Regulation of C-C chemokine receptor 5 (CCR5) stability by Lys197 and by transmembrane protein aptamers that target it for lysosomal degradation.

C-C motif chemokine receptor 5 (CCR5) is a cell surface-associated, immune-regulatory G protein-coupled receptor (GCPR) with seven transmembrane helices. We previously reported the isolation and initial characterization of short artificial transmembrane protein aptamers, named "traptamers," that specifically down-regulate CCR5 expression and inhibit infection of human T cells by HIV strains that use CCR5 as a co-receptor. Here, we investigated the mechanism of traptamer-mediated CCR5 down-regulation and show that most of the traptamers (designated class 1 traptamers) form a stable complex with CCR5 and target it for lysosome-mediated degradation. The ability of these traptamers to down-regulate CCR5 depended on Lys197 in the fifth transmembrane helix of CCR5. In the absence of traptamers, substitution of Lys197 to an uncharged amino acid increased CCR5 stability, and introduction of a lysine at the homologous position in CCR2b, a related chemokine receptor, decreased CCR2b levels. The prototypic class 2 traptamer BY6M4 also formed a complex with CCR5, but CCR5 down-regulation caused by class 2 traptamers did not depend on the lysosome or on Lys197 These results demonstrate that traptamers use diverse mechanisms to down-regulate CCR5 and identify a specific amino acid that plays a central role in controlling chemokine receptor stability. Further studies of these traptamers are likely to provide new insights into CCR5 metabolism and biology and may suggest new therapeutic approaches to modulate the levels of CCR5 and other GPCRs.

De novo selection of oncogenes.

All cellular proteins are derived from preexisting ones by natural selection. Because of the random nature of this process, many potentially useful protein structures never arose or were discarded during evolution. Here, we used a single round of genetic selection in mouse cells to isolate chemically simple, biologically active transmembrane proteins that do not contain any amino acid sequences from preexisting proteins. We screened a retroviral library expressing hundreds of thousands of proteins consisting of hydrophobic amino acids in random order to isolate four 29-aa proteins that induced focus formation in mouse and human fibroblasts and tumors in mice. These proteins share no amino acid sequences with known cellular or viral proteins, and the simplest of them contains only seven different amino acids. They transformed cells by forming a stable complex with the platelet-derived growth factor ß receptor transmembrane domain and causing ligand-independent receptor activation. We term this approach de novo selection and suggest that it can be used to generate structures and activities not observed in nature, create prototypes for novel research reagents and therapeutics, and provide insight into cell biology, transmembrane protein-protein interactions, and possibly virus evolution and the origin of life.

Transmembrane protein aptamers that inhibit CCR5 expression and HIV coreceptor function.

We have exploited the ability of transmembrane domains to engage in highly specific protein-protein interactions to construct a new class of small proteins that inhibit HIV infection. By screening a library encoding hundreds of thousands of artificial transmembrane proteins with randomized transmembrane domains (termed "traptamers," for transmembrane aptamers), we isolated six 44- or 45-amino-acid proteins with completely different transmembrane sequences that inhibited cell surface and total expression of the HIV coreceptor CCR5. The traptamers inhibited transduction of human T cells by HIV reporter viruses pseudotyped with R5-tropic gp120 envelope proteins but had minimal effects on reporter viruses with X4-tropic gp120. Optimization of two traptamers significantly increased their activity and resulted in greater than 95% inhibition of R5-tropic reporter virus transduction without inhibiting expression of CD4, the primary HIV receptor, or CXCR4, another HIV coreceptor. In addition, traptamers inhibited transduction mediated by a mutant R5-tropic gp120 protein resistant to maraviroc, a small-molecule CCR5 inhibitor, and they dramatically inhibited replication of an R5-tropic laboratory strain of HIV in a multicycle infection assay. Genetic experiments suggested that the active traptamers specifically interacted with the transmembrane domains of CCR5 and that some of the traptamers interacted with different portions of CCR5. Thus, we have constructed multiple proteins not found in nature that interfere with CCR5 expression and inhibit HIV infection. These proteins may be valuable tools to probe the organization of the transmembrane domains of CCR5 and their relationship to its biological activities, and they may serve as starting points to develop new strategies to inhibit HIV infection.