The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 suppressor pathway promoting cell survival. Targeting has emerged as validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking N-terminal fragment have been shown be potent Hdm2/HdmX antagonists. potential use these peptides, however, limited their poor stability bioavailability. Here, we report engineering cyclotide MCoTI-I efficiently...

Herein, we report for the first time design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished grafting series topologically modified CVX15 based peptides onto loop 6 MCoTI-I. The most active compound produced in this study potent CXCR4 antagonist (EC50 ≈ 20 nM) an efficient cell-entry blocker 2 nM). also showed high stability human serum, thereby providing promising lead type...

Historically introduced by McConkey to explain the slow mutation rate of highly abundant proteins, weak protein (quinary) interactions are an emergent property living cells. The complexes that result from quinary transient and thus difficult study biochemically in vitro. Cross-correlated relaxation-induced polarization transfer-based in-cell nuclear magnetic resonance allows characterization with atomic resolution inside live prokaryotic eukaryotic We show RNAs important component...

Going in circles: Expression of cyclotides containing non-natural amino acids (see scheme; gray sphere) inside live bacterial cells was accomplished using a highly efficient split intein combination with nonsense codon suppressor tRNA technology. Cyclotides p-azidophenylalanine can be expressed and easily labeled copper-free click chemistry to monitor the cyclotide–protein interactions.

Abstract We report for the first time recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans‐splicing in combination with a highly efficient split‐intein. This approach was successfully used to produce naturally occurring cyclotide MCoTI‐I and engineered MCoCP4. Cyclotide MCoCP4 shown reduce toxicity human α‐synuclein cells. selected phenotypic screening from transformed mixture plasmids encoding inactive ratio 1:5×10 4 ....

We report for the first time design and synthesis of a novel cyclotide able to activate unique receptor angiotensin (1-7) (AT1-7), MAS1 receptor. This was accomplished by grafting an AT1-7 peptide analog onto loop 6 MCoTI-I using isopeptide bonds preserve α-amino C-terminal carboxylate groups AT1-7, which are required activity. The resulting construct adopt cyclotide-like conformation showed similar activity that AT1-7. also high stability in human serum thereby providing promising lead...

Ribosomes are present inside bacterial cells at micromolar concentrations and occupy up to 20% of the cell volume. Under these conditions, even weak quinary interactions between ribosomes cytosolic proteins can affect protein activity. By using in-cell in vitro NMR spectroscopy, biophysical techniques, we show that enzymes, adenylate kinase dihydrofolate reductase, respective coenzymes, ATP NADPH, bind with affinity, this interaction suppresses enzymatic activities both enzymes. Conversely,...

Defensins are antimicrobial peptides that important in the innate immune defense of mammals. In contrast to mammalian α- and β-defensins, rhesus θ-defensin-1 (RTD-1) comprises only 18 amino acids stabilized by three disulfide bonds an unusual backbone cyclic topology. this work we report for first time recombinant expression fully folded θ-defensin RTD-1 using a bacterial system. This was accomplished intramolecular native chemical ligation combination with modified protein-splicing unit....

Abstract Distinct differences between how model proteins interact in‐cell and in vitro suggest that the cytosol might have a profound effect modulating protein–protein and/or protein–ligand interactions are not observed vitro. Analyses of NMR spectra target interacting with physiological partners further complicated by low signal‐to‐noise ratios, long overexpression times used interaction studies may lead to changes over course experiment. To unambiguously resolve principal binding mode two...

Abstract Intrinsically disordered proteins (IDPs) or unstructured segments within play an important role in cellular physiology and pathology. Low concentration, multiple binding partners, frequent post-translational modifications the presence of conformations make it difficult to characterize IDP interactions intact cells. We used peptide aptamers selected by using yeast-two-hybrid scheme in-cell NMR identify high affinity binders transiently structured at atomic resolution. Since both...

RNA constitutes up to 20% of a cell's dry weight, corresponding ∼20 mg/mL. This high concentration facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, level ubiquitin-RNA colocalization increases when cells are grown presence dextrose methanol instead as sole carbon source. Total isolated from β-galactosidase activity relative that seen with methanol. Because total...

Fremdling im Ring: Die Expression von Cyclotiden mit nichtnatürlichen Aminosäuren (grauer Kreis Schema) in lebenden Bakterienzellen gelingt mithilfe eines hoch effizienten getrennten Inteins Kombination Nonsense-Codon-Suppressor-tRNA-Technik. p-Azidophenylalanin-haltige Cyclotide können Bakterien exprimiert und leicht über kupferfreie Klick-Reaktionen markiert werden, um Cyclotid-Protein-Wechselwirkungen nachzuvollziehen.

RNA G-quadruplex (rG4) structures can influence the fate and functions of mRNAs, especially translation process. The presence rG4 in 5′-untranslated regions (5′-UTRs) mRNAs generally represses translation. However, also promote internal ribosome entry site (IRES)-mediated as one its determinants. Here, we report identification an evolutionary conserved rG4-forming sequence motif at extreme 5′-end unusually long 5′-UTR (1.7 kb) transcript human cIAP1 gene encoding cellular inhibitor apoptosis...

PS-15, a new dihydrofolate reductase inhibitor, and its cyclic metabolite were evaluated for in vitro activity against 31 clinical Mycobacterium avium complex isolates. Broth dilution MICs of PS-15 ranged from 16 to 64 micrograms/ml. The was three five times more active than the parent compound. Further evaluation these compounds an M. avium-infected murine test system will be interest.

Abstract We report for the first time recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans‐splicing in combination with a highly efficient split‐intein. This approach was successfully used to produce naturally occurring cyclotide MCoTI‐I and engineered MCoCP4. Cyclotide MCoCP4 shown reduce toxicity human α‐synuclein cells. selected phenotypic screening from transformed mixture plasmids encoding inactive ratio 1:5×10 4 ....

l-asparaginase II (MW 135 kDa) from E. coli is an FDA-approved protein drug used for the treatment of childhood leukemia. Despite its long history as a chemotherapeutic, structural basis enzyme action, in solution, remains widely contested. In this work, methyl-based 2D [1H-13C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural abundance, has been to profile enzymatic activity commercially available drug. The [1H-13C]-HSQC NMR spectra reveal role flexible loop segment enzyme,...

Cyclotide sind natürliche pflanzliche Mikroproteine mit zirkulärer Kopf-Schwanz-Verknüpfung und Cys-Knoten. In ihrer Zuschrift auf S. 3208 ff. beschreiben J. A. Camarero et al. die rekombinative Produktion eines fluoreszenzmarkierten Cyclotids in lebenden Bakterienzellen. Das Gegenwart von p-Azidophenylalanin einem Dibenzocyclooctin-Derivat des Fluoreszenzfarbstoffs Aminomethyl-Cumarinacetat (AMCA) synthetisierte Peptid wird vivo markiert. Bildgestaltung: Isaac Mora. Sauerstoffreduktion...

Graphical Abstract Cyclotide sind pflanzliche Mikroproteine mit zirkularer Cys-Knotentopologie. J. A. Camarero et al. zeigen in der Zuschrift auf S. 8510 ff. die heterologe Expression von Cyclotiden innerhalb lebender Hefezellen. Dieser Ansatz wurde zur Produktion eines Cyclotids verwendet, das α-syn-induzierte Zytotoxizität einem Synucleopathiemodell inhibiert.