Self-association Ability Research Articles

Activation and Autoinhibition Mechanisms of NLR Immune Receptor Pi36 in Rice.

Nucleotide-binding and leucine-rich repeat receptors (NLRs) are the most important and largest class of immune receptors in plants. The Pi36 gene encodes a canonical CC-NBS-LRR protein that confers resistance to rice blast fungal infections. Here, we show that the CC domain of Pi36 plays a role in cell death induction. Furthermore, self-association is required for the CC domain-mediated cell death, and the self-association ability is correlated with the cell death level. In addition, the NB-ARC domain may suppress the activity of the CC domain through intramolecular interaction. The mutations D440G next to the RNBS-D motif and D503V in the MHD motif autoactivated Pi36, but the mutation K212 in the P-loop motif inhibited this autoactivation, indicating that nucleotide binding of the NB-ARC domain is essential for Pi36 activation. We also found that the LRR domain is required for D503V- and D440G-mediated Pi36 autoactivation. Interestingly, several mutations in the CC domain compromised the CC domain-mediated cell death without affecting the D440G- or D503V-mediated Pi36 autoactivation. The autoactivate Pi36 variants exhibited stronger self-associations than the inactive variants. Taken together, we speculated that the CC domain of Pi36 executes cell death activities, whereas the NB-ARC domain suppressed CC-mediated cell death via intermolecular interaction. The NB-ARC domain releases its suppression of the CC domain and strengthens the self-association of Pi36 to support the CC domain, possibly through nucleotide exchange.

Tri-network, physical cross-linked sodium alginate hydrogel with strongly self-healing capacity and highly tough, for wearable flexible devices

Physical cross-linked hydrogels offer advantageous self-healing and easy preparation for broad application in wearable flexible devices. However, compared to chemical crosslinking, physical crosslinking is relatively fragile, yielding weak mechanical properties that hinder further development. To solve this problem, we polymerized [2-(methacryloyloxy)ethyl]dimethyl-(propyl3-sulfonate) ammonium hydroxide (DMAPS) into SA-PMAA hydrogels to form a three-network physical cross-linked hydrogel with high strength and superior self-healing ability. PDMAPS possesses dipolar-dipole action between lateral base units and self-associating ability. By introducing PDMAPS into the SA-PMAA double network structure, we created a hydrogel with excellent self-healing properties and mechanical strength. The hydrogel boasts a mechanical strength of 41 KPa, a maximum elongation at break of 820 %, and 94.7 % self-healing efficiency after 12 h, surpassing most hydrogels. With its polar groups, the hydrogel offers great compatibility with most aqueous slurries containing inorganic fillers, making it a material with immense potential as a polyelectrolyte and broad applications in the field of biosensing and wearable flexible devices.

Liquid Crystalline and Gel Properties of Luminescent Cyclometalated Palladium Complexes with Benzoylthiourea Ligands.

The design and development of new luminescent metallogels formed by cyclometalated palladium(II) complexes in protic solvents were investigated by a combination of differential scanning calorimetry (DSC), polarized optical microscopy (POM), and rheology. Cyclometalated palladium(II) complexes based on imine ligand and ancillary benzoylthiourea (BTU) ligand showed red emission in solid and gel states. The formation of a lyotropic liquid crystal phase was observed for the complex bearing shorter alkyl groups on the BTU ligand. This complex also behaved as a thermotropic liquid crystal that displays a monotropic smectic A phase (SmA). Dynamic rheology measurements (frequency sweep in the 5-90 °C range) of the 1-decanol solution of palladium(II) complexes highlighted their supramolecular self-association ability to generate 3D networks and form gels as a final result.

Formation of Protamine and Zn-Insulin Assembly: Exploring Biophysical Consequences.

The insulin-protamine interaction is at the core of the mode of action in many insulin formulations (Zn + insulin + protamine) and to treat diabetes, in which protamine is added to the stable form of hexameric insulin (Zn-insulin). However, due to the unavailability of quantitative data and a high-resolution structure, the binding mechanism of the insulin-protamine complex remains unknown. In this study, it was observed that Zn-insulin experiences destabilization as observed by the loss of secondary structure in circular dichroism (CD), and reduction in thermal stability in melting study, upon protamine binding. In isothermal titration calorimetry (ITC), it was found that the interactions were mostly enthalpically driven. This is in line with the positive ΔC m value (+880 cal mol-1), indicating the role of hydrophilic interactions in the complex formation, with the exposure of hydrophobic residues to the solvent, which was firmly supported by the 8-anilino-1-naphthalene sulfonate (ANS) binding study. The stoichiometry (N) value in ITC suggests the multiple insulin molecules binding to the protamine chain, which is consistent with the picture of the condensation of insulin in the presence of protamine. Atomic force microscopy (AFM) suggested the formation of a heterogeneous Zn-insulin-protamine complex. In fluorescence, Zn-insulin experiences strong Tyr quenching, suggesting that the location of the protamine-binding site is near Tyr, which is also supported by the molecular docking study. Since Tyr is critical in the stabilization of insulin self-assembly, its interaction with protamine may impair insulin's self-association ability and thermodynamic stability while at the same time promoting its flexible conformation desired for better biological activity.

The role of weak intermolecular interactions in photophysical behavior of isocoumarins on the example of their interaction with cyclic trinuclear silver(I) pyrazolate

The complexation of 6-substituted isocoumarins with cyclic trinuclear silver pyrazolate was studied in solution and solid state. Spectral analysis of isocoumarins in concentrated solutions revealed the formation of dimers featuring π-π stacking supported by CH⋯O or CH⋯Cl hydrogen bonds. This behaviour has been proven by DFT calculations at ωB97XD level. The center of coordination of isocoumarine to silver-containing macrocycle is the carbonyl group. Experimental data demonstrate that CO groups interact with the silver macrocycle in the 1:1 ratio. The coordination of the carbonyl group to metallacycle observed in the solid state via cooperative CO⋯Ag3 interactions is typical for ketones. Different self-association abilities of isocoumarins lead to different coordination polymers of their complexes with the macrocycle: infinite stacks of {ABBA}∞ and {AB}∞ pattern were be obtained. The differences in supramolecular packing affect the emission properties of isocoumarins. Furthermore, the coordination of the carbonyl group to silver atoms leads to phosphorescence.

Distinguishing Libyan Educational Leadership of Developing Skills Required in the Digital Age

It is just a little exploration that has been led on how these difficulties and innovation-driven changes are related to modified necessities for the ability set required by chiefs. In this paper, the researcher tries to overcome that issue by applying three-stage research plans. To start with, the researcher fosters an original calculated system wherein we order authority abilities and partner them with errands, the executives’ level, and authority experience. The method that the researcher builds on this is to lead semi-organized meetings with chiefs and methodically research work promotions at the administration level. Our meeting results show that correspondence abilities, subject-explicit information, self-association abilities, and self-reflection abilities are viewed as especially significant amid fast innovation-driven change. The results have shown that the interview participants in the education minister identified the three most significant challenges (in descending order) as being personnel acquisition, digitalization, and pressure for innovation. In the context of personnel acquisition, demographic change in connection with a shortage of qualified applicants is seen as particularly challenging. The researcher concluded that key authority abilities are relational abilities, language information, authoritative abilities, subject-explicit information, advanced proficiency, and self-reflection.

AMBRA1 Negatively Regulates the Function of ALDH1B1, a Cancer Stem Cell Marker, by Controlling Its Ubiquitination.

Activating molecule in Beclin-1-regulated autophagy (AMBRA1), a negative regulator of tumorigenesis, is a substrate receptor of the ubiquitin conjugation system. ALDH1B1, an aldehyde dehydrogenase, is a cancer stem cell (CSC) marker that is required for carcinogenesis via upregulation of the β-catenin pathway. Although accumulating evidence suggests a role for ubiquitination in the regulation of CSC markers, the ubiquitination-mediated regulation of ALDH1B1 has not been unraveled. While proteome analysis has suggested that AMBRA1 and ALDH1B1 can interact, their interaction has not been validated. Here, we show that AMBRA1 is a negative regulator of ALDH1B1. The expression of ALDH1B1-regulated genes, including PTEN, CTNNB1 (β-catenin), and CSC-related β-catenin target genes, is inversely regulated by AMBRA1, suggesting a negative regulatory role of AMBRA1 in the expression of ALDH1B1-regulated genes. We found that the K27- and K33-linked ubiquitination of ALDH1B1 is mediated via the cooperation of AMBRA1 with other E3 ligases, such as TRAF6. Importantly, ubiquitination site mapping revealed that K506, K511, and K515 are important for the K27-linked ubiquitination of ALDH1B1, while K33-linked ubiquitination occurs at K506. A ubiquitination-defective mutant of ALDH1B1 increased the self-association ability of ALDH1B1, suggesting a negative correlation between the ubiquitination and self-association of ALDH1B1. Together, our findings indicate that ALDH1B1 is negatively regulated by AMBRA1-mediated noncanonical ubiquitination.

Pullulan derivative with cationic and hydrophobic moieties as an appropriate macromolecule in the synthesis of nanoparticles for drug delivery

A new amphiphilic pullulan derivative (DBAP-PO) was obtained by grafting tertiary butyl amine and octanoyl groups on the pullulan backbone as cationic and hydrophobic moieties, respectively. The structural characteristics of the modified polymer were investigated by FT-IR and 1H and 13C NMR spectroscopy.The self-association ability in aqueous solution of DBAP-PO was studied by viscosity and fluorescence methods. The intrinsic viscosity of the polymer was determined by Wolf model. The critical aggregation concentration (CAC) value of 0.028 g/dL, determined by fluorescence measurements in the presence of pyrene, was confirmed by capillary viscosimetry and dynamic laser scattering (DLS). Dialysis method was used to demonstrate the capacity of the pullulan derivative to form spherical nanoparticles (d ~ 200 nm) loaded with model drug, sodium diclofenac (DF) (74% entrapment efficiency). The DF release was sustained and pH-dependent. In vitro cytotoxicity as well as morphological studies conducted on the human skin fibroblasts showed that DBAP-PO/DF nanoparticles do not exhibit cytotoxic effects at the pharmacologically relevant concentration of DF, maintaining the typical morphology of the cells.

Separation of Cenospheres from Lignite Fly Ash Using Acetone–Water Mixture

This work reports on the separation of cenospheres from lignite fly ash through a wet separation process-the sink-float method. A better quality of cenospheres could be achieved through a physical–chemical approach using an acetone–water mixture as a medium. This work aimed to elucidate the correlation between the structure, morphology, and composition and medium fraction variables, with data for the freshly prepared and the reused mixtures presented for comparison. The work covers a study of the macrocomponent composition of an Fe2O3–SiO2–Al2O3 system, highlighting the pair dependences of SiO2–Al2O3, Al2O3–Fe2O3, and SiO2/Al2O3–Al2O3 and revealing an interesting result in terms of geochemical characteristics categorizing the collected cenosphere fractions separated from high-calcium class C fly ash produced from a lignite coal power plant in Thailand (as magnetic cenospheres). The CaO and SO3 contents increased monotonically with increased water content, particularly for the CaO composition profile, which was found to be similar to the increased carbonate concentration measured from the mixtures after use. The physicochemical properties in terms of the self-association ability of the acetone–water mixing phase is believed to have played an important role in determining the intermolecular interactions and reactivity of ions in the liquid phase, consequently affecting the separation efficiency, recovery yield, and quality of cenospheres.

Synthesis, Self-Association, and Solubilizing Ability of an Amphiphilic Derivative of Poly(ethylene glycol) Methyl Ether

A new amphiphilic derivative–methoxy(polyethoxy)ethyl stearate has been synthesized with the goal of creating therefrom systems for delivery and enhancement of bioavailability of piperine. The aggregation and solubilizing properties of ω-methylpoly(oxyethylene) stearate have been studied by means of a set of physicochemical methods.

New insights into protamine-like component organization in Mytilus galloprovincialis' sperm chromatin.

We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.

Tryptophan as a probe to study the anticancer mechanism of action and specificity of α-helical anticancer peptides.

In the present study, a single tryptophan, as a fluorescence probe, was shifted from the N-terminus to the middle and to the C-terminus of a 26-residue α-helical anticancer peptide sequence to study the mechanism of action and specificity. The hydrophobicity of peptides, as well as peptide helicity and self-associating ability, were slightly influenced by the position change of tryptophan in the peptide sequence, while the hemolytic activity and anticancer activity of the peptide analogs remained the same. The tryptophan fluorescence experiment demonstrated that peptide analogs were more selective against LUVs mimicking cancer cell membranes than LUVs mimicking normal cell membranes. During the interaction with target membranes, the N-terminus of an anticancer peptide may be inserted vertically or tilted into the hydrophobic components of the phospholipid bilayer first. The thermodynamic parameters of the peptides PNW and PCW, when interacting with zwitterionic DMPC or negatively charged DMPS, were determined by ITC. DSC experiments showed that peptide analogs significantly altered the phase transition profiles of DMPC, but did not dramatically modify the phase transition of DMPS. It is demonstrated that hydrophobic interactions are the main driving force for peptides interacting with normal cell membranes, whilst, electrostatic interactions dominate the interactions between peptides and cancer cell membranes. Utilizing tryptophan as a fluorescence probe molecule appears to be a practicable approach to determine the interaction of peptides with phospholipid bilayers.

Mapping the Interacting Sites Mediating Tetramerisation of Ryanodine Receptor Amino-Terminus

The cardiac ryanodine receptor (RyR2) is an intracellular calcium release channel composed of four ∼560kDa subunits. It mediates sarcoplasmic reticulum calcium release playing a critical role in cardiac excitation-contraction coupling. Recently, we presented evidence that the RyR2 N-terminus self-associates into a tetrameric form, which stabilises the closed conformation of the channel. In order to identify the interacting domains mediating RyR2 N-terminus tetramerisation, we used truncated constructs in yeast two-hybrid and chemical cross-linking assays. RyR2 residues 1-418 remained in monomeric form, whereas residues 1-530 retained self-association ability although it was much weaker compared to longer fragments. Tetramerisation was not abolished by removal of the N-terminal 160 amino acids but further truncations were detrimental. A targeted approach for putative inter-subunit contact sites was also employed testing the effect of small internal deletions within RyR2 residues 1-906. We found that two small deletions (167-178 or 335-358 amino acids) severely compromised tetramerisation ability. These findings suggest that residues 167-178 and 335-358 are the primary interacting sites, whereas additional downstream determinants further strengthen the RyR2 N-terminus self-association.

An integrated proteomics and bioinformatics analyses of hepatitis B virus X interacting proteins and identification of a novel interactor apoA-I

HBx is well-known to be a multifunctional protein encoded by HBV and its biological functions are mainly dependent on pleiotropic protein-protein interactions (PPIs); however, the global mapping of HBx-interactome has not been established so far. Thus, in this study, we have identified 127 HBx-interacting proteins by a profound GST pull-down assay coupled with mass spectrometry, and constructed an HBx-interactome network and core apoA-I pathways with a series of bioinformatics approaches. One of the identified HBx-binding partners is apolipoprotein A-I (apoA-I), which has a specific role in lipid and cholesterol metabolism. The HBx-apoA-I protein interaction was confirmed by both GST pull-down and co-immunoprecipitation. The ectopic overexpression of apoA-I can lead to a significant inhibition on HBV secretion concomitant with the reduction of cellular cholesterol level. In addition, HBV can modulate the function of apoA-I through HBx which might interact with the 44-189 residues of apoA-I and result in dysfunction of apoA-I such as decreased self-association ability, increased carbonyl level and impaired lipid-binding ability. Our results demonstrate an integrated physical association of HBx and host proteins, especially a novel interactor apoA-I that may influence the HBV secretion, which would shed new light on exploring the complicated mechanisms of HBV manipulation on host cellular functions. HBx is well-known to be a multifunctional protein encoded by HBV and its biological functions are mainly dependent on pleiotropic protein-protein interactions. Although a series of HBx-interacting proteins have been identified, a global characterization of HBx interactome has not been reported. In this study, we have identified a total of 127 HBx-interacting proteins by a profound GST pull-down assay coupled with mass spectrometry, and constructed an HBx-interactome network with a series of bioinformatics approaches. Our results demonstrate an integrated physical association of HBx and host proteins which may help us explore the complicated mechanisms of HBV manipulation on host cellular functions. In addition, we validated one of the identified HBx-binding partners, apolipoprotein A-I (apoA-I), which played a significant inhibitory effect on HBV secretion, indicating a crucial role of the HBx-apoA-I axis in HBV life cycle.

Assessing the effect of RET Transmembrane Domain Mutations in receptor self-association capability using the in vivo TOXCAT System

Mutations of c-RET proto-oncogene with a unique localization within the human transmembrane receptor represent a challenge for contemporary molecular oncology techniques. RET transmembrane domain (TMD)-driven dimerization of the receptor leads to its permanent activation that eventually results in the development of medullary thyroid neoplasia. In this study, we describe the employment of the TOXCAT system which enables to investigate mutation-induced alterations in the strength of RET TMD dimerization in vivo. We suggest an improvement of the method by adding reporter gene quantification at the mRNA levels as a support to the commonly used reporter protein level. We have investigated possible changes in RET TMD dimerization in case of two germline RET TMD mutations found in in several individual cases and MEN2 families worldwide, p.Ala641Ser and p.Ser649Leu. According to our results, substitution of Ser-649 residue by leucine, found as a result of germline mutation, caused a significant decrease of RET TMD self-association in comparison to RET wild-type transmembrane domain. The impaired ability of self-association suggests a novel, yet unknown mechanism of tyrosine kinase domain activation, possibly independent of RET homodimerization.

Yersinia pestis autoagglutination factor is a component of the type six secretion system

Autoagglutination (AA) is a protective phenotypic trait facilitating survival of bacteria in hostile environments and in the host during infection. Autoagglutination factors (AFs) that possess self-associating ability are currently characterized in many Gram-negative bacteria, but Yersinia pestis AFs are still a matter of debate. Previously, we have shown that AF of Hms − strain Y. pestis EV76 is a complex of the 17,485-kDa protein and a low-molecular-weight component with siderophore activity. Here, we identified the protein moiety of AF and examined its role in AA of Hms + and Hms − Y. pestis strains. Using MALDI-TOF MS of trypsin-hydrolyzed AF, we unambiguously identified the protein as YPO0502, which belongs to a family of Hcp-proteins forming pilus-like structures of the type six secretion system (T6SS). To address the role of YPO0502 in AA, we cloned ypo0502 in E. coli, overexpressed it in Y. pestis and constructed its knock-out mutant in Y. pestis. However, all these approaches failed: YPO0502 was not secreted in E. coli, formed inclusion bodies when overexpressed in Y. pestis, and could probably be compensated by other Hcp-like proteins in Y. pestis. In contrast, downregulation of ypo0502 expression by its antisense RNA supported the contribution of YPO0502 in AA of Hms + and Hms − Y. pestis strains. The results of the present study indicate that the Hcp-like component of T6SS encoded by ypo502 is involved in Y. pestis AA and suggest that at least one ( ypo0499-0516) of the 6 T6SS clusters of Y. pestis is involved in bacterial interaction.

Hydrogen-bonded supramolecular polymers from derivatives of α-amino-ε-caprolactam: A bio-based material

Hydrogen-bonded supramolecular polymers were prepared from the derivatives of α-amino-e-caprolactam (ACL), obtained from a renewable resource. Several self-complimentary bis- or tetra-caprolactam monomers were synthesized by varying the number of carbons of the spacer between the hydrogen-bonding end groups. Physical properties of these hydrogen-bonded polymers were clearly demonstrated by differential scanning colorimetry, solid-state NMR, and X-ray powder diffraction analyses. The supramolecular behavior was also supported by fiber formation from the melt for several of these compounds, and stable glassy materials were prepared from the physical mixtures of two different biscaprolactams. The self-association ability of ACL was also used by incorporating ACL at the chain ends of low-molecular weight Jeffamine (M n = 900 g/mol) using urea and amide linkages. The transformation of this liquid oligomer at room temperature into a self-standing, transparent film clearly showed the improvement in mechanical properties obtained by the introduction of terminal hydrogen-bonding groups. Finally, the use of monomers with a functionality of four gave rise to network formation either alone or combination with bifunctional monomers.

Mucoadhesive polyelectrolyte microparticles containing recombinant human insulin and its analogs aspart and lispro

Microparticles containing recombinant human insulin and its analogs aspart and lispro were prepared using an alternate adsorption of chitosan and dextran sulfate from solutions onto microaggregates of protein-dextran sulfate insoluble complex. The following properties of polyelectrolyte hormone-containing microparticles were studied: pH stability, surface charge, mucoadhesive properties, Ca(2+) binding, degradation under the influence of proteases (trypsin, chymotrypsin). The influence of the self-association ability of encapsulated insulins on the form of protein releasing from microparticles was studied. Insulins aspart and lispro released from the microparticles as monomers were more liable to proteolysis than human insulin released as a hexamer. The combined effect of properties of polyelectrolyte microparticles and of encapsulated recombinant proteins on the bioavailability of insulin under peroral administration is discussed.

Studies on Mechanism of Action of Anticancer Peptides by Modulation of Hydrophobicity Within a Defined Structural Framework

In the present study, the hydrophobicity of a 26-residue α-helical peptide (peptide P) was altered to study the effects of peptide hydrophobicity on the mechanism of action of cationic anticancer peptides. Hydrophobicity of the nonpolar face of the peptides was shown to correlate with peptide helicity. The self-association ability of peptides in aqueous environment, determined by the reversed-phase high performance liquid chromatography temperature profiling, showed strong influence on anticancer activity. The peptide analogues with greater hydrophobicity showed stronger anticancer activity determined by IC(50) values with a necrotic-like membrane disruption mechanism. Peptide analogues exhibited high specificity against cancer cells and much higher anticancer activity than widely-used anticancer chemical drugs. The mechanism of action of anticancer peptides was also investigated. The hydrophobicity of peptides plays a crucial role in the mechanism of action against cancer cells, which could present a way, using a de novo design approach, to create anticancer peptides as potential therapeutics in clinical practices.

Structure-guided de novo design of α-helical antimicrobial peptide with enhanced specificity

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAKKTVLHTALKAISS-amide (peptide P) was utilized as the framework to study the effects of introducing hydrophilic amino acid lysine on the nonpolar face of the helix on peptide biological activities. Lysine residue was systematically used to substitute original hydrophobic amino acid at the selected locations on the nonpolar face of peptide P. In order to compensate for the loss of hydrophobicity caused by lysine substitution, leucine was also used to replace original alanine to increase peptide overall hydrophobicity. Hemolytic activity is correlated with peptide hydrophobicity. By introducing lysine on the nonpolar face, we significantly weaken peptide hemolytic activity as well as antimicrobial activity. However, by utilizing leucine to compensate the hydrophobicity, we improve antimicrobial activity against both Gram-negative and -positive bacteria. Peptide self-association ability and hydrophobicity were also determined. This specific rational approach of peptide design could be a powerful method to optimize antimicrobial peptides with clinical potential as peptide antibiotics.