Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein

Freeman-Cook, Lisa L.; DiMaio, Daniel

doi:10.1038/sj.onc.1209039

Cited by 25 publications

(17 citation statements)

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“…By screening libraries that express thousands of small proteins with randomized transmembrane domains, we identified small transmembrane proteins that induced focus formation in murine C127 fibroblasts by activating the endogenous PDGF ␤ receptor (10,12,39). These proteins were designed to retain the N-terminal and C-terminal segments of BPV E5, including the cysteines important for dimerization and Asp33, and all of the active proteins shared at least 50% sequence identity with BPV E5 (11,39).…”

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confidence: 99%

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

Talbert-Slagle¹,

Marlatt²,

Barrera

et al. 2009

J Virol

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The bovine papillomavirus E5 protein (BPV E5) is a 44-amino-acid homodimeric transmembrane protein that binds directly to the transmembrane domain of the platelet-derived growth factor (PDGF) ␤ receptor and induces ligand-independent receptor activation. Three specific features of BPV E5 are considered important for its ability to activate the PDGF ␤ receptor and transform mouse fibroblasts: a pair of C-terminal cysteines, a transmembrane glutamine, and a juxtamembrane aspartic acid. By using a new genetic technique to screen libraries expressing artificial transmembrane proteins for activators of the PDGF ␤ receptor, we isolated much smaller proteins, from 32 to 36 residues, that lack all three of these features yet still dimerize noncovalently, specifically activate the PDGF ␤ receptor via its transmembrane domain, and transform cells efficiently. The primary amino acid sequence of BPV E5 is virtually unrecognizable in some of these proteins, which share as few as seven consecutive amino acids with the viral protein. Thus, small artificial proteins that bear little resemblance to a viral oncoprotein can nevertheless productively interact with the same cellular target. We speculate that similar cellular proteins may exist but have been overlooked due to their small size and hydrophobicity.Viruses are tiny relative to the cells they infect. The largest animal viruses encode at most a few hundred genes, in contrast to the tens of thousands of genes expressed by the host cell. To overcome the constraints imposed by their small size, viruses use multiple or even overlapping reading frames and alternative splicing to produce more than one protein from single transcripts, build capsids from repeating subunits, harness cellular mechanisms to produce viral products, and express extremely small proteins. Several of these small viral proteins are membrane anchored, including the 44-residue SH protein of parainfluenza virus 5, which blocks apoptosis of infected cells (16), and the 96-residue M2 protein of influenza A, which forms an ion channel in endosomal membranes of infected cells and is required for infectivity (reviewed in reference 38). One of the best-characterized small viral transmembrane proteins is the E5 protein encoded by bovine papillomavirus type 1 (BPV E5). BPV E5 contains only 44 amino acids and is thus essentially an isolated transmembrane domain (Fig. 1) (reviewed in reference 47). It is the primary translation product from a small open reading frame and is sufficient to cause tumorigenic cell transformation, making it the smallest known autonomous oncoprotein.BPV E5 induces cell transformation by specifically activating a much larger cellular target, the platelet-derived growth factor (PDGF) ␤ receptor tyrosine kinase (8,14,30,35). The PDGF ␤ receptor, a single-span transmembrane protein of more than 1,000 amino acids, is normally activated by the binding of its dimeric ligand, PDGF, to the extracellular domain of the receptor. In contrast, a dimer of the BPV E5 protein binds to the transmembrane doma...

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confidence: 99%

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

Talbert-Slagle¹,

Marlatt²,

Barrera

et al. 2009

J Virol

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“…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 independence in mouse cells (13)(14)(15)(16).…”

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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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“…We have developed genetic methods to select short biologically active transmembrane proteins modeled on the E5 protein from libraries expressing many thousands of artificial proteins with randomized hydrophobic segments (named transmembrane protein aptamers or "traptamers") (22)(23)(24)(25). Because of the relative simplicity of transmembrane domains, we reasoned that this approach could be used to define the minimal chemical complexity sufficient to construct biologically active proteins.…”

Section: Significancementioning

confidence: 99%

Biologically active LIL proteins built with minimal chemical diversity

Heim

Marston

Federman

et al. 2015

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

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We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context.T he chemical diversity of the 20 standard amino acid sidechains found in proteins supports myriad biochemical activities essential for life, and additional chemical diversity is generated by posttranslational amino acid modifications. The number of potential protein sequences is enormous: for proteins only 300 amino acids long, ∼10 400 possible sequences exist, a number larger by many orders-of-magnitude than the number of atoms in the known universe. This immense number of sequences and the chemical and conformational complexity of naturally occurring proteins hinder our ability to understand protein structure, folding, and function, and complicate protein engineering efforts. In addition, many different related amino acid sequences can fold into nearly identical structures, and even proteins with quite divergent sequences or protein folds can use similar chemistry to carry out related functions or display the same catalytic activity (e.g., refs. 1-3). These considerations further complicate attempts to clearly identify and understand the key structural features of proteins. Thus, the isolation of biologically active proteins with drastically reduced chemical complexity would represent a significant advance in protein science by facilitating the correlation of specific structural features with function.Up to 30% of all proteins contain ∼20-25-amino acid, primarily hydrophobic segments that span cell membranes (4). These transmembrane domains often play critical roles in cellular processes by engaging in highly specific protein-protein and protein-lipid interactions required for the proper folding, oligomerization, and function of transmembrane proteins (5-8). For example, receptor tyrosine kinases (RTKs) such as the PDGF-β receptor (PDGFβR) usually consist of an extracellular ligand binding do...

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Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein

Cited by 25 publications

References 85 publications

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

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

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

Biologically active LIL proteins built with minimal chemical diversity

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