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.

• Keywords
• Cervical cancer, Endometrium, Fallopian tube, Multiciliated cells, Squamous epithelial stem cells, p73 isoforms,
• MeSH
• Epithelial Cells metabolism   MeSH
• Humans MeSH
• Antibodies, Monoclonal MeSH
• Cell Line, Tumor MeSH
• Uterine Cervical Neoplasms metabolism   pathology   genetics   MeSH
• Neoplasms metabolism   pathology   genetics   MeSH
• Protein Isoforms * metabolism   genetics   MeSH
• Tumor Protein p73 * metabolism   genetics   MeSH
• Carcinoma, Squamous Cell metabolism   pathology   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• delta Np73 protein, human MeSH Browser
• Antibodies, Monoclonal MeSH
• Protein Isoforms * MeSH
• Tumor Protein p73 * MeSH
• TP73 protein, human MeSH Browser

The p53 family of proteins evolved from a common ancestor into three separate genes encoding proteins that act as transcription factors with distinct cellular roles. Isoforms of each member that lack specific regions or domains are suggested to result from alternative transcription start sites, alternative splicing or alternative translation initiation, and have the potential to exponentially increase the functional repertoire of each gene. However, evidence supporting the presence of individual protein variants at functional levels is often limited and is inferred by mRNA detection using highly sensitive amplification techniques. We provide a critical appraisal of the current evidence for the origins, expression, functions and regulation of p53-family isoforms. We conclude that despite the wealth of publications, several putative isoforms remain poorly established. Future research with improved technical approaches and the generation of isoform-specific protein detection reagents is required to establish the physiological relevance of p53-family isoforms in health and disease. In addition, our analyses suggest that p53-family variants evolved partly through convergent rather than divergent evolution from the ancestral gene.

• MeSH
• Alternative Splicing * MeSH
• Humans MeSH
• RNA, Messenger metabolism   genetics   MeSH
• Evolution, Molecular MeSH
• Tumor Suppressor Protein p53 * metabolism   genetics   MeSH
• Transcription Initiation Site MeSH
• Protein Isoforms * genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 * MeSH
• Protein Isoforms * MeSH

In the last decade, significant advances have been made towards the rational design of proteins, DNA, and other organic nanostructures. The emerging possibility to precisely engineer molecular structures resulted in a wide range of new applications in fields such as biotechnology or medicine. The complexity and size of the artificial molecular systems as well as the number of interactions are greatly increasing and are manifesting the need for computational design support. In addition, a new generation of AI-based structure prediction tools provides researchers with completely new possibilities to generate recombinant proteins and functionalized DNA nanostructures. In this study, we present Catana, a web-based modelling environment suited for proteins and DNA nanostructures. User-friendly features were developed to create and modify recombinant fusion proteins, predict protein structures based on the amino acid sequence, and manipulate DNA origami structures. Moreover, Catana was jointly developed with the novel Unified Nanotechnology Format (UNF). Therefore, it employs a state-of-the-art coarse-grained data model, that is compatible with other established and upcoming applications. A particular focus was put on an effortless data export to allow even inexperienced users to perform in silico evaluations of their designs by means of molecular dynamics simulations. Catana is freely available at http://catana.ait.ac.at/.

• MeSH
• DNA chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Nanostructures * chemistry   MeSH
• Nanotechnology methods   MeSH
• Nucleic Acids * MeSH
• Recombinant Fusion Proteins MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Nucleic Acids * MeSH
• Recombinant Fusion Proteins MeSH

The p53 and Mouse double minute 2 (MDM2) proteins are hubs in extensive networks of interactions with multiple partners and functions. Intrinsically disordered regions help to adopt function-specific structural conformations in response to ligand binding and post-translational modifications. Different techniques have been used to dissect interactions of the p53-MDM2 pathway, in vitro, in vivo, and in situ each having its own advantages and disadvantages. This review uses the p53-MDM2 to show how different techniques can be employed, illustrating how a combination of in vitro and in vivo techniques is highly recommended to study the spatio-temporal location and dynamics of interactions, and to address their regulation mechanisms and functions. By using well-established techniques in combination with more recent advances, it is possible to rapidly decipher complex mechanisms, such as the p53 regulatory pathway, and to demonstrate how protein and nucleotide ligands in combination with post-translational modifications, result in inter-allosteric and intra-allosteric interactions that govern the activity of the protein complexes and their specific roles in oncogenesis. This promotes elegant therapeutic strategies that exploit protein dynamics to target specific interactions.

• Keywords
• ATM *, DNA damage response *, MDM2 *, MDMX *, p53 *, p53 mRNA *, post-translational modification *, protein-RNA interactions *, protein-protein interactions *,
• MeSH
• Phosphorylation genetics   MeSH
• Nuclear Proteins MeSH
• Humans MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• DNA Damage genetics   MeSH
• Protein Processing, Post-Translational genetics   MeSH
• Cell Cycle Proteins genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   MeSH
• Protein Binding genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Nuclear Proteins MeSH
• MDM2 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH

BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells. METHODS: We generated a non-shed CA IX mutant by deletion of amino acids 393-402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. CONCLUSIONS: Our results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.

• MeSH
• Phenotype MeSH
• Neoplasm Invasiveness pathology   MeSH
• Carbonic Anhydrase IX metabolism   MeSH
• Carcinogenesis metabolism   pathology   MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Neoplasms metabolism   pathology   MeSH
• ADAM17 Protein metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Carbonic Anhydrase IX MeSH
• ADAM17 Protein MeSH

Interleukin-1α (IL-1α) is a dual-function proinflammatory mediator. In addition to its role in the canonical IL-1 signaling pathway, which employs membrane-bound receptors, a growing body of evidence shows that IL-1α has some additional intracellular functions. We identified the interaction of IL-1α with the tumor suppressor p53 in the nuclei and cytoplasm of both malignant and noncancerous mammalian cell lines using immunoprecipitation and the in situ proximity ligation assay (PLA). This interaction was enhanced by treatment with the antineoplastic drug etoposide, which suggests a role for the IL-1α•p53 interaction in genotoxic stress.

• MeSH
• Cytoplasm metabolism   MeSH
• DNA Breaks, Double-Stranded MeSH
• Microscopy, Fluorescence MeSH
• HeLa Cells MeSH
• Immunoprecipitation MeSH
• Interleukin-1alpha genetics   metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• DNA Damage genetics   physiology   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interleukin-1alpha MeSH
• Tumor Suppressor Protein p53 MeSH

Structured RNA regulatory motifs exist from the prebiotic stages of the RNA world to the more complex eukaryotic systems. In cases where a functional RNA structure is within the coding sequence a selective pressure drives a parallel co-evolution of the RNA structure and the encoded peptide domain. The p53-MDM2 axis, describing the interactions between the p53 tumor suppressor and the MDM2 E3 ubiquitin ligase, serves as particularly useful model revealing how secondary RNA structures have co-evolved along with corresponding interacting protein motifs, thus having an impact on protein - RNA and protein - protein interactions; and how such structures developed signal-dependent regulation in mammalian systems. The p53(BOX-I) RNA sequence binds the C-terminus of MDM2 and controls p53 synthesis while the encoded peptide domain binds MDM2 and controls p53 degradation. The BOX-I peptide domain is also located within p53 transcription activation domain. The folding of the p53 mRNA structure has evolved from temperature-regulated in pre-vertebrates to an ATM kinase signal-dependent pathway in mammalian cells. The protein - protein interaction evolved in vertebrates and became regulated by the same signaling pathway. At the same time the protein - RNA and protein - protein interactions evolved, the p53 trans-activation domain progressed to become integrated into a range of cellular pathways. We discuss how a single synonymous mutation in the BOX-1, the p53(L22 L), observed in a chronic lymphocyte leukaemia patient, prevents the activation of p53 following DNA damage. The concepts analysed and discussed in this review may serve as a conceptual mechanistic paradigm of the co-evolution and function of molecules having roles in cellular regulation, or the aetiology of genetic diseases and how synonymous mutations can affect the encoded protein.

• Keywords
• Ciona intestinalis, Intrinsically disordered proteins, Molecular basis of disease, Protein-RNA interactions, RNA world, Transcription factor, mRNA translation,
• MeSH
• Genetic Predisposition to Disease MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Tumor Suppressor Proteins genetics   metabolism   MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Neoplasms genetics   metabolism   pathology   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• Gene Expression Profiling MeSH
• Transcriptome MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH
• Tumor Suppressor Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• RNA-Binding Proteins MeSH

p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

• Keywords
• ATM kinase, MDM2, cell signaling, intrinsically disordered proteins, p53 messenger RNA, synonymous mutations,
• MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• A549 Cells MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Phosphorylation genetics   physiology   MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Mutation genetics   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Polyribosomes metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   metabolism   MeSH
• Protein Stability MeSH
• Intrinsically Disordered Proteins genetics   metabolism   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ataxia Telangiectasia Mutated Proteins MeSH
• RNA, Small Interfering MeSH
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• TP53 protein, human MeSH Browser
• Intrinsically Disordered Proteins MeSH

Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.

• Keywords
• EBNA1, Epstein-Barr virus (EBV), G-quadruplexes, PhenDC3, protein-mRNA interactions, pyridostatin, structure-activity relationship,
• MeSH
• Aminoquinolines chemistry   MeSH
• Biological Assay methods   MeSH
• Phosphoproteins metabolism   MeSH
• G-Quadruplexes * MeSH
• Picolinic Acids chemistry   MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Cell Line, Tumor MeSH
• Nucleolin MeSH
• RNA-Binding Proteins metabolism   MeSH
• Epstein-Barr Virus Nuclear Antigens genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aminoquinolines MeSH
• EBV-encoded nuclear antigen 1 MeSH Browser
• Phosphoproteins MeSH
• Picolinic Acids MeSH
• RNA, Messenger MeSH
• RNA-Binding Proteins MeSH
• pyridostatin MeSH Browser
• Epstein-Barr Virus Nuclear Antigens MeSH

BACKGROUND: Increased levels of the pathogenic amyloid β-peptide (Aβ), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aβ in neurons. METHODS: MAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aβ production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aβ42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis. RESULTS: Immunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aβ42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aβ42. Furthermore, overexpression of MAO-B enhanced Aβ production. CONCLUSIONS: This study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aβ production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aβ levels using protein-protein interaction breakers.

• Keywords
• Alzheimer disease, Alzheimer disease pathogenesis, Amyloid β-peptide, Intraneuronal amyloid β-peptide, Monoamine oxidase B, γ-Secretase,
• MeSH
• Alzheimer Disease metabolism   pathology   MeSH
• Amyloid beta-Peptides metabolism   MeSH
• Axons metabolism   MeSH
• Dendrites metabolism   MeSH
• Rats MeSH
• Middle Aged MeSH
• Humans MeSH
• RNA, Small Interfering genetics   metabolism   MeSH
• Models, Molecular MeSH
• Monoamine Oxidase genetics   metabolism   MeSH
• Brain metabolism   pathology   MeSH
• Mice MeSH
• Neurons metabolism   ultrastructure   MeSH
• Presenilin-1 genetics   MeSH
• Receptors, N-Methyl-D-Aspartate metabolism   MeSH
• Gene Expression Regulation genetics   MeSH
• Amyloid Precursor Protein Secretases metabolism   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Synapses metabolism   MeSH
• Transfection MeSH
• Cell Line, Transformed MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Mice MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amyloid beta-Peptides MeSH
• RNA, Small Interfering MeSH
• Monoamine Oxidase MeSH
• NR2B NMDA receptor MeSH Browser
• Presenilin-1 MeSH
• Receptors, N-Methyl-D-Aspartate MeSH
• Amyloid Precursor Protein Secretases MeSH