Control of the chemical composition and thickness of deposited coatings over carbon nanotubes using acrylic acid plasma

In this study, it is demonstrated that the surface of carbonnanotubes can be coated with a polymer nanometer size film (nanocoating) with tailored surface polar behavior when treated with acrylic acid plasma. The polar behavior of the polymer nanocoating can be hydrophilic or hydrophobic depending deposition and erosion processes caused by ionized species in the plasma. In turn, deposition and erosion can be controlled by plasma power. Deposition dominates at 20 W power, where a significant amount of polymer nanocoating is produced with carboxylic acid functional groups in the surface thus having an hydrophilic behavior. On the contrary, a smaller amount of polymer nanocoating with hydrophobic behavior (i.e. without any functional groups on its surface) suggests that erosion isthe dominant process when 40 W power is used. Finally, a competition between deposition and erosion processes results in different polar behavior and amount of polymer nanocoating depending of the treatment time

Residual helicity at the active site of the histidine phosphocarrier, HPr, modulates binding affinity to its natural partners

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first a-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first a-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. The refore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

A phosphorylation-induced switch in the nuclear localization sequence of the intrinsically disordered nupr1 hampers binding to importin

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin a, of which there are several human isoforms; among them, importin a3 (Impa3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impa3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impa3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impa3 with low micromolar affinity (1.7–27 µM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i, i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impa3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure

Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ''fuzzy'' interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs

Characterization of carbon nanofibers superficially modified with ethylene plasma

Nanofibras de carbón (CNFs) han sido tratadas superficialmente mediante polimerización por plasma de etileno. Después del tratamiento, las CNFs fueron caracterizadas por Espectroscopía Infrarroja de Transformada de Fourier (FTIR), Análisis Termogravimétrico (TGA), Difracción de Rayos X (WAXS) y Microscopía Electrónica de Barrido de Emisión de Campo (FESEM- Jeol JSM7401F) en modalidad de transmisión (STEM). Finalmente, las CNFs tratadas y no tratadas fueron probadas en agua, cloroformo y triclorobenceno (TCB) con la finalidad de observar el grado de dispersión de las fibras en estos solventes. El análisis por FTIR muestra señales correspondientes a la parte orgánica depositada sobre la superficie de las CNFs. En el análisis por TGA se puede apreciar una mayor pérdida en peso para las CNFs tratadas en comparación con las CNFs no tratadas. Las pruebas de dispersión muestran que las CNFs tratadas y no tratadas exhiben un comportamiento distinto en cada uno de los solventes usados. Además, los resultados obtenidos por STEM indican que las CNFs tratadas tienen diferentes características morfológicas respecto a las no tratadas.Abstract: Carbon nanofibers (CNFs) have been surface modified by plasma polymerization of ethylene. After treatment, the CNFs were characterized by spectroscopy Fourier Transform Infrared (FTIR), Thermogravimetric Analysis (TGA), X-ray diffraction (WAXS) and Scanning Electron Microscopy Field Emission (FESEM- Jeol JSM7401F) in transmission mode (STEM). Finally, the CNFs treated and untreated were tested in water, chloroform and trichlorobenzene (TCB) in order to observe the degree of dispersion of the fibers in these solvents. The FTIR analysis shows signals corresponding to the organic part deposited on the surface of the CNFs. In the TGA analysis can be seen a greater loss weight for treated CNFs compared to untreated CNFs. Dispersion test show that the treated and untreated CNFs exhibit a different behavior in each of the solvents used. Moreover, the results obtained by STEM indicated that treated CNFs have different morphological characteristics compared to the untreated

Cysteine mutational studies provide insight into a thiol-based redox switch mechanism of metal and DNA binding in FurA from Anabaena sp. PCC 7120

Aims: The ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding. Results: Residue C101 that belongs to a conserved CXXC motif plays an essential role in both metal and DNA binding activities in vitro. Substitution of C101 by serine impairs DNA and metal binding abilities of FurA. Isothermal titration calorimetry measurements show that the redox state of C101 is responsible for the protein ability to coordinate the metal corepressor. Moreover, the redox state of C101 varies with the presence or absence of C104 or C133, suggesting that the environments of these cysteines are mutually interdependent. Innovation: We propose that C101 is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor. Conclusion: This mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria

ZZW-115-dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents

Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR

Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity

Caracterización de nanofibras de carbón modificadas superficialmente con plasma de etileno

Carbon nanofibers (CNFs) have been surface modified by plasma polymerization of ethylene. After treatment, the CNFs were characterized by spectroscopy Fourier Transform Infrared (FTIR), Thermogravimetric Analysis (TGA), X-ray diffraction (WAXS) and Scanning Electron Microscopy Field Emission (FESEM- Jeol JSM7401F) in transmission mode (STEM). Finally, the CNFs treated and untreated were tested in water, chloroform and trichlorobenzene (TCB) in order to observe the degree of dispersion of the fibers in these solvents. The FTIR analysis shows signals corresponding to the organic part deposited on the surface of the CNFs. In the TGA analysis can be seen a greater loss weight for treated CNFs compared to untreated CNFs. Dispersion test show that the treated and untreated CNFs exhibit a different behavior in each of the solvents used. Moreover, the results obtained by STEM indicated that treated CNFs have different morphological characteristics compared to the untreated.Nanofibras de carbón (CNFs) han sido tratadas superficialmente mediante polimerización por plasma de etileno. Después del tratamiento, las CNFs fueron caracterizadas por Espectroscopía Infrarroja de Transformada de Fourier (FTIR), Análisis Termogravimétrico (TGA), Difracción de Rayos X (WAXS) y Microscopía Electrónica de Barrido de Emisión de Campo (FESEM- Jeol JSM7401F) en modalidad de transmisión (STEM). Finalmente, las CNFs tratadas y no tratadas fueron probadas en agua, cloroformo y triclorobenceno (TCB) con la finalidad de observar el grado de dispersión de las fibras en estos solventes. El análisis por FTIR muestra señales correspondientes a la parte orgánica depositada sobre la superficie de las CNFs. En el análisis por TGA se puede apreciar una mayor pérdida en peso para las CNFs tratadas en comparación con las CNFs no tratadas. Las pruebas de dispersión muestran que las CNFs tratadas y no tratadas exhiben un comportamiento distinto en cada uno de los solventes usados. Además, los resultados obtenidos por STEM indican que las CNFs tratadas tienen diferentes características morfológicas respecto a las no tratadas

Residual helicity at the active site of the histidine phosphocarrier, HPr, modulates binding affinity to its natural partners

18 pags., 4 figs., 5 tabs. -- This article belongs to the Special Issue Folding and Design of α-Helical Proteins and Peptides: Theory Meets Nanomaterials, Biotechnology and HealthThe phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr is mainly disordered. We attempted to improve the affinity of HPr to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. The refore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI2018-097991-B-I00 to J.L.N., BFU2016-78232-P to A.V.-C., BES-2017-080739 to D.O.-A., and RTI2018-101969-J-I00 to A.F.). The NMR equipment used in this work was funded by Generalitat Valenciana (Spain) and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana (Spain) 2014–2020)