D. C. Anderson

Dominant effector genetics in mammalian cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example—and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance—we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells

BACKGROUND Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes. RESULTS Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides. CONCLUSIONS Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.

Characterization and Use of Green Fluorescent Proteins from Renilla mulleri and Ptilosarcus guernyi for the Human Cell Display of Functional Peptides

Green fluorescent protein (GFP) is useful as an intracellular scaffold for the display of random peptide libraries in yeast. GFPs with a different sequence from Aequorea victoria have recently been identified from Renilla mulleri and Ptilosarcus gurneyi. To examine these proteins as intracellular scaffolds for peptide display in human cells, we have determined the expression level of retrovirally delivered human codon-optimized versions in Jurkat-E acute lymphoblastic leukemia cells using fluorescence activated cell sorting and Western blots. Each wild type protein is expressed at 40% higher levels than A. victoria mutants optimized for maximum fluorescence. We have compared the secondary structure and stability of these GFPs with A. victoria GFP using circular dichroism (CD). All three GFPs essentially showed a perfect β-strand conformation and their melting temperatures (Tm) are very similar, giving an experimental evidence of a similar overall structure. Folded Renilla GFP allows display of an influenza hemagglutinin epitope tag in several internal insertion sites, including one which is not permissive for such display in Aequorea GFP, giving greater flexibility in peptide display options. To test display of a functional peptide, we show that the SV-40 derived nuclear localization sequence PPKKKRKV, when inserted into two different potential loops, results in the complete localization of Renilla GFP to the nucleus of human A549 cells.

A novel artificial loop scaffold for the noncovalent constraint of peptides.

BACKGROUND Few examples exist of peptides of < 35 residues that form a stable tertiary structure without disulfide bonds. A method for stabilization and noncovalent constraint of relatively short peptides may allow the construction and use of intracellular peptide libraries containing protein minidomains. RESULTS We have examined a novel method for the noncovalent constraint of peptides by attaching the peptide EFLIVKS (single-letter amino acid code), which forms dimers, to the amino and carboxyl termini of different peptide inserts. An 18 residue random coil taken from the inhibitor loop of barley chymotrypsin inhibitor 2 was inserted between the peptides to produce a 32-mer minidomain that is attacked only slowly by elastase, has numerous slowly exchanging protons, contains a high beta-structure content and has a T(m) above 37 degrees C. A point mutation disrupting the hydrophobic interior in both dimerizing peptides causes a loss of all slowly exchanging protons and of secondary structure. Adding specific charged residues to each terminus substantially increased the T(m), as did point mutants designed to add interdimerizer ion pairs. Three flexible epitope tag inserts and a nonamer insert do not appear to be folded in a stable structure by EFLIVKS. The properties of two peptides selected for expression in HeLa cells suggest they do form a stable tertiary structure. CONCLUSIONS Attaching short dimerizing peptides to both the amino and carboxyl termini of several 18-mer peptides appears to create stable monomeric tertiary structures. Mutations in the dimerizers can either destabilize or significantly stabilize a standard 18-mer insert. Dimerizing peptides flanking random insert sequences could be used as a strategy to generate heterogeneous peptide libraries with both extended and folded members.

Intracellular Combinatorial Chemistry with Peptides in Selection of Caspase-like Inhibitors

Fas is a cell surface receptor that can transmit signals for programmed cell death. Using a retroviral expression system, we have demonstrated that a short peptide derived from the cleavage site in a cellular target of a pro-apoptotic cysteine protease can be expressed within intact cells with sufficient activity to inhibit Fas-mediated apoptosis. In vitro analysis demonstrates that this retrovirally-expressed peptide is as potent as 150uM levels of the chemically synthesized peptide. Furthermore, using retroviral peptide library-based functional cloning we identified variants of this peptide with apparent anti-apoptotic activity. This approach is likely to lead to the identification of peptide variants with activity against a variety of signaling processes, both normal and pathological.

Nucleophile Labeling of Cysteine and Serine Protease Substrates

Dipeptides containing fluorescein or biotin have been incorporated into proteolytic substrate cleavage products of bovine serum albumin generated by human cathepsin S or neutrophil elastase and into a fragment of the 31-kDa interleukin 1β precursor by human interleukin 1β-converting enzyme. Incorporation of the nucleophile is blocked by prior inhibition of the enzymes, and is not seen when proteolysis occurs in the absence of label, and the protease is then inhibited before the addition of label. Labeling is dependent on the pH, the time of reaction, and the concentrations of the nucleophile and substrate. Labeling of proteins can be readily detected by SDS-polyacrylamide gel electrophoresis. The pattern of elastase-labeled bovine serum albumin bands differs among P1′ Phe, Ala, and Gly, suggesting that nucleophilic attack on acyl enzyme intermediates derived from a large protein may differ from attack on small intermediates. The only observed labeled fragment catalyzed by interleukin 1β-converting enzyme is fragment 28-116 from the interleukin 1β precursor, suggesting that the cleavage between residues 27 and 28 is at least as efficient as between residues 116 and 117. This labeling method does not require organic solvent or nonphysiological pH values and thus may be useful for the discovery of novel protease substrates in cells or other in vivo systems or for diagnostic applications.

Structural implications of a novel peptide dimerizer sequence when anchored to terminal ends of different peptide motifs

Imposing constraints on displayed peptides in peptide libraries has been found to confer a number of important advantages over unstructured peptides used for binding target proteins [1]. Relative to linear peptides, constrained peptides are more stable to proteolysis and possess a more restricted conformation space that can allow a higher affinity for cognate binding proteins due to reduced conformational entropy. Standard covalent constructs such as disulfide bonds are unstable inside the cells due to a high content of reduced glutathione and the presence of thioredoxin reductase. In an effort to develop a mini protein scaffold for the retroviral display of peptide libraries, we fused a short peptide sequence, EFLIVKS (with self binding constant of 0.33 μM at pH 7.5) to the Nand C-termini of a variety of peptides. Structural studies were performed on these constructs. One is an 18-mer sequence derived from the protease contact loop of barley chymotrypsin inhibitor 2 (Ci2b) [2]. When cyclized with a disulfide bond, this is thought to act as a potent inhibitor of proteases such as elastase [3]. Here we report evidence of folding and stability of several members of this family of EFLIVKS-folded mini-domains studied by CD spectroscopy, deuterium exchange kinetics, and quenched molecular dynamics conformation searches.

Proteomics Approach for Identifying Interacting Partners of a C-Terminal Functional Peptide Derived from the Tumor Suppressor, p21 cip/waf1

With the recent technological innovations of proteomics, protein separation followed by mass spectrometry (MS) has become the technique of choice in identifying and validating potential drug targets. As a part of our drug discovery program, we tested the validity of this approach by conducting a simple affinity extraction followed by mass spectrometric (MS) analysis of the components isolated. For this purpose, we chose the C-terminal peptide of the tumor suppressor p21cip/waf1, which is known to bind proliferating cell nuclear antigen (PCNA) and cause cell cycle arrest [1]. Using Jurkat-E cell lysate, affinity extraction of PCNA and its binding partners was carried out by spiking streptavidin agarose beads pre-conjugated with biotinylated p21-derived peptide(s). Using tryptic digests of entire affinity extracts and differential micro-capillary LC/MS/MS, or difference 2D gels combined with in-gel tryptic digests and MALDI-TOF MS, we have identified binding partners of the p21 C-terminal peptide, or of its complex with PCNA. Results from the above experiments were confirmed either by reciprocal affinity extraction and/or Western blotting with respective antibodies. This study suggests that peptides obtained from intracellular functional screens could also serve as efficient baits to affinity extract target proteins and map mammalian cell protein interaction networks.