Parsing
Intermolecular Interactions
Identification
of Enzyme Inhibitors from Phage-Displayed Combinatorial Peptide Libraries Pp. 535-543
Brian K. Kay
and Paul T. Hamilton
[Abstract] [Purchase Issue/Articles]
Identification
of a Novel Human Peroxisomal 2,4-Dienoyl-CoA Reductase Related Protein Using
the M13
Phage
Protein VI Phage Display Technology Pp. 545-552
L. Amery, G.
P. Mannaerts, S. Subramani, P. P. Van Veldhoven
and M. Fransen
[Abstract] [Purchase Issue/Articles]
Identification of Small Molecule Binding Sites within Proteins Using Phage Display Technology Pp. 553-572
D. J. Rodi,
G. E. Agoston, R. Manon, R. Lapcevich, S. J. Green and L. Makowski
[Abstract] [Purchase Issue/Articles]
Substrate Phage
as a Tool to Identify Novel Substrate Sequences of Proteases Pp. 573-583
Shuichi
Ohkubo, Kazutaka Miyadera, Yoshikazu Sugimoto, Ken-ichi Matsuo, Konstanty
Wierzba and Yuji Yamada
[Abstract] [Purchase Issue/Articles]
Predicting In Vivo Protein-Peptide Interactions
with Random Phage Display Pp.
100%-591
James F.
Smothers and Steven Henikoff
[Abstract] [Purchase Issue/Articles]
The Cloning
of Human Genes Using cDNA Phage Display and Small-molecule Chemical Probes Pp. 593-597
Sergey N. Savinov
and David J. Austin
[Abstract] [Purchase Issue/Articles]
Caveat
Receptor: Proteomes on Display Pp.
599-602
F. J.
Stevens
[Abstract] [Purchase Issue/Articles]
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Identification of Enzyme Inhibitors from Phage-Displayed Combinatorial Peptide
Libraries
Brian K. Kay
and Paul T. Hamilton
In recent years,
there have been a growing number of examples of the successful isolation of peptide
ligands for enzymes from phage-displayed combinatorial peptide libraries. These
peptides typically bind at or near the active site of the enzymes and can
inhibit their activity. We review the literature on peptide ligands that have
been isolated for enzymes other than proteases as well as present data on
peptide ligands we have identified for E. coli dihydrofolate reductase (DHFR)
which bind at, or near, the same site as the known inhibitors methotrexate or
trimethoprim. Thus, while the peptide ligand isolated from phage-displayed
libraries may not resemble the chemical structure of the normal substrate of
the enzyme, the peptide can be used as an inhibitor to evaluate the function of
the enzyme or for drug discovery efforts (i.e., as a lead compound for
peptidomimetic design or as displaceable probe in high-throughput screens of
libraries of small molecules).
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Identification
of a Novel Human Peroxisomal 2,4-Dienoyl-CoA Reductase Related Protein Using
the M13 Phage Protein VI Phage Display Technology
L. Amery, G.
P. Mannaerts, S. Subramani, P. P. Van Veldhoven
and M. Fransen
Recently, we
reported the successful use of the gVI-cDNA phage display technology to clone
cDNAs coding for novel peroxisomal enzymes by affinity selection using
immobilized antisera directed against peroxisomal subfractions (Fransen, M.;
Van Veldhoven, P.P.; Subramani, S. Biochem. J., 1999, 340, 561‑568). To
identify other unknown peroxisomal enzymes, we further exploited this promising
approach. Here we report the isolation and cloning of another novel human cDNA
encoding a protein ending in the tripeptide AKL, a C-terminal peroxisomal
targeting signal (PTS1). Primary structure analysis revealed that this molecule
shared the highest sequence similarity to members of the 2,4-dienoyl-CoA
reductase (DCR) family. However, functional analysis indicated that a
recombinantly expressed version of the novel protein did not possess DCR
activity with either 2-trans,4-trans-hexadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA
as a substrate. The recombinant protein interacted with HsPex5p, the human
PTS1-binding protein. Binding was competitively inhibited by a PTS1-containing
peptide and was abolished when the last amino acid of the PTS1 signal was
deleted. Transfection of mammalian cells with gene fusions between green
fluorescent protein (GFP) and the human cDNA confirmed a peroxisomal
localization and, therefore, the functionality of the PTS1. These results
further demonstrate the suitability of the gVI-cDNA phage display technology
for cDNA expression cloning using an antibody as a probe.
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Identification
of Small Molecule Binding Sites within Proteins Using Phage Display Technology
Affinity selection
of peptides displayed on phage particles was used as the basis for mapping
molecular contacts between small molecule ligands and their protein targets.
Analysis of the crystal structures of complexes between proteins and small
molecule ligands revealed that virtually all ligands of molecular weight 300 Da
or greater have a continuous binding epitope of 5 residues or more. This
observation led to the development of a technique for binding site
identification which involves statistical analysis of an affinity-selected set
of peptides obtained by screening of libraries of random, phage-displayed
peptides against small molecules attached to solid surfaces. A random sample of
the selected peptides is sequenced and used as input for a similarity scanning
program which calculates cumulative similarity scores along the length of the
putative receptor. Regions of the protein sequence exhibiting the highest
similarity with the selected peptides proved to have a high probability of
being involved in ligand binding. This technique has been employed successfully
to map the contact residues in multiple known targets of the anticancer drugs
paclitaxel (Taxol™), docetaxel (Taxotere™) and 2-methoxyestradiol and the
glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1].
These data corroborate the observation that the binding properties of peptides
displayed on the surface of phage particles can mimic the binding properties of
peptides in naturally occurring proteins. It follows directly that structural
context is relatively unimportant for determining the binding properties of
these disordered peptides. This technique represents a novel, rapid, high
resolution method for identifying potential ligand binding sites in the absence
of three-dimensional information and has the potential to greatly enhance the
speed of development of novel small molecule pharmaceuticals.
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Substrate
Phage as a Tool to Identify Novel Substrate Sequences of Proteases
Shuichi
Ohkubo, Kazutaka Miyadera, Yoshikazu Sugimoto, Ken-ichi Matsuo, Konstanty
Wierzba and Yuji Yamada
Combinatorial
phage peptide libraries have been used to identify the ligands for specific
target molecules. These libraries are also useful for identification of the
specific substrates of various proteases. A “substrate phage library” has a
random peptide sequence at the N-terminus of the phage coat protein and an
additional tag sequence that enables attachment of the phage to an immobile
phase. When these libraries are incubated with a specific enzyme, such as a
protease, the uncleaved phage is excluded from the solution with tag-binding
macromolecules. This provides a novel approach to define substrate specificity.
The aim of this review is to summarize recent progress on the application of
the substrate phage technique to identify specific substrates of proteolytic
enzymes. As an example, some of our own experimental data on the selection and
characterization of substrate sequences for thrombin, a serine protease, and
membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using
this approach, the canonical consensus substrate sequence for thrombin was
deduced from the selected clones. As expected from the collagenolytic activity
of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A
more selective substrate sequence for MT1-MMP was identified during a substrate
phage screen. The delineation of the substrate specificity of proteases will
help to elucidate the enzymatic properties and the physiological roles of these
enzymes. Comprehensive screening of very large numbers of potential substrate
sequences is possible with substrate phage libraries. Thus, this approach
allows novel substrate sequences and previously unknown target molecules to be
defined.
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Predicting In Vivo Protein-Peptide Interactions with Random Phage Display
James F.
Smothers and Steven Henikoff
Binding sites in
protein complexes occasionally map to small peptides within one or more
proteins. Random peptide display methods simulate binding interactions by
providing all possible peptide combinations with an equal opportunity to bind a
protein of interest. The natural substrates for the protein are typically known
in advance. However, it is often the case that such substrates are identified
as putative partner proteins by using in vivo methods such as yeast two‑hybrid
screening. Unfortunately, such methods often produce lengthy datasets of
protein sequences and offer little mechanistic insight into how such
interactions might take place in vivo. Here, we review an approach that
addresses this problem. First, sequence alignment tools identify and
characterize blocks of conserved sequences among peptides recovered during
random peptide display. Next, searching programs detect similar blocks of
conserved sequences within naturally‑occurring proteins to predict
partner proteins. Finally, the significance of an interaction is tested using
site‑specific mutagenesis, binding competition or co‑immunoprecipitation
experiments. This strategy should become increasingly powerful with the growing
popularity of interaction studies, sequencing projects and microarray analyses
in modern biology.
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The Cloning
of Human Genes Using cDNA Phage Display and Small-molecule Chemical Probes
The cloning of genes
based on protein function has become a powerful tool for protein discovery and
should play an important role in proteomics in general. We have recently
reported a technique for the functional identification of protein targets by
combining traditional affinity chromatography with cDNA phage display. This
procedure, referred to as display cloning, directly couples biologically active
natural products to the gene of their protein cellular target. We now report
the cloning of a human gene, the a domain of F1 ATP synthase, using a synthetic
scaffold molecule which serves as a prototype for a diverse chemical library.
The ability to select genes from cDNA libraries using probes from combinatorial
libraries would greatly increase the number of small molecule/protein
interactions that can be identified. This method might prove valuable in
furthering our understanding of biology and its application toward drug
development.
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Caveat Receptor: Proteomes on Display
Display methods
development is currently extending the application of this strategy beyond the
generation of ligand binding reagents for research, clinical, or
biotechnological purposes to its use as a primary research tool. Peptide- and
cDNA display methods have the potential to contribute to understanding the
mechanisms of certain classes of drugs and to help map protein-protein
interactions of physiological importance. Although the critical contribution of
comprehensive amino acid sequence databases has been recognized, of equal
importance might be structural genomics concepts in the application of display
system technology to proteomics research. Lessons learned from the study of
antibody-antigen interactions are reviewed here and applied to the field of
display technology with the goal of delineating major factors involved in the
successful mimicry of natural protein-ligand interactions.