The role of peptide motifs in the evolution of a protein network

Abstract

Naturally occurring proteins in cellular networks often share peptide motifs. These motifs have been known to play a pivotal role in protein interactions among the components of a network. However, it remains unknown how these motifs have contributed to the evolution of the protein network. Here we addressed this issue by a synthetic biology approach. Through the motif programming method, we have constructed an artificial protein library by mixing four peptide motifs shared among the Bcl-2 family proteins that positively or negatively regulate the apoptosis networks. We found one strong pro-apoptotic protein, d29, and two proteins having moderate, but unambiguous anti-apoptotic functions, a10 and d16, from the 28 tested clones. Thus both the pro- and anti-apoptotic modulators were present in the library, demonstrating that functional proteins with opposing effects can emerge from a single pool prepared from common motifs. Motif programming studies have exhibited that the annotated function of the motifs were significantly influenced by the context that the motifs embedded. The results further revealed that reshuffling of a set of motifs realized the promiscuous state of protein, from which disparate functions could emerge. Our finding suggests that motifs contributed to the plastic evolvability of the protein network.

Figure 1.

Exploring apoptotic regulatory networks by motif programming. (A) Schematic diagram of the roles of motifs in network evolution and network dynamics. (B) (left) Apoptotic regulatory networks regulated by Bcl-2 family proteins. (right) Both anti-apoptotic and pro-apoptotic proteins share the BH1-4 peptide motifs but the proteins vary with respect to the number of the four motifs. (C) Screening strategy of cell death modulators utilized in this study. (left) Structure of human Bcl-xL, an anti-apoptotic multidomain protein [based on the (1R2D)]. The core regions of the BH1, BH2, BH3 and BH4 motifs focused on in this study are shown in cyan, orange, red and green, respectively, though we used the BH3 motif from Noxa instead of Bcl-xL. (middle) Four designed microgenes that encode BH peptides in their first reading frames. (right) Cell-based screening to identify artificial cell death modulators that oppositely regulate the apoptotic circuits (cell-killer or cell-protector).

Figure 2.

A pro-apoptotic clone d29 created from a mixture of BH motifs. (A) Screening for pro-apoptotic clones. MCF-7 cells were transfected with DNA (0.16 μg) encoding the target protein (each of 28 clones) or empty vector (control). Cell viability was then evaluated using WST-1 assays. Bars depict means ± SD from at least four independent experiments. (B) Schematic drawing of d29, a10 and d16 used in this study. (C) Expressions of d29, a10 and d16. MCF-7 cells were transfected with DNA encoding d29, a10, d16 or empty vector and analyzed by western blotting. (D) Pro-apoptotic function of d29. MCF-7 cells were initially transfected with DNA (0.8 μg) encoding empty vector (control), Bax, d29 or a10. After 48 h, the cells were fixed and changes in the cell morphology were correlated with TUNEL staining. (E) The incidence of apoptotic cells was counted using TUNEL staining. Bars depict means ± SD from three independent experiments. (F) Expression of synthetic proteins was visualized using a confocal microscope and anti-c-myc antibody (red). A good correlation was observed between TUNEL positivity and d29 expression.

Figure 3.

d29 modulates mitochondria-dependent apoptosis regulated by Bcl-xL. (A) Schematic drawing of mutant derivatives of d29. The lower panel shows western blots of mutants proteins expressed in MCF-7 cells detected by c-myc antibody; Lane 1; d29, Lanes 2–4; d29(NΔ26), d29(NΔ52) and d29(NΔ77). (B) Apoptotic inductions by d29 and its derivatives were scored by TUNEL assay. Bars depict means ± SD from three independent experiments. (C) Immunohistochemical analysis showing the release of cytochrome c from mitochondria in cells accumulating d29. MCF-7 cells were transfected with DNA encoding d29, Bax or empty vector (control), after which they were fixed in 4% PFA, and d29 and cytochrome c were immunostained using anti-c-myc and anti-cyto-c monoclonal antibodies, respectively. Bax was detected using anti-Bax polyclonal antibody. Cytochrome c was diffusely distributed within cells accumulating d29 or Bax, but showed a punctate distribution pattern corresponding to the mitochondria in cells not accumulating d29 or Bax. (D) Bcl-xL rescued cells from d29-induced growth inhibition. MCF-7 cells were transfected with DNA encoding d29, with or without a Bcl-xL expression vector. Empty vector served as a control. WST-1 assays were carried out to evaluate the cell viability. Bars depict means ± SD from three independent experiments. (E) MCF-7 cells were transfected as in (D). The incidence of cell death determined by counting cells floating in the medium and then dividing their number by the total number of cells (1 × 10⁶ cell/well). Bars depict means ± SD from three independent experiments. (F) Bcl-xL inhibits d29-dependent PARP cleavage. Western blots of c-myc (for d29), Bcl-xL and poly (ADP-ribose) polymerase (PARP). PARP cleavage was induced by d29, and this pro-apoptotic activity was inhibited by the co-expression of Bcl-xL.

Figure 4.

Anti-apoptotic d16 created from the same BH motifs-mixing library. (A) Transfection of clones a10 or d16 protected cells against apoptosis induced by Bim or anticancer drugs. HeLa cells transfected with DNA (0.1 μg) encoding empty vector, anti-apoptotic Bcl-xL, a10 or d16 were cotransfected with a Bim expression construct (10 or 50 ng; top) or were treated with VP-16 (50 or 200 μM; middle) or STS (62.5 or 1000 nM; bottom). Cell viability was then measured (WST-1 assays); bars depict means ± SD from three independent experiments. (B) Cell morphological and TUNEL analyses showing the anti-apoptotic activity of d16. (C) Anti-apoptotic effect of d16. Transfected HeLa cells were treated for 23 h with 125 nM STS before TUNEL analysis (red) and immunostaining of synthetic or Bcl-xL proteins (green). The same cells seen in Figure 4B were used to evaluate the correlation between the changes in the cell morphology and TUNEL staining. (D) Schematic diagram of two protein modulators (d29 and a10/d16) that oppositely regulate apoptotic regulatory networks.

Figure 5.

Investigating the relationship between protein sequences and cellular localizations. For analysis of the protein localizations, the MCF-7 cells were plated and pre-incubated for 24 h at 37°C. After transfection with the respective plasmids (0.8 μg) using lipofectamine (2 μl), the cells were incubated for 24 h and then fixed in 4% PFA. The localizations of the synthetic proteins were analyzed using the anti-myc antibody (see also Materials and Methods section for Immunohistochemical Analysis). Schematic drawing of artificial proteins is also shown.