De novo selection of oncogenes

Abstract

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.

Keywords: E5 protein; protein engineering; receptor tyrosine kinase; synthetic biology; traptamer.

Fig. 1.

Design of the UDv3 library and sequences of active small transmembrane proteins. (A) Amino acid sequence of the wild-type BPV E5 protein. (B) Design of the UDv3 library. Randomized positions are indicated by “X,” flanked by the amino acids encoded by the Kozak start site (MA) and the C-terminal aromatic anchor (YW). (C) Amino acid sequences of the four transforming clones recovered from the library. All randomized segments are shown in bold.

Fig. 2.

Traptamers transform C127 mouse cells and HFFs. (A) C127 cells were infected with empty retroviral vector or a virus expressing E5 or the indicated traptamer and incubated for 14 d. Stained plates for the controls and two representative traptamers are shown. (B) Percentage of focus-forming activity relative to E5 in C127 cells (dark and light gray bars) and HFFs (cross-hatched bars), normalized for viral titer. Wild-type traptamers are shown with dark gray and cross-hatched bars and FF mutants with light gray bars. Results shown are the average of multiple independent experiments, with SE. The E5 FF mutant was not tested (nt), so the activity of the FF mutants was normalized to wild-type E5. (C) Focus formation by 9C-3 mutants. C127 cells were infected with retroviruses expressing wild-type 9C-3 or 9C-3 containing substitutions at position 17 (all containing an N-terminal FLAG tag). The graph is representative of two independent experiments and shows the percentage of focus-formation activity normalized to virus titer, relative to wild-type tagged 9C-3.

Fig. 3.

Traptamers activate the PDGF β receptor. (A) Detergent extracts were prepared from C127 cells (left blots) and HFFs (right blots) expressing empty vector, E5, or the indicated traptamer. Extracts were immunoprecipitated with anti-PDGF β receptor antibody and immunoblotted with the indicated antibodies (PR, PDGF β receptor; PY, phosphotyrosine) to detect expression and tyrosine phosphorylation of the PDGF β receptor, respectively, or with antibody recognizing PI3K and SHP2 to assess association of these substrates with the receptor. (B) C127 cells expressing vector (V) or the indicated wild-type (YW) or mutant (FF) traptamers were analyzed as above for PDGF β receptor expression and tyrosine phosphorylation. The mature (m) and precursor (p) forms of the PDGF β receptor are indicated.

Fig. 4.

Traptamers are expressed in C127 cells and form a stable complex with the PDGF β receptor. Detergent extracts were prepared from C127 cells expressing empty vector (V), E5, or an untagged or a FLAG-tagged traptamer, as indicated (12, 12A-5; 3, 3A-2; 6, 6A-1; 9, 9C-3). In the upper two blots, extracts were immunoprecipitated with anti-PDGF β receptor antibody and immunoblotted with the same antibody (PR) or with a phosphotyrosine (PY) antibody. In the lower four blots, extracts were immunoprecipitated with an anti-FLAG antibody and immunoblotted with either the FLAG antibody to detect expression of the tagged traptamers or the PDGF β receptor antibody to detect PDGF β receptor in complex with the traptamer. The lanes in the bottom blots were all from a single gel and exposed the same amount of time; an empty lane was removed as indicated by the space between the blots. A slower migrating band marked by an asterisk in the 9C-3 lane blotted with the FLAG antibody may represent a dimeric form of this traptamer. The mature (m) and precursor (p) forms of the mPDGF β receptor are indicated.

Fig. 5.

Genetic requirements for traptamer activity. BaF3 cells expressing empty vector (LXSN) (A), the mouse PDGF β receptor (mPRβ) (B), or the chimeric βαβ receptor (C) were infected with empty retroviral vector (square) or retroviruses expressing E5 (no symbol), the v-sis oncogene (triangle), 6A-1 (+), 9C-3 (X), 12A-5 (circle), or 3A-2 (diamond). After selection for puromycin resistance, cells were incubated in medium lacking IL-3, and live cells were counted on the indicated days. Results shown are representative of multiple independent experiments. (D) BaF3 cells expressing the truncated PDGF β receptor (TPR) were infected with empty retroviral vector or retrovirus expressing E5 or the indicated traptamer. After selection for puromycin resistance, cells were incubated in the absence of IL-3 for between 5 and 7 d in different experiments, and live cells were counted. The graph shows results from three independent experiments, expressed as the average percentage of live cells relative to the number of live cells in E5-expressing cultures, with SEM shown.

Fig. 6.

C127 cells expressing traptamers are tumorigenic. (A) C127 cells expressing the empty vector, E5, or the indicated traptamer were injected into nude mice, and tumor formation was scored after 4 wk. (B) C127 cells expressing E5 (purple X), 9C-3 (green triangle), 12A-5 (blue diamond), or 3A-2 (red square) were injected s.c. into nude mice (two sites per mouse). At various times, palpable masses were measured in two dimensions, and tumor volume was calculated. The average tumor volume per mouse was used to calculate the group mean and SD, which are shown. (C) Tumors were excised, imbedded in paraffin, sectioned, and stained with hematoxylin/eosin. (Scale bar: 50 μm.) (D) RIPA extracts prepared from tumors induced by 12A-5, 9C-3, or 3A-2 were immunoprecipitated with antibody recognizing the PDGF β receptor and immunoblotted for phosphotyrosine (Upper) or PDGF β receptor (Lower). The same amount of extracted protein was immunoprecipitated for each lane. Molecular mass markers (in kilodaltons) and the mature (m) and precursor (p) forms of the mPDGF β receptor are indicated.