Use of the Yeast Three-Hybrid System as a Tool to Study Caspases

Criekinge, Wim Van; Gurp, Maria van; Decoster, Els; Schotte, Peter; Craen, Marc Van de; Fiers, Walter; Vandenabeele, Peter; Beyaert, Rudi

doi:10.1006/abio.1998.2823

Cited by 11 publications

(4 citation statements)

References 19 publications

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“…The strongest otubain/actin interaction observed was between the actin cytoskeleton and the C91S mutant of otubain 1. The C91S mutant may act as a substrate‐trapping mutant as has been suggested for other mutated cysteine proteases including caspase‐1 [10], suggesting that a substrate for otubain is either actin or an actin‐associated protein.…”

Section: Resultsmentioning

confidence: 76%

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry

et al. 2005

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Yersinia encodes a protein kinase, YpkA, which disrupts the actin cytoskeleton. Using an approach termed chemical genetics, we identified a 36-kDa substrate for YpkA in both J774 lysates and bovine brain cytosol. Mass spectrometry analysis identified this substrate as FLJ20113, an open reading frame that corresponds to otubain 1, a deubiquitinating enzyme implicated in immune cell clonal anergy. We demonstrate that otubain 1 is phosphorylated by YpkA in vitro and interacts with YpkA and actin in vivo. Identification of otubain 1 as a YpkA substrate suggests that regulation of immune cell anergy may be a survival mechanism for Yersinia.

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Section: Resultsmentioning

confidence: 76%

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry

et al. 2005

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“…Yeast hybrid assays were performed by the regular method ( James et al, 1996 ) or with a three-hybrid approach ( Van Criekinge et al, 1998 ). In the latter case, two plasmids containing the GAL4 DNA-binding domain and nutritionally selective genes of TRP1 or URA3 (pGBD or pGBDU, respectively) fused with keratin 18 or keratin 8, or without them, were transformed into Saccharomyces cerevisiae PJ69-4A and selected.…”

Section: Yeast Hybrid Assaymentioning

confidence: 99%

The keratin-binding protein Albatross regulates polarization of epithelial cells

Sugimoto

Inoko

Shiromizu

et al. 2008

The Journal of Cell Biology

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The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell–cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.

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“…In order to adapt the two-hybrid system to the needs of caspases we devised in parallel our own three-hybrid system. This system can be used to study any hetero-dimeric target (Figure 9)(68). …”

Section: New Developmentsmentioning

confidence: 99%

Yeast two-hybrid: State of the art

Criekinge¹,

Beyaert

1999

Biol Proced Online

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111

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Genome projects are approaching completion and are saturating sequence databases. This paper discusses the role of the two-hybrid system as a generator of hypotheses. Apart from this rather exhaustive, financially and labour intensive procedure, more refined functional studies can be undertaken. Indeed, by making hybrids of two-hybrid systems, customised approaches can be developed in order to attack specific function-related problems. For example, one could set-up a "differential" screen by combining a forward and a reverse approach in a three-hybrid set-up. Another very interesting project is the use of peptide libraries in two-hybrid approaches. This could enable the identification of peptides with very high specificity comparable to "real" antibodies. With the technology available, the only limitation is imagination.

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Use of the Yeast Three-Hybrid System as a Tool to Study Caspases

Cited by 11 publications

References 19 publications

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry

The keratin-binding protein Albatross regulates polarization of epithelial cells

Yeast two-hybrid: State of the art

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