The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.
- Publication type
- Journal Article MeSH
Radiation and chemotherapy represent standard-of-care cancer treatments. However, most patients eventually experience tumour recurrence, treatment failure and metastatic dissemination with fatal consequences. To elucidate the molecular mechanisms of resistance to radio- and chemotherapy, we exposed human cancer cell lines (HeLa, MCF-7 and DU145) to clinically relevant doses of 5-azacytidine or ionizing radiation and compared the transcript profiles of all surviving cell subpopulations, including low-adherent stem-like cells. Stress-mobilized low-adherent cell fractions differed from other survivors in terms of deregulation of hundreds of genes, including those involved in interferon response. Exposure of cancer cells to interferon-gamma but not interferon-beta resulted in the development of a heterogeneous, low-adherent fraction comprising not only apoptotic/necrotic cells but also live cells exhibiting active Notch signalling and expressing stem-cell markers. Chemical inhibition of mitogen-activated protein kinase/ERK kinase (MEK) or siRNA-mediated knockdown of extracellular signal-regulated kinase 1/2 (Erk1/2) and interferon responsible factor 1 (IRF1) prevented mobilization of the surviving low-adherent population, indicating that interferon-gamma-mediated loss of adhesion and anoikis resistance required an active Erk pathway interlinked with interferon signalling by transcription factor IRF1. Notably, a skin-specific protein suprabasin (SBSN), a recently identified oncoprotein, was among the top scoring genes upregulated in surviving low-adherent cancer cells induced by 5-azacytidine or irradiation. SBSN expression required the activity of the MEK/Erk pathway, and siRNA-mediated knockdown of SBSN suppressed the low-adherent fraction in irradiated, interferon-gamma- and 5-azacytidine-treated cells, respectively, implicating SBSN in genotoxic stress-induced phenotypic plasticity and stress resistance. Importantly, SBSN expression was observed in human clinical specimens of colon and ovarian carcinomas, as well as in circulating tumour cells and metastases of the 4T1 mouse model. The association of SBSN expression with progressive stages of cancer development indicates its role in cancer evolution and therapy resistance.
- MeSH
- Anoikis drug effects radiation effects MeSH
- Azacitidine pharmacology MeSH
- Drug Resistance, Neoplasm MeSH
- Antigens, Differentiation genetics MeSH
- Interferons pharmacology MeSH
- Humans MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Neoplastic Stem Cells drug effects metabolism radiation effects MeSH
- Neoplasm Proteins genetics MeSH
- Neoplasms drug therapy genetics radiotherapy MeSH
- Antineoplastic Agents pharmacology MeSH
- Gene Expression Regulation, Neoplastic drug effects radiation effects MeSH
- Up-Regulation drug effects radiation effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Inverse-electron-demand Diels-Alder (iEDDA) cycloaddition between 1,2,4,5-tetrazines and strained dienophiles belongs among the most popular bioconjugation reactions. In addition to its fast kinetics, this cycloaddition can be tailored to produce fluorescent products from non-fluorescent starting materials. Here we show that even the reaction intermediates formed in iEDDA cycloaddition can lead to the formation of new types of fluorophores. The influence of various substituents on their photophysical properties and the generality of the approach with use of various trans-cyclooctene derivatives were studied. Model bioimaging experiments demonstrate the application potential of fluorogenic iEDDA cycloaddition.
- MeSH
- Cycloaddition Reaction MeSH
- Cyclooctanes chemistry MeSH
- Fluorescent Dyes chemical synthesis chemistry MeSH
- Microscopy, Fluorescence methods MeSH
- HeLa Cells MeSH
- Heterocyclic Compounds, 2-Ring chemical synthesis chemistry MeSH
- Heterocyclic Compounds, 1-Ring chemistry MeSH
- Microscopy, Confocal methods MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Aging involves tissue accumulation of senescent cells (SC) whose elimination through senolytic approaches may evoke organismal rejuvenation. SC also contribute to aging-associated pathologies including cancer, hence it is imperative to better identify and target SC. Here, we aimed to identify new cell-surface proteins differentially expressed on human SC. Besides previously reported proteins enriched on SC, we identified 78 proteins enriched and 73 proteins underrepresented in replicatively senescent BJ fibroblasts, including L1CAM, whose expression is normally restricted to the neural system and kidneys. L1CAM was: 1) induced in premature forms of cellular senescence triggered chemically and by gamma-radiation, but not in Ras-induced senescence; 2) induced upon inhibition of cyclin-dependent kinases by p16INK4a; 3) induced by TGFbeta and suppressed by RAS/MAPK(Erk) signaling (the latter explaining the lack of L1CAM induction in RAS-induced senescence); and 4) induced upon downregulation of growth-associated gene ANT2, growth in low-glucose medium or inhibition of the mevalonate pathway. These data indicate that L1CAM is controlled by a number of cell growth- and metabolism-related pathways during SC development. Functionally, SC with enhanced surface L1CAM showed increased adhesion to extracellular matrix and migrated faster. Our results provide mechanistic insights into senescence of human cells, with implications for future senolytic strategies.
- MeSH
- Cell Adhesion physiology MeSH
- Cell Cycle MeSH
- Down-Regulation MeSH
- Fibroblasts MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Humans MeSH
- Neural Cell Adhesion Molecule L1 genetics metabolism MeSH
- Cell Line, Tumor MeSH
- Cell Movement physiology MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Gene Expression Regulation drug effects radiation effects MeSH
- RNA Interference MeSH
- Signal Transduction MeSH
- Cellular Senescence MeSH
- Transforming Growth Factor beta metabolism pharmacology MeSH
- Gamma Rays MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications. Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (MTABGNRs). Interestingly, even after cellular accumulation of high amounts of MTABGNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton, or in cell migration could be detected in vitro. In vivo, after intravenous administration, the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of MTABGNRs administered as 10 or 50 μg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of MTABGNRs by living systems.
- MeSH
- Autophagy drug effects MeSH
- Quaternary Ammonium Compounds metabolism MeSH
- Humans MeSH
- Lysosomes drug effects metabolism MeSH
- Mesenchymal Stem Cells cytology drug effects MeSH
- Actin Cytoskeleton drug effects metabolism MeSH
- Mutagens toxicity MeSH
- Mice, Inbred C57BL MeSH
- Cell Line, Tumor MeSH
- Nanotubes chemistry toxicity ultrastructure MeSH
- Podocytes drug effects metabolism MeSH
- Cell Movement drug effects MeSH
- DNA Damage MeSH
- Spleen drug effects pathology MeSH
- Sulfhydryl Compounds metabolism MeSH
- Tissue Distribution MeSH
- Blood Platelets drug effects pathology ultrastructure MeSH
- Gold metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Cationic colloidal gold nanorods (GNRs) have a great potential as a theranostic tool for diverse medical applications. GNRs' properties such as cellular internalization and stability are determined by physicochemical characteristics of their surface coating. GNRs modified by (16-mercaptohexadecyl)trimethylammonium bromide (MTAB), MTABGNRs, show excellent cellular uptake. Despite their promise for biomedicine, however, relatively little is known about the cellular pathways that facilitate the uptake of GNRs, their subcellular fate and intracellular persistence. Here we studied the mechanism of cellular internalization and long-term fate of GNRs coated with MTAB, for which the synthesis was optimized to give higher yield, in various human cell types including normal diploid versus cancerous, and dividing versus nondividing (senescent) cells. The process of MTABGNRs internalization into their final destination in lysosomes proceeds in two steps: (1) fast passive adhesion to cell membrane mediated by sulfated proteoglycans occurring within minutes and (2) slower active transmembrane and intracellular transport of individual nanorods via clathrin-mediated endocytosis and of aggregated nanorods via macropinocytosis. The expression of sulfated proteoglycans was the major factor determining the extent of uptake by the respective cell types. Upon uptake into proliferating cells, MTABGNRs were diluted equally and relatively rapidly into daughter cells; however, in nondividing/senescent cells the loss of MTABGNRs was gradual and very modest, attributable mainly to exocytosis. Exocytosed MTABGNRs can again be internalized. These findings broaden our knowledge about cellular uptake of gold nanorods, a crucial prerequisite for future successful engineering of nanoparticles for biomedical applications such as photothermal cancer therapy or elimination of senescent cells as part of the emerging rejuvenation approach.
- MeSH
- Cell Membrane drug effects metabolism MeSH
- Endocytosis drug effects physiology MeSH
- Exocytosis * drug effects physiology MeSH
- Microscopy, Confocal MeSH
- Culture Media MeSH
- Quaternary Ammonium Compounds chemical synthesis chemistry MeSH
- Humans MeSH
- Lysosomes drug effects MeSH
- Microscopy, Electron, Scanning MeSH
- Cell Line, Tumor MeSH
- Nanotubes analysis chemistry MeSH
- Polylysine chemistry pharmacokinetics MeSH
- Cell Proliferation drug effects MeSH
- Proteoglycans chemistry metabolism MeSH
- Flow Cytometry MeSH
- Drug Stability MeSH
- Sulfhydryl Compounds chemistry MeSH
- Chemistry Techniques, Synthetic MeSH
- Gold chemistry pharmacokinetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.
- MeSH
- Cell Membrane metabolism MeSH
- Cell Nucleus metabolism MeSH
- Exocytosis physiology MeSH
- Fibroblasts cytology metabolism MeSH
- HeLa Cells MeSH
- Cells, Cultured MeSH
- Skin cytology metabolism MeSH
- Humans MeSH
- Actin Cytoskeleton metabolism MeSH
- Myosin Type I metabolism MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Cell Movement MeSH
- Cell Shape MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Paxillin (PXN) is a focal adhesion protein that has been implicated in signal transduction from the extracellular matrix. Recently, it has been shown to shuttle between the cytoplasm and the nucleus. When inside the nucleus, paxillin promotes cell proliferation. Here, we introduce paxillin as a transcriptional regulator of IGF2 and H19 genes. It does not affect the allelic expression of the two genes; rather, it regulates long-range chromosomal interactions between the IGF2 or H19 promoter and a shared distal enhancer on an active allele. Specifically, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, thus activating IGF2 gene transcription, whereas it restrains the interaction between the enhancer and the H19 promoter, downregulating the H19 gene. We found that paxillin interacts with cohesin and the mediator complex, which have been shown to mediate long-range chromosomal looping. We propose that these interactions occur at the IGF2 and H19 gene cluster and are involved in the formation of loops between the IGF2 and H19 promoters and the enhancer, and thus the expression of the corresponding genes. These observations contribute to a mechanistic explanation of the role of paxillin in proliferation and fetal development.
- MeSH
- Hep G2 Cells MeSH
- Chromosomal Proteins, Non-Histone genetics MeSH
- Extracellular Matrix genetics MeSH
- Focal Adhesions genetics MeSH
- Genomic Imprinting genetics MeSH
- Insulin-Like Growth Factor II biosynthesis genetics MeSH
- Humans MeSH
- DNA Methylation genetics MeSH
- Paxillin administration & dosage MeSH
- Cell Proliferation drug effects genetics MeSH
- Promoter Regions, Genetic MeSH
- Cell Cycle Proteins genetics MeSH
- RNA, Long Noncoding biosynthesis genetics MeSH
- Signal Transduction drug effects MeSH
- Fetal Development genetics MeSH
- Gene Expression Regulation, Developmental MeSH
- Enhancer Elements, Genetic MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
RNA polymerase I (Pol I) transcription is essential for the cell cycle, growth and protein synthesis in eukaryotes. In the present study, we found that phosphatidylinositol 4,5-bisphosphate (PIP2) is a part of the protein complex on the active ribosomal promoter during transcription. PIP2 makes a complex with Pol I and the Pol I transcription factor UBF in the nucleolus. PIP2 depletion reduces Pol I transcription, which can be rescued by the addition of exogenous PIP2. In addition, PIP2 also binds directly to the pre-rRNA processing factor fibrillarin (Fib), and co-localizes with nascent transcripts in the nucleolus. PIP2 binding to UBF and Fib modulates their binding to DNA and RNA, respectively. In conclusion, PIP2 interacts with a subset of Pol I transcription machinery, and promotes Pol I transcription.
- MeSH
- Cell Nucleolus genetics metabolism MeSH
- Chromosomal Proteins, Non-Histone genetics metabolism MeSH
- DNA-Binding Proteins genetics metabolism MeSH
- Phosphatidylinositol 4,5-Diphosphate genetics metabolism MeSH
- Transcription, Genetic genetics MeSH
- HeLa Cells MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- RNA Precursors genetics metabolism MeSH
- Promoter Regions, Genetic genetics MeSH
- RNA Polymerase I genetics metabolism MeSH
- Pol1 Transcription Initiation Complex Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
