Activation of the PDGF ß Receptor by a Persistent Artificial Signal Peptide.

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF ß receptor (PDGFßR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFßR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFßR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFßR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFßR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFßR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.

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.

Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF ß receptor in an active dimeric conformation.

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor ß receptor (PDGFßR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFßR complex. In this model, an active dimer of the PDGFßR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFßR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFßR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.

Biologically active LIL proteins built with minimal chemical diversity.

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 ß-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.

Detection and quantification of bovine papillomavirus type 2 in urinary bladders and lymph nodes in cases of Bovine Enzootic Hematuria from the endemic region of Azores.

Bovine Enzootic Hematuria (BEH) is a disease with a severe impact on production indexes and characterized by the development of bovine urinary bladder tumors, particularly in the Azores archipelago. The purpose of this study was to investigate and quantify BPV2 tissue distribution in bovine urinary bladder tumors, normal bladders, and iliac lymph nodes of cattle from the Azores. A real-time PCR system targeting the L1 gene was developed and allowed for the specific detection of the virus. BPV2 DNA was detected in a large proportion of the samples tested, both from neoplastic and healthy tissues, indicating that this virus is very prevalent in the bovine population of the Azores. Moreover, all types of tissues tested were positive, confirming a wide viral distribution within the infected animal. Bovine cutaneous papillomas sampled from Portuguese mainland dairy cattle were used as controls. Viral load ranged between 2.2×10(4) copies/cell in the skin papillomas, and 0.0002 copies/cell in the urinary bladders tumors from the Azores. This is the first report presenting quantitative data on BPV2 infection in bovine urinary bladder lesions from the Azores. This approach will provide a useful tool to evaluate the role of BPV2 not only in the pathogenesis BEH but also in cell transformation mechanisms.

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.

A single amino acid substitution converts a transmembrane protein activator of the platelet-derived growth factor ß receptor into an inhibitor.

Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFß receptor (PDGFßR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFßR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFßR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFßR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFßR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFßR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFßR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFßR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFßR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins.

The E5 proteins.

The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 protein is the PDGF ß receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis.

Transforming signals resulting from sustained activation of the PDGFbeta receptor in mortal human fibroblasts.

The platelet-derived growth factor beta receptor (PDGFbetaR) plays an important role in proliferation and motility of fibroblasts. We have been investigating the effects of sustained PDGFbetaR activation in mortal human diploid fibroblasts (HDFs), which are typically difficult to transform. We have previously shown that the bovine papillomavirus E5 protein, through its ability to crosslink and constitutively activate the PDGFbetaR, induces morphological transformation, enhanced growth and loss of contact inhibition (focus formation) in HDFs. Here, we characterized two E5 mutants as being severely defective for focus formation but still competent for enhanced growth, suggesting that proliferation is insufficient for loss of contact inhibition. These E5 mutants were then used in a comparative study to distinguish the PDGFbetaR signaling intermediates required for the enhanced growth phenotype from those required for focus formation. Our data suggested that a PI 3-kinase (PI3K)-AKT-cyclin D3 pathway, a Grb2-Gab1-SHP2 complex and JNK played a role in the enhanced growth phenotype. However, a SHP2-p66Shc-p190BRhoGAP complex and ROCK were implicated exclusively in focus formation. We speculate that a SHP2-p66Shc-p190BRhoGAP signaling complex recruited to the activated PDGFbetaR promotes a distinct Rho-dependent process required for focus formation but not growth of HDFs.

Oxidative and calcium stress regulate DSCR1 (Adapt78/MCIP1) protein.

DSCR1 (adapt78) is a stress-inducible gene and cytoprotectant. Its protein product, DSCR1 (Adapt78), also referred to as MCIP1, inhibits intracellular calcineurin, a phosphatase that mediates many cellular responses to calcium. Exposure of human U251 and HeLa cells to hydrogen peroxide led to a rapid hyperphosphorylation of DSCR1 (Adapt78). Inhibitor and agonist studies revealed that a broad range of kinases were not responsible for DSCR1 (Adapt78) hyperphosphorylation, including ERK1/2, although parallel activation of the latter was observed. Phosphorylation of both DSCR1 (Adapt78) and ERK1/2 was attenuated by inhibitors of tyrosine phosphatase, suggesting the common upstream involvement of tyrosine dephosphorylation. The hyperphosphorylation electrophoretic shift in DSCR1 (Adapt78) mobility was also observed with other oxidizing agents (peroxynitrite and menadione) but not nonoxidants. Calcium ionophores strongly induced the levels of both hypo- and hyper-phosphorylated DSCR1 (Adapt78) but did not alter phosphorylation status. Calcium-dependent growth factor- and angiotensin II-stimulation also induced both DSCR1 (Adapt78) species. Phosphorylation of either or both serines in a 13-amino acid peptide made to a calcineurin-interacting conserved region of DSCR1 (Adapt78) attenuated inhibition of calcineurin. These data indicate that DSCR1 (Adapt78) protein is a novel, early stage oxidative stress-activated phosphorylation target and newly identified calcium-inducible protein, and suggest that these response mechanisms may contribute to the known cytoprotective and calcineurin-inhibitory activities of DSCR1 (Adapt78).

Apoptosis of mortal human fibroblasts transformed by the bovine papillomavirus E5 oncoprotein.

Mortal human fibroblasts can be partially transformed by the bovine papillomavirus E5 oncoprotein through activation of the platelet-derived growth factor beta receptor. Here, we report that these cells undergo massive apoptosis 2 weeks after confluence. Although activation of caspase 3 was observed in the apoptotic cells, it was not required for apoptosis. The appearance of the mitochondrial proteins cytochrome c and apoptosis-inducing factor in cytosolic and nuclear compartments, respectively, provided a basis for mitochondrial dysfunction and a caspase-independent mechanism of apoptosis in these cells. Although an activating conformational change in Bax also was evident in the apoptotic cells, enforced overexpression of Bcl-2 was insufficient to prevent apoptosis. Finally, a small peptide present in the conditioned medium from dying transformed cells appeared responsible for inducing apoptosis through affecting a conformational change in Bax and eventual relocalization of apoptosis-inducing factor to the nucleus. Thus, an atypical apoptotic pathway is activated in mortal human fibroblasts in response to transformation induced by sustained receptor tyrosine kinase activation.

Molecular examination of the transmembrane requirements of the platelet-derived growth factor beta receptor for a productive interaction with the bovine papillomavirus E5 oncoprotein.

The small transmembrane E5 protein of bovine papillomavirus (BPV) transforms cells by forming a stable complex with and activating the platelet-derived growth factor beta receptor (PDGFbetaR). The E5/PDGFbetaR interaction is thought to involve specific physical contacts between the transmembrane domains of the two proteins. Lys(499) at the extracellular juxtamembrane position and Thr(513) within the transmembrane domain of the PDGFbetaR are required for the interaction and are predicted to contact analogously positioned residues in the E5 protein. Here, mutagenic analysis of the transmembrane region of the PDGFbetaR was performed to further characterize the nature of the E5/PDGFbetaR interaction. We show that the receptor transmembrane domain, with minimal extracellular and intracellular sequence, is sufficient for the interaction. In addition, we provide evidence that the polar nature of Thr(513) as well as its positioning along the transmembrane alpha-helix is important for the interaction. We also identify the receptor transmembrane amino acids Ile(506) and Leu(520) as additional requirements for the interaction. Because Lys(499), Thr(513), Ile(506), and Leu(520) all align along the same face of the predicted PDGFbetaR transmembrane alpha-helix, our data support the model that the PDGFbetaR contacts the E5 protein via multiple amino acids along a single alpha-helical interface.

Multiple transmembrane amino acid requirements suggest a highly specific interaction between the bovine papillomavirus E5 oncoprotein and the platelet-derived growth factor beta receptor.

The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor beta receptor (PDGFbetaR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFbetaR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFbetaR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFbetaR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFbetaR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.