A Functional Proteomic Method for Biomarker Discovery

The sequencing of the human genome holds out the hope for personalized medicine, but it is clear that analysis of DNA or RNA content alone is not sufficient to understand most disease processes. Proteomic strategies that allow unbiased identification of proteins and their post-transcriptional and -translation modifications are an essential complement to genomic strategies. However, the enormity of the proteome and limitations in proteomic methods make it difficult to determine the targets that are particularly relevant to human disease. Methods are therefore needed that allow rational identification of targets based on function and relevance to disease. Screening methodologies such as phage display, SELEX, and small-molecule combinatorial chemistry have been widely used to discover specific ligands for cells or tissues of interest, such as tumors. Those ligands can be used in turn as affinity probes to identify their cognate molecular targets when they are not known in advance. Here we report an easy, robust and generally applicable approach in which phage particles bearing cell- or tissue-specific peptides serve directly as the affinity probes for their molecular targets. For proof of principle, the method successfully identified molecular binding partners, three of them novel, for 15 peptides specific for pancreatic cancer

Peptides Derived from HIV-1 Integrase that Bind Rev Stimulate Viral Genome Integration

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3′-end processing in which IN removes a pGT dinucleotide from the 3′ end of each viral long terminal repeat (LTR). Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication. assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR.The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events

Targeting of Nonkaryophilic Cell-Permeable Peptides into the Nuclei of Intact Cells by Covalently Attached Nuclear Localization Signals.

Dermaseptins are a family of antimicrobial peptides that lyse target bacterial cells by destabilization of their membranes. Here we present a novel application of a peptide derived from the dermaseptin S4, S413. At nontoxic concentrations, fluorescently labeled S413 was able to penetrate intact cultured HeLa cells but essentially failed to enter their nuclei despite its low molecular weight. Covalent attachment of nuclear localization signal (NLS) motifs of the SV40-T-antigen and of the HIV-1 Rev protein (ARM) conferred karyophilic properties upon the S413. The resulting peptides, which were designated as PV-S413 and RR-S413 penetrated into intact HeLa cells and were able to accumulate within the cells' nuclei. In studies with digitonin-permeabilized cells, nuclear uptake of the PV-S413 and the RR-S413 peptides showed the same features that characterize active nuclear import. Nuclear import was observed at 37 °C, was ATP-dependent, and was inhibited by the free peptides bearing the SV40 NLS and the Rev and Tat ARMs. Microinjected S413 remained in the cytoplasm while microinjected RR-S413 was translocated into the cells' nuclei. The new type of cell-permeable “karyophilic” peptides described here may be of potential application as a lead compound for therapeutic purposes, as a tool to study nucleocytoplasmic shuttling in intact cells, and for the delivery of peptides to the nucleus

Functional analysis of backbone cyclic peptides bearing the arm domain of the HIV-1 Rev protein: Characterization of the karyophilic and inhibition of Rev-induced gene expression.

This work describes the synthesis and activity of a novel backbone cyclic (BC) peptide library based on the sequence of the HIV-1 Rev arginine-rich motif (ARM). All the peptides in the library possess the same sequence but differ in their ring-moiety properties. The BC peptides were synthesized using simultaneous multiple-peptide synthesis and were fully assembled using bis(trichloromethyl)carbonate as a coupling agent. All the peptides in the library had inhibitory effects on the binding of Rev-GFP to importin β in vitro. Studies performed with one of the BC Rev-ARM analogues, Rev-13, demonstrated that, like its parental linear peptide, it is karyophilic; i.e., it is able to mediate the nuclear import of conjugated bovine serum albumin (BSA) molecules. The cell penetrating properties of the BC peptides were assessed utilizing an ELISA-based system. This assay provides a quantitative evaluation of cell penetration. Most of the peptides from the library were able to penetrate intact Colo-205 cells to varying degrees. Furthermore, these BC peptides were able to carry BSA into intact Colo-205 cells. In addition to its cell penetrating and binding properties, the BC Rev-13 analogue inhibited Rev-induced gene expression in HeLa cells by 60−70% in the low micromolar range and exhibited no cell toxicity. The potential of BC peptides bearing ARM domains as lead compounds for the production of anti-HIV drugs is discussed