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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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Search of putative substrates of L protease by homology to the motif identified in Gemin5. (A) Alignment of amino acid sequences present in Gemin5 and Daxx around the RKAR motif. Subscript symbols indicate amino acid positions. (B) Proteolysis of Daxx in FMDV-infected cells. Extracts of FMDV-infected cells (IBRS-2 and BHK-21 cells) immunoblotted with α-Daxx; the unspecific band detected in each of this cell line (depicted by thin lines) was not observed in Hela cell extract. (C) Extracts of BHK-21 cells transfected with pcDNA3.1-HIS6CDaxx (expressing the human Daxx protein) 24 h prior to transfection of Lb plasmid, immunoblotted with α-Xpress (left panel); two polypeptides of ∼116 and 100 kDa are detected by immunodetection of the endogenous human Daxx in HeLa cells (right panel). Arrows depict the Daxx protein and the p60 proteolysis product; tubulin was used as loading control.
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gks172-F7: Search of putative substrates of L protease by homology to the motif identified in Gemin5. (A) Alignment of amino acid sequences present in Gemin5 and Daxx around the RKAR motif. Subscript symbols indicate amino acid positions. (B) Proteolysis of Daxx in FMDV-infected cells. Extracts of FMDV-infected cells (IBRS-2 and BHK-21 cells) immunoblotted with α-Daxx; the unspecific band detected in each of this cell line (depicted by thin lines) was not observed in Hela cell extract. (C) Extracts of BHK-21 cells transfected with pcDNA3.1-HIS6CDaxx (expressing the human Daxx protein) 24 h prior to transfection of Lb plasmid, immunoblotted with α-Xpress (left panel); two polypeptides of ∼116 and 100 kDa are detected by immunodetection of the endogenous human Daxx in HeLa cells (right panel). Arrows depict the Daxx protein and the p60 proteolysis product; tubulin was used as loading control.

Mentions: Comparison of the Daxx sequence to Gemin5/p85 (Figure 7A) showed two closely located motifs, RRLR and RRAR, embedded in a larger sequence that fits all the criteria mentioned before (Figure 4A). To test the hypothesis that proteins retrieved in the search might be authentic targets of Lpro, we analysed extracts prepared from either BHK-21 or IBRS-2 cells infected with FMDV by western blot using α-Daxx antibody (Figure 7B). Remarkably, Daxx protein was degraded during FMDV infection although no stable proteolysis products were detected (two unspecific bands that appeared depending on the cell line analysed, were undetected in HeLa cell extracts included for antibody specificity (Figure 7C)). Confirming the proteolysis of Daxx during FMDV infection, we found that overexpression of N-terminal Xpress-tagged Daxx (45) prior to Lpro expression allowed us to observe a p60 band, coincident with the molecular weight of the expected cleavage products (Figure 7C). Similar results were observed with α-Daxx antibody overexpressing Daxx in transfected cells (Supplementary Figure S3). By contrast, we have not been able to detect cleavage products in EMCV or SVDV infected cells (Supplementary Figure S4). These results were in agreement with the hypothesis that proteins carrying a motif similar to RKAR are candidate substrates of the L protease.Figure 7.


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

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

Search of putative substrates of L protease by homology to the motif identified in Gemin5. (A) Alignment of amino acid sequences present in Gemin5 and Daxx around the RKAR motif. Subscript symbols indicate amino acid positions. (B) Proteolysis of Daxx in FMDV-infected cells. Extracts of FMDV-infected cells (IBRS-2 and BHK-21 cells) immunoblotted with α-Daxx; the unspecific band detected in each of this cell line (depicted by thin lines) was not observed in Hela cell extract. (C) Extracts of BHK-21 cells transfected with pcDNA3.1-HIS6CDaxx (expressing the human Daxx protein) 24 h prior to transfection of Lb plasmid, immunoblotted with α-Xpress (left panel); two polypeptides of ∼116 and 100 kDa are detected by immunodetection of the endogenous human Daxx in HeLa cells (right panel). Arrows depict the Daxx protein and the p60 proteolysis product; tubulin was used as loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks172-F7: Search of putative substrates of L protease by homology to the motif identified in Gemin5. (A) Alignment of amino acid sequences present in Gemin5 and Daxx around the RKAR motif. Subscript symbols indicate amino acid positions. (B) Proteolysis of Daxx in FMDV-infected cells. Extracts of FMDV-infected cells (IBRS-2 and BHK-21 cells) immunoblotted with α-Daxx; the unspecific band detected in each of this cell line (depicted by thin lines) was not observed in Hela cell extract. (C) Extracts of BHK-21 cells transfected with pcDNA3.1-HIS6CDaxx (expressing the human Daxx protein) 24 h prior to transfection of Lb plasmid, immunoblotted with α-Xpress (left panel); two polypeptides of ∼116 and 100 kDa are detected by immunodetection of the endogenous human Daxx in HeLa cells (right panel). Arrows depict the Daxx protein and the p60 proteolysis product; tubulin was used as loading control.
Mentions: Comparison of the Daxx sequence to Gemin5/p85 (Figure 7A) showed two closely located motifs, RRLR and RRAR, embedded in a larger sequence that fits all the criteria mentioned before (Figure 4A). To test the hypothesis that proteins retrieved in the search might be authentic targets of Lpro, we analysed extracts prepared from either BHK-21 or IBRS-2 cells infected with FMDV by western blot using α-Daxx antibody (Figure 7B). Remarkably, Daxx protein was degraded during FMDV infection although no stable proteolysis products were detected (two unspecific bands that appeared depending on the cell line analysed, were undetected in HeLa cell extracts included for antibody specificity (Figure 7C)). Confirming the proteolysis of Daxx during FMDV infection, we found that overexpression of N-terminal Xpress-tagged Daxx (45) prior to Lpro expression allowed us to observe a p60 band, coincident with the molecular weight of the expected cleavage products (Figure 7C). Similar results were observed with α-Daxx antibody overexpressing Daxx in transfected cells (Supplementary Figure S3). By contrast, we have not been able to detect cleavage products in EMCV or SVDV infected cells (Supplementary Figure S4). These results were in agreement with the hypothesis that proteins carrying a motif similar to RKAR are candidate substrates of the L protease.Figure 7.

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