (three out of eight) residues. Sequences meeting these criteria but having proline or E, D residues in the four upstream positions were excluded. Two sequences where the third residue was hydrophobic were selected, 838–854 (A) and 1226–1241 (B). (C) Comparison of the Gemin5 amino acid sequence 838–854 with that of the L-VP4 junction in the viral polyprotein, eIF4GII and eIF4GI. A broken line rectangle depicts the motif of higher resemblance between Gemin5 and L-VP4 junction. Subscript symbols indicate amino acid positions.

Mentions: Based on a study of synthetic substrate specificity (48) as well as amino acid sequences hydrolysed by Lpro in the L-VP4 junction, eIF4GI and eIF4GII (38,39), we developed criteria to search for candidate Lpro target sites in Gemin5. Basically, targets of 12 residues were first selected if they contain positively charged (K or R) amino acids upstream of polar (three out of eight) residues. Sequences meeting these criteria but having proline or negatively charged residues in the four upstream positions were excluded. From this group, we selected sequences where the third residue was hydrophobic. Thus, in silico search of Lpro target candidates was carried out dividing Gemin5 polypeptide sequences into all possible overlapping subsets of 12 amino acids. Only four sequences met the criteria mentioned earlier having the peculiarity of being two separate partially overlapping sequences (Figure 4A and B). These potential targets were located within residues 838–854 and 1226–1241, respectively. In support of the functional relevance of our prediction, the size of the cleavage products estimated from the predicted targets matched very closely the observed size of Gemin5 fragments (p85 and p57) in infected cells (Figure 1B), as well as the size of the Lb-mediated proteolysis products observed in the coexpression of truncated polypeptides (p37 and p25 in Figure 3A).Figure 4.


Gemin5 proteolysis reveals a novel motif to identify L protease targets.

Piñeiro D, Ramajo J, Bradrick SS, Martínez-Salas E - Nucleic Acids Res. (2012)

Gemin5 amino acid sequences retrieved in the search in silico of putative substrates of L protease. The Gemin5 open reading frame (1508 amino acids) was subdivided into overlapping subsets of 12 amino acids. Putative targets were selected if they contain K or R amino acids upstream of polar (three out of eight) residues. Sequences meeting these criteria but having proline or E, D residues in the four upstream positions were excluded. Two sequences where the third residue was hydrophobic were selected, 838–854 (A) and 1226–1241 (B). (C) Comparison of the Gemin5 amino acid sequence 838–854 with that of the L-VP4 junction in the viral polyprotein, eIF4GII and eIF4GI. A broken line rectangle depicts the motif of higher resemblance between Gemin5 and L-VP4 junction. Subscript symbols indicate amino acid positions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367203&req=5

gks172-F4: Gemin5 amino acid sequences retrieved in the search in silico of putative substrates of L protease. The Gemin5 open reading frame (1508 amino acids) was subdivided into overlapping subsets of 12 amino acids. Putative targets were selected if they contain K or R amino acids upstream of polar (three out of eight) residues. Sequences meeting these criteria but having proline or E, D residues in the four upstream positions were excluded. Two sequences where the third residue was hydrophobic were selected, 838–854 (A) and 1226–1241 (B). (C) Comparison of the Gemin5 amino acid sequence 838–854 with that of the L-VP4 junction in the viral polyprotein, eIF4GII and eIF4GI. A broken line rectangle depicts the motif of higher resemblance between Gemin5 and L-VP4 junction. Subscript symbols indicate amino acid positions.
Mentions: Based on a study of synthetic substrate specificity (48) as well as amino acid sequences hydrolysed by Lpro in the L-VP4 junction, eIF4GI and eIF4GII (38,39), we developed criteria to search for candidate Lpro target sites in Gemin5. Basically, targets of 12 residues were first selected if they contain positively charged (K or R) amino acids upstream of polar (three out of eight) residues. Sequences meeting these criteria but having proline or negatively charged residues in the four upstream positions were excluded. From this group, we selected sequences where the third residue was hydrophobic. Thus, in silico search of Lpro target candidates was carried out dividing Gemin5 polypeptide sequences into all possible overlapping subsets of 12 amino acids. Only four sequences met the criteria mentioned earlier having the peculiarity of being two separate partially overlapping sequences (Figure 4A and B). These potential targets were located within residues 838–854 and 1226–1241, respectively. In support of the functional relevance of our prediction, the size of the cleavage products estimated from the predicted targets matched very closely the observed size of Gemin5 fragments (p85 and p57) in infected cells (Figure 1B), as well as the size of the Lb-mediated proteolysis products observed in the coexpression of truncated polypeptides (p37 and p25 in Figure 3A).Figure 4.

Bottom Line: Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57.Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells.Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

ABSTRACT
Translation of picornavirus RNA is governed by the internal ribosome entry site (IRES) element, directing the synthesis of a single polyprotein. Processing of the polyprotein is performed by viral proteases that also recognize as substrates host factors. Among these substrates are translation initiation factors and RNA-binding proteins whose cleavage is responsible for inactivation of cellular gene expression. Foot-and-mouth disease virus (FMDV) encodes two proteases, L(pro) and 3C(pro). Widespread definition of L(pro) targets suffers from the lack of a sufficient number of characterized substrates. Here, we report the proteolysis of the IRES-binding protein Gemin5 in FMDV-infected cells, but not in cells infected by other picornaviruses. Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57. In silico search of putative L targets within Gemin5 identified two sequences whose potential recognition was in agreement with proteolysis products observed in infected cells. Mutational analysis revealed a novel L(pro) target sequence that included the RKAR motif. Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells. This protein carries a RRLR motif whose substitution to EELR abrogated L(pro) recognition. Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

Show MeSH
Related in: MedlinePlus