The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival "holding" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.
- MeSH
- antigeny CD279 imunologie MeSH
- jednořetězcové protilátky * imunologie genetika MeSH
- lidé MeSH
- monoklonální protilátky imunologie genetika MeSH
- nádory imunologie terapie MeSH
- peptidová knihovna MeSH
- psi MeSH
- rekombinantní proteiny imunologie genetika MeSH
- translační biomedicínský výzkum MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
TP73 is a member of the TP53 gene family and produces N- and C-terminal protein isoforms through alternative promoters, alternative translation initiation and alternative splicing. Most notably, p73 protein isoforms may either contain a p53-like transactivation domain (TAp73 isoforms) or lack this domain (ΔTAp73 isoforms) and these variants have opposing or independent functions. To date, there is a lack of well-characterised isoform-specific p73 antibodies. Here, we produced polyclonal and monoclonal antibodies to N-terminal p73 variants and the C-terminal p73α isoform, the most common variant in human tissues. These reagents show that TAp73 is a marker of multiciliated epithelial cells, while ΔTAp73 is a marker of non-proliferative basal/reserve cells in squamous epithelium. We were unable to detect ΔNp73 variant proteins, in keeping with recent data that this is a minor form in human tissues. Most cervical squamous cell carcinomas (79%) express p73α, and the distribution of staining in basal cells correlated with lower tumour grade. TAp73 was found in 17% of these tumours, with a random distribution and no association with clinicopathological features. These data indicate roles for ΔTAp73 in maintaining a non-proliferative state of undifferentiated squamous epithelial cells and for TAp73 in the production of differentiated multiciliated cells.
- MeSH
- epitelové buňky metabolismus MeSH
- lidé MeSH
- monoklonální protilátky MeSH
- nádorové buněčné linie MeSH
- nádory děložního čípku metabolismus patologie genetika MeSH
- nádory metabolismus patologie genetika MeSH
- protein - isoformy * metabolismus genetika MeSH
- protein p73 * metabolismus genetika MeSH
- spinocelulární karcinom metabolismus patologie genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Monoclonal antibodies targeting immune checkpoints have revolutionized oncology. Yet, the effectiveness of these treatments varies significantly among patients, and they are associated with unexpected adverse events, including hyperprogression. The murine research model used in drug development fails to recapitulate both the functional human immune system and the population heterogeneity. Hence, a novel model is urgently needed to study the consequences of immune checkpoint blockade. Dogs appear to be uniquely suited for this role. Approximately 1 in 4 companion dogs dies from cancer, yet no antibodies are commercially available for use in veterinary oncology. Here we characterize two novel antibodies that bind canine PD-1 with sub-nanomolar affinity as measured by SPR. Both antibodies block the clinically crucial PD-1/PD-L1 interaction in a competitive ELISA assay. Additionally, the antibodies were tested with a broad range of assays including Western Blot, ELISA, flow cytometry, immunofluorescence and immunohistochemistry. The antibodies appear to bind two distinct epitopes as predicted by molecular modeling and peptide phage display. Our study provides new tools for canine oncology research and a potential veterinary therapeutic.
- MeSH
- antigeny CD274 imunologie antagonisté a inhibitory metabolismus MeSH
- antigeny CD279 * imunologie antagonisté a inhibitory metabolismus MeSH
- epitopy imunologie MeSH
- inhibitory kontrolních bodů imunologie farmakologie MeSH
- lidé MeSH
- monoklonální protilátky * imunologie MeSH
- nádory imunologie veterinární farmakoterapie MeSH
- nemoci psů imunologie farmakoterapie MeSH
- psi MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.
- MeSH
- antivirové látky farmakologie chemie metabolismus MeSH
- diferenciační antigeny * metabolismus chemie MeSH
- konformace proteinů * MeSH
- lidé MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus biosyntéza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
HDMX and its homologue HDM2 are two essential proteins for the cell; after genotoxic stress, both are phosphorylated near to their RING domain, specifically at serine 403 and 395, respectively. Once phosphorylated, both can bind the p53 mRNA and enhance its translation; however, both recognize p53 protein and provoke its degradation under normal conditions. HDM2 has been well-recognized as an E3 ubiquitin ligase, whereas it has been reported that even with the high similarity between the RING domains of the two homologs, HDMX does not have the E3 ligase activity. Despite this, HDMX is needed for the proper p53 poly-ubiquitination. Phosphorylation at serine 395 changes the conformation of HDM2, helping to explain the switch in its activity, but no information on HDMX has been published. Here, we study the conformation of HDMX and its phospho-mimetic mutant S403D, investigate its E3 ligase activity and dissect its binding with p53. We show that phospho-mutation does not change the conformation of the protein, but HDMX is indeed an E3 ubiquitin ligase in vitro; however, in vivo, no activity was found. We speculated that HDMX is regulated by induced fit, being able to switch activity accordingly to the specific partner as p53 protein, p53 mRNA or HDM2. Our results aim to contribute to the elucidation of the contribution of the HDMX to p53 regulation.
- MeSH
- jaderné proteiny genetika MeSH
- messenger RNA metabolismus MeSH
- nádorový supresorový protein p53 * genetika metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- protoonkogenní proteiny c-mdm2 * genetika metabolismus MeSH
- protoonkogenní proteiny genetika MeSH
- serin metabolismus MeSH
- ubikvitin genetika MeSH
- ubikvitinace MeSH
- ubikvitinligasy genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The TP63 gene encodes two major protein variants; TAp63 contains a p53-like transcription domain and consequently has tumor suppressor activities whereas ΔNp63 lacks this domain and acts as an oncogene. The two variants show distinct expression patterns in normal tissues and tumors, with lymphocytes and lymphomas/leukemias expressing TAp63, and basal epithelial cells and some carcinomas expressing high levels of ΔNp63, most notably squamous cell carcinomas (SCC). Whilst the transcriptional functions of TAp63 and ΔNp63 isoforms are known, the mechanisms involved in their regulation are poorly understood. Using squamous epithelial cells that contain high levels of ΔNp63 and low/undetectable TAp63, the DNA demethylating agent decitabine (5-aza-2'-deoxycytidine, 5-dAza) caused a dose-dependent increase in TAp63, with a simultaneous reduction in ΔNp63, indicating DNA methylation-dependent regulation at the isoform-specific promoters. The basal cytokeratin KRT5, a direct ΔNp63 transcriptional target, was also reduced, confirming functional alteration of p63 activity after DNA demethylation. We also showed high level methylation of three CpG sites in the TAP63 promoter in these cells, which was reduced by decitabine. DNMT1 depletion using inducible shRNAs partially replicated these effects, including an increase in the ratio of TAP63:ΔNP63 mRNAs, a reduction in ΔNp63 protein and reduced KRT5 mRNA levels. Finally, high DNA methylation levels were found at the TAP63 promoter in clinical SCC samples and matched normal tissues. We conclude that DNA methylation at the TAP63 promoter normally silences transcription in squamous epithelial cells, indicating DNA methylation as a therapeutic approach to induce this tumor suppressor in cancer. That decitabine simultaneously reduced the oncogenic activity of ΔNp63 provides a "double whammy" for SCC and other p63-positive carcinomas. Whilst a variety of mechanisms may be involved in producing the opposite effects of DNA demethylation on TAp63 and ΔNp63, we propose an "either or" mechanism in which TAP63 transcription physically interferes with the ability to initiate transcription from the downstream ΔNP63 promoter on the same DNA strand. This mechanism can explain the observed inverse expression of p63 isoforms in normal cells and cancer.
- Publikační typ
- časopisecké články MeSH
The p53 family member p63 exists as two major protein variants (TAp63 and ΔNp63) with distinct expression patterns and functional properties. Whilst downstream target genes of p63 have been studied intensively, how p63 variants are themselves controlled has been relatively neglected. Here, we review advances in understanding ΔNp63 and TAp63 regulation, highlighting their distinct pathways. TAp63 has roles in senescence and metabolism, and in germ cell genome maintenance, where it is activated post-transcriptionally by phosphorylation cascades after DNA damage. The function and regulation of TAp63 in mesenchymal and haematopoietic cells is less clear but may involve epigenetic control through DNA methylation. ΔNp63 functions to maintain stem/progenitor cells in various epithelia and is overexpressed in squamous and certain other cancers. ΔNp63 is transcriptionally regulated through multiple enhancers in concert with chromatin modifying proteins. Many signalling pathways including growth factors, morphogens, inflammation, and the extracellular matrix influence ΔNp63 levels, with inconsistent results reported. There is also evidence for reciprocal regulation, including ΔNp63 activating its own transcription. ΔNp63 is downregulated during cell differentiation through transcriptional regulation, while post-transcriptional events cause proteasomal degradation. Throughout the review, we identify knowledge gaps and highlight discordances, providing potential explanations including cell-context and cell-matrix interactions. Identifying individual p63 variants has roles in differential diagnosis and prognosis, and understanding their regulation suggests clinically approved agents for targeting p63 that may be useful combination therapies for selected cancer patients. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
- MeSH
- lidé MeSH
- nádorové supresorové proteiny * MeSH
- nádory * MeSH
- protein - isoformy MeSH
- transkripční faktory * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
