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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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Mutational analysis of the sequence recognized as substrate of Lpro in Daxx protein. (A) Diagram of the double and triple amino acid substitutions introduced in the RRLR motif of the wild-type Daxx. A dot in the sequence alignment is used to indicate no change with respect to the wild-type sequence. Plasmids expressing the Daxx (WT) or the substitution mutants (M, carrying substitutions indicated on the left of each panel) were transfected 24 h prior to transfection of Lb plasmid in BHK-21 cells. Cell extracts were analysed by 10% SDS–PAGE and western blot using α-Xpress antibody. Arrows indicate the position of L-induced products (p60), which was always detected in the WT sequence loaded in parallel. The bottom panel shows a tubulin immunoblot as loading control. (B) Diagram of the amino acid substitutions introduced in the KKRRAR motif of the wild-type Daxx. Transfections and immunoblots were carried out as in (A).
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gks172-F8: Mutational analysis of the sequence recognized as substrate of Lpro in Daxx protein. (A) Diagram of the double and triple amino acid substitutions introduced in the RRLR motif of the wild-type Daxx. A dot in the sequence alignment is used to indicate no change with respect to the wild-type sequence. Plasmids expressing the Daxx (WT) or the substitution mutants (M, carrying substitutions indicated on the left of each panel) were transfected 24 h prior to transfection of Lb plasmid in BHK-21 cells. Cell extracts were analysed by 10% SDS–PAGE and western blot using α-Xpress antibody. Arrows indicate the position of L-induced products (p60), which was always detected in the WT sequence loaded in parallel. The bottom panel shows a tubulin immunoblot as loading control. (B) Diagram of the amino acid substitutions introduced in the KKRRAR motif of the wild-type Daxx. Transfections and immunoblots were carried out as in (A).

Mentions: To validate this hypothesis, we generated multiple mutants in both motifs (RRLR and RRAR) substituting basic amino acids, R or K, by E (Figure 8A and B). Transfection of Daxx constructs expressing these substitution mutants with the L protease construct indicated that substitution of the RRLR motif by EELR or EELE fully abrogated the recognition of the protein by Lpro (Figure 8A). Substitution of the second site RRAR to EEAR did not affect proteolysis, whereas substitution to EEAE diminished the intensity of the proteolysis products (Figure 8B). Similarly, and in agreement with the results obtained with Gemin5/p85, the triple mutant EERRAE interfered with the recognition of the protein by Lpro while the double mutant EERRAR was not affected (Figure 8B). Therefore, we conclude that the sequence 354-VLARRLRENRSLAMS-370 of Daxx protein constitutes the preferential recognition motif for the L protease.Figure 8.


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

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

Mutational analysis of the sequence recognized as substrate of Lpro in Daxx protein. (A) Diagram of the double and triple amino acid substitutions introduced in the RRLR motif of the wild-type Daxx. A dot in the sequence alignment is used to indicate no change with respect to the wild-type sequence. Plasmids expressing the Daxx (WT) or the substitution mutants (M, carrying substitutions indicated on the left of each panel) were transfected 24 h prior to transfection of Lb plasmid in BHK-21 cells. Cell extracts were analysed by 10% SDS–PAGE and western blot using α-Xpress antibody. Arrows indicate the position of L-induced products (p60), which was always detected in the WT sequence loaded in parallel. The bottom panel shows a tubulin immunoblot as loading control. (B) Diagram of the amino acid substitutions introduced in the KKRRAR motif of the wild-type Daxx. Transfections and immunoblots were carried out as in (A).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3367203&req=5

gks172-F8: Mutational analysis of the sequence recognized as substrate of Lpro in Daxx protein. (A) Diagram of the double and triple amino acid substitutions introduced in the RRLR motif of the wild-type Daxx. A dot in the sequence alignment is used to indicate no change with respect to the wild-type sequence. Plasmids expressing the Daxx (WT) or the substitution mutants (M, carrying substitutions indicated on the left of each panel) were transfected 24 h prior to transfection of Lb plasmid in BHK-21 cells. Cell extracts were analysed by 10% SDS–PAGE and western blot using α-Xpress antibody. Arrows indicate the position of L-induced products (p60), which was always detected in the WT sequence loaded in parallel. The bottom panel shows a tubulin immunoblot as loading control. (B) Diagram of the amino acid substitutions introduced in the KKRRAR motif of the wild-type Daxx. Transfections and immunoblots were carried out as in (A).
Mentions: To validate this hypothesis, we generated multiple mutants in both motifs (RRLR and RRAR) substituting basic amino acids, R or K, by E (Figure 8A and B). Transfection of Daxx constructs expressing these substitution mutants with the L protease construct indicated that substitution of the RRLR motif by EELR or EELE fully abrogated the recognition of the protein by Lpro (Figure 8A). Substitution of the second site RRAR to EEAR did not affect proteolysis, whereas substitution to EEAE diminished the intensity of the proteolysis products (Figure 8B). Similarly, and in agreement with the results obtained with Gemin5/p85, the triple mutant EERRAE interfered with the recognition of the protein by Lpro while the double mutant EERRAR was not affected (Figure 8B). Therefore, we conclude that the sequence 354-VLARRLRENRSLAMS-370 of Daxx protein constitutes the preferential recognition motif for the L protease.Figure 8.

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