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Targeting the INCENP IN-box-Aurora B interaction to inhibit CPC activity in vivo.

Gohard FH, St-Cyr DJ, Tyers M, Earnshaw WC - Open Biol (2014)

Bottom Line: We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function.The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo.Our results provide proof of concept that inhibition of the Aurora B-IN-box interaction is a viable strategy for interfering with CPC function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.

ABSTRACT
The chromosome passenger complex (CPC) is an essential regulator of mitosis and cytokinesis. The CPC consists of Aurora B kinase, inner centromere protein (INCENP), and the targeting subunits survivin and borealin/Dasra B. INCENP is a scaffolding subunit for the CPC and activates Aurora B via its conserved IN-box domain. We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function. The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo. We then screened a targeted library of small (five to nine residues long) circular peptide (CP) IN-box fragments generated using split intein circular ligation of proteins and peptides (SICLOPPS) methodology. We identified a number of CPs that caused modest but reproducible increases in rates of multinucleated and micronucleated cells. Our results provide proof of concept that inhibition of the Aurora B-IN-box interaction is a viable strategy for interfering with CPC function in vivo.

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Expression and processing of SICLOPPS in HeLa cells. (a) Translation of SICLOPPS constructs produces a linear precursor that undergoes post-translational splicing to yield a linear product and a circularized peptide or protein (adapted from [30]). (b) Experimental timeline for testing SICLOPPS expression by transient transfection in this study. (c) Immunoblot detection of the SICLOPPS linear precursor (P) and the faster migrating linear product (L).
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RSOB140163F1: Expression and processing of SICLOPPS in HeLa cells. (a) Translation of SICLOPPS constructs produces a linear precursor that undergoes post-translational splicing to yield a linear product and a circularized peptide or protein (adapted from [30]). (b) Experimental timeline for testing SICLOPPS expression by transient transfection in this study. (c) Immunoblot detection of the SICLOPPS linear precursor (P) and the faster migrating linear product (L).

Mentions: For the purpose of this study, we wished to genetically express the hINCENP IN-box domain and smaller fragments thereof as soluble peptides in vivo. As small, unmodified peptides tend to be unstable in cells, we sought to stabilize putative inhibitory peptides via head-to-tail circularization using split intein circular ligation of protein and peptides (SICLOPPS) [29]. This approach relies on the in-frame insertion of a nucleotide sequence encoding the desired protein or peptide as a linker between two halves of a split intein. The two halves of the split intein are oriented so that their post-translational splicing in cis leads to the excision of the linker region as a cyclic peptide (CP; figure 1a).FigureĀ 1.


Targeting the INCENP IN-box-Aurora B interaction to inhibit CPC activity in vivo.

Gohard FH, St-Cyr DJ, Tyers M, Earnshaw WC - Open Biol (2014)

Expression and processing of SICLOPPS in HeLa cells. (a) Translation of SICLOPPS constructs produces a linear precursor that undergoes post-translational splicing to yield a linear product and a circularized peptide or protein (adapted from [30]). (b) Experimental timeline for testing SICLOPPS expression by transient transfection in this study. (c) Immunoblot detection of the SICLOPPS linear precursor (P) and the faster migrating linear product (L).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4248066&req=5

RSOB140163F1: Expression and processing of SICLOPPS in HeLa cells. (a) Translation of SICLOPPS constructs produces a linear precursor that undergoes post-translational splicing to yield a linear product and a circularized peptide or protein (adapted from [30]). (b) Experimental timeline for testing SICLOPPS expression by transient transfection in this study. (c) Immunoblot detection of the SICLOPPS linear precursor (P) and the faster migrating linear product (L).
Mentions: For the purpose of this study, we wished to genetically express the hINCENP IN-box domain and smaller fragments thereof as soluble peptides in vivo. As small, unmodified peptides tend to be unstable in cells, we sought to stabilize putative inhibitory peptides via head-to-tail circularization using split intein circular ligation of protein and peptides (SICLOPPS) [29]. This approach relies on the in-frame insertion of a nucleotide sequence encoding the desired protein or peptide as a linker between two halves of a split intein. The two halves of the split intein are oriented so that their post-translational splicing in cis leads to the excision of the linker region as a cyclic peptide (CP; figure 1a).FigureĀ 1.

Bottom Line: We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function.The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo.Our results provide proof of concept that inhibition of the Aurora B-IN-box interaction is a viable strategy for interfering with CPC function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.

ABSTRACT
The chromosome passenger complex (CPC) is an essential regulator of mitosis and cytokinesis. The CPC consists of Aurora B kinase, inner centromere protein (INCENP), and the targeting subunits survivin and borealin/Dasra B. INCENP is a scaffolding subunit for the CPC and activates Aurora B via its conserved IN-box domain. We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function. The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo. We then screened a targeted library of small (five to nine residues long) circular peptide (CP) IN-box fragments generated using split intein circular ligation of proteins and peptides (SICLOPPS) methodology. We identified a number of CPs that caused modest but reproducible increases in rates of multinucleated and micronucleated cells. Our results provide proof of concept that inhibition of the Aurora B-IN-box interaction is a viable strategy for interfering with CPC function in vivo.

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