Biology subjects

Dayer, G.

Publications and source records attributed to Dayer, G..

3 recordsLinked to original sources

bioRxiv2022

Targeting the Human Papillomavirus 16 E6 Oncoprotein with Antibodies

The human papillomavirus (HPV) 16 genome encodes two oncoproteins, E6 and E7, which are essential for viral carcinogenesis. While E7 promotes cell proliferation, E6 abolishes the resulting p53-dependent apoptotic response. Due to this specific function, E6 is considered a suitable target for the development of a variety of therapeutic agents such as antibodies. Here, we review anti-E6 antibodies/antibody fragments generated by us and others, as well as present our latest results with Camelidae-derived single-domain antibodies (sdAbs). We had previously isolated a pool of anti-E6 sdAbs to identify E6 binders with the potential to be used clinically and in research. While our previous work has focused on recombinant E6 proteins, here we evaluated these sdAbs binding capacity to the endogenous E6 protein using co-immunoprecipitation and immunofluorescence. We obtained reproducible results in these applications with two sdAbs, filling a gap in HPV research. Despite their apparent E6 binding ability, these sdAbs do not raise p53 levels or induce apoptosis. Thus, while these reagents are valuable diagnostic and detection tools, identifying their therapeutic potential will require further development and testing.

cancer biology↗

bioRxiv2020

Virus-host protein-protein interactions between human papillomavirus 16 E6 A1 and D2/D3 sub-lineages: variances and similarities

High-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers with type 16 being the most frequent. Compared to the European Prototype (A1, denoted "EP"), the Asian-American (D2/D3, denoted "AA") sub-lineage or "variant" is reported to have increased abilities to promote carcinogenesis. Few global interactome studies have looked at protein-protein interactions (PPIs) between host proteins and variants of the key transforming E6 protein. We applied a primary human foreskin keratinocyte model transduced with EP and AA variant E6 genes and co-immunoprecipitated expressed E6 proteins along with interacting cellular proteins to detect virus-host binding partners. We reasoned that, due to single nucleotide polymorphisms, AAE6 and EPE6 may have unique PPIs with host cellular proteins--conferring gain or loss of function--resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to each of AAE6 and EPE6, along with 13 E6 targets common to both AAE6 and EPE6. We also applied a more inclusive process based on pathway selection and discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets between the two sub-lineages. Interpretations for both approaches were made using databases such as UniProt, BioGRID and Reactome. Detected E6 targets are implicated in important hallmarks of cancer: deregulating Notch and other signaling, energetics and hypoxia, DNA replication and repair, and immune response. Validation experiments, such as reverse co-immunoprecipitation and RNA interference, are required to substantiate these findings. Here, we provide an unprecedented resource for new research questions in HR HPV biology. The current data also underline our labs driving hypothesis that E6, being a "master regulator" in HPV-positive cancers, is an excellent candidate for anti-cancer treatment strategies. Author SummaryChronic infection with high-risk human papillomavirus (HPV) type 16 is the most prevalent cause of cervical and other mucosal cancers. The E6 oncoproteins of the European Prototype (EP) and the Asian-American (AA) HPV variants differentially promote carcinogenesis. We looked at protein-protein interactions between host proteins and two key HPV variant E6 proteins of these strains to reveal how high risk HPVs cause cancer, based on the proteins they bind to in infected cells. Our methodology combined molecular biology and data mining techniques using widely available databases. We confirmed and discovered novel virus-host associations that explained how HPV AA and EP variants differ in their carcinogenic capabilities, and confirmed the candidacy of the E6 protein as a viable target for HPV therapies.

molecular biology↗

bioRxiv2018

Single-domain antibodies represent novel alternatives to monoclonal antibodies as targeting agents against the human papillomavirus 16 E6 protein

Approximately one-fifth of all malignancies worldwide are etiologically-associated with a persistent viral or bacterial infection. Thus, there is particular interest in therapeutic molecules which utilize components of a natural immune response to specifically inhibit oncogenic microbial proteins, as it is anticipated they will elicit fewer off-target effects than conventional treatments. This concept has been explored in the context of human papillomavirus type 16 (HPV16)-related cancers, through the development of monoclonal antibodies and fragments thereof against the viral E6 oncoprotein. However, challenges related to the biology of E6 as well as the functional properties of the antibodies themselves appear to have precluded their clinical translation. In this study, we attempted to address these issues by exploring the utility of the variable domains of camelid heavy-chain-only antibodies (denoted as VHHs). Through the construction and panning of two llama immune VHH phage display libraries, a pool of potential VHHs was isolated. The interactions of these VHHs with recombinant E6 protein were further characterized using ELISA, Western blotting under both denaturing and native conditions, as well as surface plasmon resonance, and three antibodies were identified that bound recombinant E6 with affinities in the nanomolar range. Our results now lead the way for subsequent studies into the ability of these novel molecules to inhibit HPV16-infected cells in vitro and in vivo.

cancer biology↗