ΔNp63 transcriptionally regulates ATM to control p53 Serine-15 phosphorylation

Background: Delta Np63 alpha is an epithelial progenitor cell marker that maintains epidermal stem cell self-renewal capacity. Previous studies revealed that UV-damage induced p53 phosphorylation is confined to Delta Np63 alpha-positive cells in the basal layer of human epithelium. Results: We now report that phosphorylation of the p53 tumour suppressor is positively regulated by Delta Np63 alpha in immortalised human keratinocytes. Delta Np63 alpha depletion by RNAi reduces steady-state ATM mRNA and protein levels, and attenuates p53 Serine-15 phosphorylation. Conversely, ectopic expression of Delta Np63 alpha in p63-null tumour cells stimulates ATM transcription and p53 Serine-15 phosphorylation. We show that ATM is a direct Delta Np63 alpha transcriptional target and that the Delta Np63 alpha response element localizes to the ATM promoter CCAAT sequence. Structure-function analysis revealed that the Delta Np63-specific TA2 transactivation domain mediates ATM transcription in coordination with the DNA binding and SAM domains. Conclusions: Germline p63 point mutations are associated with a range of ectodermal developmental disorders, and targeted p63 deletion in the skin causes premature ageing. The Delta Np63 alpha-ATM-p53 damage-response pathway may therefore function in epithelial development, carcinogenesis and the ageing processes

Dual detection system for cancer-associated point mutations assisted by a multiplexed LNA-based amperometric bioplatform coupled with rolling circle amplification

DNA point mutation in a BRAF proto-oncogene, V600E, is considered an important prognostic and predictive biomarker in various types of cancer, such as melanoma or colorectal cancer. We report here a novel electrochemical (EC) bioplatform for the analysis of BRAF V600E mutation coupled with rolling circle amplification (RCA) and locked nucleic acid (LNA) capture probes. A dual detection system was implemented, whereby two padlock probes complementary to either wild-type (wt) BRAF gene or DNA with V600E mutation (mut) led to amplification of wt or mut variant, respectively. Hybridization with specific LNA capture probes then increased the assay specificity, while EC detection provided rapid measurement times. The bioplatform was applied to analyze BRAF V600E mutation of cancer cells and tumor tissues from patients with melanoma or colorectal cancer. This is the first RCA-based EC bioplatform for BRAF analysis in a dual format without using PCR or sophisticated instrumentation.The financial support of the Czech Health Research Council (No. NU21-08-00078), National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU, BBMRI.cz (No. LM2023033), MH CZ - DRO (MMCI, 00209805), PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovacion), ´ TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (S2018/NMT-4349) and PI20CIII/00019 Grants from the AES-ISCIII Program co-founded by FEDER funds, are gratefully acknowledged. A. Valverde acknowledges a predoctoral contract from Complutense University of Madrid. Authors would like to thank Nina Libova for her technical support.S

Comparison of metal nanoparticles (Au, Ag, Eu, Cd) used for immunoanalysis using LA-ICP-MS detection

Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1-14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein

Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation

International audienceAnterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses

Identification of AGR2 binding partners by protein-protein interaction screen.

AGR2 protein is known to be overexpressed in many human cancers, and its expression levels can be used to predict patient outcome. This protein inhibits activity of tumor suppressor p53, mediates cell migration, cellular transformation, angiogenesis and metastasis development. Biochemically, it is an endoplasmic reticulum localized molecular chaperone, classified into protein disulfide isomerase family, responsible for the folding and assembling of client proteins. Two different mass spectrometry approaches LC-MS/MS analysis with label-free quantification and LC-MS/MS analysis with SILAC quantitation were used to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 is in particular associated with processes situated into the endoplasmic reticulum to maintain intracellular metabolic homeostasis and in response to endoplasmic reticulum stress AGR2 participates in unfolded protein response, changes in cellular metabolic, energy and redox state. Taken together, our study provides a comprehensive insight into the protein-protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells. This work was supported by the project BBMRI.cz (LM2023033), MH CZ - DRO (MMCI, 00209805) and Programme EXCELES (LX22NPO5102)

Impaired Pre-mRNA Processing and Altered Architecture of 3' Untranslated Regions Contribute to the Development of Human Disorders

The biological fate of each mRNA and consequently, the protein to be synthesised, is highly dependent on the nature of the 3' untranslated region. Despite its non-coding character, the 3' UTR may affect the final mRNA stability, the localisation, the export from the nucleus and the translation efficiency. The conserved regulatory sequences within 3' UTRs and the specific elements binding to them enable gene expression control at the posttranscriptional level and all these processes reflect the actual state of the cell including proliferation, differentiation, cellular stress or tumourigenesis. Through this article, we briefly outline how the alterations in the establishment and final architecture of 3' UTRs may contribute to the development of various disorders in humans