Artificial Transmembrane Oncoproteins Smaller than the Bovine Papillomavirus E5 Protein Redefine Sequence Requirements for Activation of the Platelet-Derived Growth Factor β Receptor

Talbert-Slagle, Kristina; Marlatt, Sara A.; Barrera, Francisco N.; Khurana, Ekta; Oates, Joanne; Gerstein, Mark; Engelman, Donald M.; Dixon, Ann M.; DiMaio, Daniel

doi:10.1128/jvi.00946-09

Cited by 23 publications

(31 citation statements)

References 49 publications

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“…The recovered clones were tested individually for activity in BaF3 cells expressing wild-type or mutant PDGF ␤ receptors, including a chimeric receptor containing the transmembrane domain of the PDGF ␣ receptor (PR-␤␣␤), which is not recognized by the wild-type E5 protein (37). As expected, the wild-type E5 protein was inactive in parental BaF3 cells lacking the PDGF ␤ receptor, BaF3-L512I cells, and BaF3-␤␣␤ cells but conferred growth factor independence in BaF3 cells expressing the wild-type PDGF ␤ receptor (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Protein-antibody mixtures were rotated overnight at 4°C, and complexes were collected with protein A-Sepharose, washed, and resuspended in 2ϫ Laemmli sample buffer. Proteins were denatured and separated on a 7.5% (for PDGF receptor and phosphotyrosine blotting) or 20% (for E5 blotting) SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) (E5) or nitrocellulose (PDGF ␤ receptor or PY) membrane for blotting, as described previously (37). No SDS was included for transfer of the E5 protein.…”

Section: Methodsmentioning

confidence: 99%

“…No SDS was included for transfer of the E5 protein. Membranes were incubated with primary antibodies overnight, washed, incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h, washed again, and detected by using West Pico chemiluminescent reagents (Pierce), as previously described (37).…”

Section: Methodsmentioning

confidence: 99%

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Compensatory Mutants of the Bovine Papillomavirus E5 Protein and the Platelet-Derived Growth Factor β Receptor Reveal a Complex Direct Transmembrane Interaction

Edwards

Xie

Bowers

et al. 2013

J Virol

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eThe 44-amino-acid E5 protein of bovine papillomavirus is a dimeric transmembrane protein that exists in a stable complex with the platelet-derived growth factor (PDGF) ␤ receptor, causing receptor activation and cell transformation. The transmembrane domain of the PDGF ␤ receptor is required for complex formation, but it is not known if the two proteins contact one another directly. Here, we studied a PDGF ␤ receptor mutant containing a leucine-to-isoleucine substitution in its transmembrane domain, which prevents complex formation with the wild-type E5 protein in mouse BaF3 cells and inhibits receptor activation by the E5 protein. We selected E5 mutants containing either a small deletion or multiple substitution mutations that restored binding to the mutant PDGF ␤ receptor, resulting in receptor activation and growth factor independence. These E5 mutants displayed lower activity with PDGF ␤ receptor mutants containing other transmembrane substitutions in the vicinity of the original mutation, and one of them cooperated with a receptor mutant containing a distal mutation in the juxtamembrane domain. These results provide strong genetic evidence that the transmembrane domains of the E5 protein and the PDGF ␤ receptor contact one another directly. They also demonstrate that different mutations in the E5 protein allow it to tolerate the same mutation in the PDGF ␤ receptor transmembrane domain and that a mutation in the E5 protein can allow it to tolerate different mutations in the PDGF ␤ receptor. Thus, the rules governing direct interactions between transmembrane helices are complex and not restricted to local interactions. Proteins that span cellular membranes are thought to comprise up to 30% of the proteome (1). The membrane-spanning segments of most of these transmembrane proteins adopt an ␣-helical conformation and can undergo highly specific, lateral interactions to form oligomeric protein complexes or properly folded multipass transmembrane proteins (2, 3). These helical interactions are often essential for biological activity, but the structural basis of the vast majority of transmembrane interactions is not understood because of difficulties in obtaining high-resolution structures of transmembrane helical bundles. Mutational analysis showed that homodimeric helix-helix interactions can be determined by highly specific interactions between amino acid side chains (4). Formation of heteromeric transmembrane complexes can be mediated by interactions between hydrophilic amino acid side chains (5-7), but the structural basis for the specificity of heteromeric transmembrane interactions has not been studied in detail. In our laboratory, we have developed genetic methods to analyze heteromeric transmembrane interactions in mammalian cells and showed that artificial transmembrane proteins can undergo specific functional interactions with native transmembrane proteins (8-12).The 44-amino-acid E5 oncoprotein of bovine papillomavirus type 1 (BPV) is essentially an isolated transmembrane domain that forms a homodime...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Compensatory Mutants of the Bovine Papillomavirus E5 Protein and the Platelet-Derived Growth Factor β Receptor Reveal a Complex Direct Transmembrane Interaction

Edwards

Xie

Bowers

et al. 2013

J Virol

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“…These proteins are designated traptamers, for transmembrane aptamers. We isolated traptamers that specifically activate the PDGF β receptor or the erythropoietin receptor or down-regulate the HIV coreceptor, C-C chemokine receptor type 5 (CCR5) (19)(20)(21)(22)(23)(24). However, because the E5 protein was selected during viral evolution for its ability to insert into cell membranes, fold properly, and interact with the PDGF β receptor, the E5 sequences retained in these traptamers may be required for biological activity.…”

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De novo selection of oncogenes

Chacón

Petti

Scheideman

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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 β 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.synthetic biology | protein engineering | receptor tyrosine kinase | E5 protein | traptamer A ll extant naturally occurring proteins are derived from preexisting ones by the incremental process of natural selection. Cellular proteins are thus the products of a long chain of evolutionary decisions driven by chance mutations and selective pressures, which allowed some profitable lineages to flourish but drove many others to extinction. Indeed, because of the immense diversity that can result from the arrangement of 20 different amino acids into long polymers, the roster of distinct proteins on earth is a miniscule fraction of possible chemical structures (1). Because they are trapped by their evolutionary history, naturally occurring proteins are not necessarily the optimal proteins for accomplishing a given task. Therefore, one of the major goals of synthetic biology is to generate artificial proteins for a variety of practical applications, such as optimizing catalytic activity and even developing entirely new catalytic activities, generating molecules suitable for industrial processes, and fabricating novel biomaterials.Computational methods have been developed to construct artificial proteins based on detailed understanding of the chemistry, energetics, and structure-function relationships of existing proteins (2-4). This process, known as de novo or computational protein design, typically starts from a known protein structure or a conserved motif and iteratively samples numerous parameters such as amino acid substitutions and side-chain rotamers to identify predicted low-energy states. Although this approach requires enormous computational power, it has been used successfully for both globular and transmemb...

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“…To identify small proteins with different TM sequences that transform cells, we constructed expression libraries in which the TM domain of the E5 protein was replaced with randomized sequences of primarily hydrophobic amino acids and isolated clones from these libraries that induced focus formation or growth-factor independence in mouse cells (13)(14)(15)(16). Although the active clones had diverse TM sequences, all of the proteins analyzed induced cell transformation by specifically activating the PDGFβR.…”

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confidence: 99%

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor

Cammett¹,

Jun²,

Cohen³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor β receptor (PDGFβR). We used the E5 protein as a scaffold to construct a retrovirus library expressing ∼500; 000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFβR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word "traptamer" for these transmembrane aptamers.bovine papilloma virus | E5 protein | expression libraries | protein engineering | traptamers I t is thought that up to 30% of all proteins span cell membranes (1). Most membrane-spanning protein segments adopt an α-helical conformation that is largely independent of their amino acid sequence and minimally influenced by extramembraneous protein domains. Although transmembrane (TM) domains were once thought to be rather nonspecific protein segments whose primary function was to anchor proteins in membranes, they can participate in highly-specific, inter-and intramolecular side-by-side interactions that control important biological processes (2).The 44-amino acid bovine papillomavirus type 1 (BPV) E5 protein is essentially an isolated TM domain. It contains a hydrophobic central segment and exists in the intracellular membranes of transformed cells as a type II TM homodimer stabilized by disulfide bonds in its carboxy-terminus (3-5). The E5 dimer binds to the TM domains of two molecules of the platelet-derived growthfactor β receptor (PDGFβR) tyrosine kinase, resulting in receptor dimerization and activation, leading to cell transformation (6-10). The E5 protein does not bind to other TM domains or to certain PDGFβR TM mutants (11).Based on our analysis of the E5 protein, we proposed that it might be possible to construct small, artificial TM proteins that regulate a variety of viral and cellular TM proteins in trans (11, 12). To identify small proteins with different TM sequences that transform cells, we constructed expression libraries in which the TM domain of the E5 protein was replaced with randomized sequences of primarily hydrophobic amino acids and isolated clones from these libraries that induced focus formation or growth-factor independe...

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Artificial Transmembrane Oncoproteins Smaller than the Bovine Papillomavirus E5 Protein Redefine Sequence Requirements for Activation of the Platelet-Derived Growth Factor β Receptor

Cited by 23 publications

References 49 publications

Compensatory Mutants of the Bovine Papillomavirus E5 Protein and the Platelet-Derived Growth Factor β Receptor Reveal a Complex Direct Transmembrane Interaction

Compensatory Mutants of the Bovine Papillomavirus E5 Protein and the Platelet-Derived Growth Factor β Receptor Reveal a Complex Direct Transmembrane Interaction

De novo selection of oncogenes

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor

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