An integrated vector system for cellular studies of phage display-derived peptides

Voss, Stephan D.; DeGrand, Alec M.; Romeo, G; Cantley, Lewis C.; Frangioni, John V.

doi:10.1016/s0003-2697(02)00268-3

Cited by 7 publications

(9 citation statements)

References 13 publications

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“…Residues in Mad1 SID which are solvent exposed, such as Glu14, show no selection for any particular amino acid. The trypto- on May 9, 2018 by guest http://mcb.asm.org/ phan present at the N terminus is common to many sequences isolated from peptide screens and likely is selected because it stabilizes the peptide in an ␣-helical structure rather than for increased binding to PAH2 (16,59). Indeed, an I9W mutation in Mad1 SID reduced binding to PAH2 (data not shown).…”

Section: Fig 2 Mutational Analysis Of the Pah2 Hydrophobic Cleft (A)mentioning

confidence: 96%

“…The L8 phage display library expresses completely degenerate 8-aa peptides in a linear sequence and was described in detail previously (16,59). PFU containing 2,000 times the library complexity (3.6 ϫ 10 11 PFU for round 1 and 5 ϫ 10 10 PFU for round 2) were applied to GST-PAH2 in a total volume of 200 l of phosphate-buffered saline-0.1% Tween 20.…”

Section: Plasmidsmentioning

confidence: 99%

“…Phage specific for GST were removed by immunodepletion with 100 g of agarose-bound GST after cycles 1 and 2 of affinity purification (differential phage display). The sequences of isolated clones were determined by initially using PCR to amplify the region containing the peptide open reading frame and then directly sequencing the PCR product (59).…”

Section: Plasmidsmentioning

confidence: 99%

“…The large variety of PAH2-interacting sequences led us to try to identify an optimal PAH2-binding peptide. To do this, we used purified GST-PAH2 to screen a phage display library containing completely degenerate peptides of 8 aa (16,59). After three rounds of selection, the interacting clones were isolated, tested for binding to PAH2, and then sequenced.…”

Section: Fig 2 Mutational Analysis Of the Pah2 Hydrophobic Cleft (A)mentioning

confidence: 99%

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Functional Analysis of the Mad1-mSin3A Repressor-Corepressor Interaction Reveals Determinants of Specificity, Affinity, and Transcriptional Response

Cowley

Kang

Frangioni

et al. 2004

Molecular and Cellular Biology

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The recruitment of corepressors by DNA-bound repressors is likely to be a critical rate-limiting step in the transcriptional regulation of many genes. An excellent paradigm for such an interaction is the association of the basic helix-loop-helix zipper protein Mad1 with the corepressor mSin3A. When bound together, the Sin3 interaction domain (SID) of Mad1 forms extensive hydrophobic contacts with the four-helix bundle formed by the paired amphipathic helix 2 (PAH2) domain of mSin3A. Using the costructure to predict the principle residues required for binding, we have carried out an extensive mutational analysis to examine the Mad1 SID-mSin3A PAH2 interaction in vitro and in vivo. Bulky hydrophobic residues in the ␣1 (I308 and V311) and ␣2 (L329 and L332) helices of the PAH2 domain are necessary to accommodate the precise arrangement of bulky (L12) and short (A15 and A16) hydrophobic residues in the amphipathic Mad1 SID. We have also used phage display to derive an optimal SID, which shows an essentially identical arrangement of key residues. By manipulating these key residues, we have generated altered-specificity Mad1 SID mutants that bind only to a PAH2 domain with a reciprocal mutation, permitting us to demonstrate for the first time that these domains interact directly in vivo. We have also found that the integrity of the PAH1 domain affects the Mad1 SID-PAH2 interaction. It is conceivable that cross talk between different PAH domains and their binding partners helps to determine the subunit composition and order of assembly of mSin3A complexes.An important mechanistic link between transcriptional regulation and chromatin structure is the association of sequencespecific DNA-binding transcription factors with coactivators and corepressors (3,11,46,57). Coactivator and corepressor recruitment is generally considered to underlie the gene-specific alterations in chromatin structure that accompany changes in gene expression during differentiation, development, and oncogenesis. An emerging theme is that many different transcription factors recruit the same set of coactivators and corepressors. For example, transcriptional activation mediated by p53, c-Myb, CREB, MyoD, MEF2, and different nuclear hormone receptors, among others, involves recruitment of the coactivator p300 or CBP, each of which possesses and binds histone acetyltransferases (for a review, see reference 54). Similarly, corepressor complexes such as mSin3 and NURD have been found to interact with many DNA-binding repressors as well as with histone deacetylases (HDACs) and other factors involved in the modification of chromatin to a repressive state (for reviews, see references 4 and 28). Given this multiplicity of associations, both the nature and the specificity of the interactions between transcription factors and their coactivators and corepressors take on considerable importance.We have been studying the interactions between Mad proteins and the mSin3A corepressor complex. Mad family proteins (comprising Mad1, Mxi1, Mad3, and Mad4) belong to the ...

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Section: Fig 2 Mutational Analysis Of the Pah2 Hydrophobic Cleft (A)mentioning

confidence: 96%

Section: Plasmidsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Section: Fig 2 Mutational Analysis Of the Pah2 Hydrophobic Cleft (A)mentioning

confidence: 99%

See 2 more Smart Citations

Functional Analysis of the Mad1-mSin3A Repressor-Corepressor Interaction Reveals Determinants of Specificity, Affinity, and Transcriptional Response

Cowley

Kang

Frangioni

et al. 2004

Molecular and Cellular Biology

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“…Each of the cell lines were electroporated with 30 μg of EGFP fused ESAP1 encoding plasmid (pGESAP1) or empty vector (pG1). 37 After selecting cells for five days with G418 (Calbiochem 345812), soft agar colony formation assay was set up. 8 The colonies were stained with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] for 2 h at 37°C.…”

Section: Disclosure Of Potential Conflicts Of Interestmentioning

confidence: 99%

Novel peptide binds EWS-FLI1 and reduces the oncogenic potential in Ewing tumors

Erkizan¹,

Scher²,

Gamble³

et al. 2011

Cell Cycle

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Translocation of green fluorescent protein by comparative analysis with multiple signal peptides

Linton

Walsh

Sims

et al. 2011

Biotechnology Journal

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Type I and II secretory pathways are used for the translocation of recombinant proteins from the cytoplasm of Escherichia coli. The purpose of this study was to evaluate four signal peptides (HlyA, TorA, GeneIII, and PelB), representing the most common secretion pathways in E. coli, for their ability to target green fluorescent protein (GFP) for membrane translocation. Signal peptide-GFP genetic fusions were designed in accordance with BioFusion standards (BBF RFC 10, BBF RFC 23). The HlyA signal peptide targeted GFP for secretion to the extracellular media via the type I secretory pathway, whereas TAT-dependent signal peptide TorA and Sec-dependent signal peptide GeneIII exported GFP to the periplasm. The PelB signal peptide was inefficient in translocating GFP. The use of biological technical standards simplified the design and construction of functional signal peptide-recombinant protein genetic devices for type I and II secretion in E. coli. The utility of the standardized parts model is further illustrated as constructed biological parts are available for direct application to other studies on recombinant protein translocation.

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An integrated vector system for cellular studies of phage display-derived peptides

Cited by 7 publications

References 13 publications

Functional Analysis of the Mad1-mSin3A Repressor-Corepressor Interaction Reveals Determinants of Specificity, Affinity, and Transcriptional Response

Functional Analysis of the Mad1-mSin3A Repressor-Corepressor Interaction Reveals Determinants of Specificity, Affinity, and Transcriptional Response

Novel peptide binds EWS-FLI1 and reduces the oncogenic potential in Ewing tumors

Translocation of green fluorescent protein by comparative analysis with multiple signal peptides

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