A FRET Sensor to Monitor Bivalent SUMO–SIM Interactions in SUMO Chain Binding

The small ubiquitin‐like modifier (SUMO) can be assembled into polymeric chains as part of its diverse biochemical signal pattern upon conjugation to substrate proteins. SUMO chain recognition is facilitated by receptor proteins that contain at least two SUMO‐interacting motifs (SIMs). Little is known about the structure of SUMO chains, both in an unliganded form and upon complexation with multi‐SIM protein partners. A FRET sensor has been developed based on a linear dimer of human SUMO‐2 as a minimal SUMO chain analogue. The synthetic acceptor and donor dyes were conjugated by maleimide and copper‐catalyzed click chemistry to each of the two SUMO subunits. FRET changes were only observed in the presence of di‐ or multi‐SIM ligands. Alteration of the short linker sequence between SIMs 2 and 3 of RNF4 showed a great tolerance, and hence, structural flexibility, of the SUMO dimer for bivalent binding of adjacent SIMs. The di‐SUMO FRET sensor reports on the binding of SIM clusters of the proteins C5orf25 and SOBP; this suggest that these can bind to adjacent subunits of a SUMO chain. The developed FRET sensor will be a useful tool to study the importance of SIM and linker sequences, as well as biochemical and structural properties of SUMO chains and multi‐SIM proteins.     

Synthesis of ultrastable eu‐complex/polystyrene composite luminescent nanoparticles using a solvent swelling method

Nanoparticles composed of Eu‐complex, Eu(TTA)₃Phen, and polystyrene (PS) were successfully synthesized through an improved solvent swelling method. The unstable Eu(TTA)₃Phen could be protected by the hydrophobic shell of PS nanoparticles. This method is very appropriate to embed unstable luminescent molecules into polymer nanoparticles. The as‐synthesized luminescent PS nanoparticles have been turned out to be water‐dispersible, strong red luminescent, ultrastable in strong acid and alkali, and luminescence lifetime enhanced. A cell imaging assay was further carried out and strong luminescent signal could be obtained, which showed that the as‐synthesized luminescent Eu‐complex/PS nanoparticles are a good candidate to be used as luminescent nanoparticles in tumor cell detection. This solvent swelling method is simple, easy to scale‐up, and has great potential in the preparation of other luminescent nanoparticles. POLYM. COMPOS., 2011. © 2011 Society of Plastics Engineers     

Application of the biodegradable diblock copolymer poly(L‐lactide)‐block‐poly(L‐cysteine): Drug delivery and protein conjugation

A novel approach to self‐assembled and shell‐crosslinked (SCL) micelles from the diblock copolymer poly(L ‐lactide)‐block‐poly(L ‐cysteine) to be used as drug and protein delivery carriers is described. Rifampicin was used as a model drug. The drug‐loaded SCL micelles were obtained by self‐assembly of the copolymer in the presence of the drug in aqueous media. Their morphology and size were studied with dynamic light scattering and field emission scanning electron microscopy. The rifampicin loading capacity and encapsulation efficiency were studied with ultraviolet–visible spectrophotometry. The drug‐release rate in vitro depended on the oxidizing and reducing environment. Moreover, a straightforward approach to the conjugation of the copolymer with bovine serum albumin (BSA) was developed, and a gel electrophoresis test demonstrated that this conjugated BSA could be reversibly released from the copolymer substrate under reducing conditions. In conclusion, this L ‐cysteine copolymer can be used in drug delivery and in protein fixation and recovery. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Reduced VDAC1, Maintained Mitochondrial Dynamics and Enhanced Mitochondrial Biogenesis in a Transgenic Tau Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is one of the most common forms of neurodegeneration, defined by reduced cognitive function, which is caused by the gradual death of neurons in the brain. Recent studies have shown an age-dependent rise in the levels of voltage-dependent anion channel 1 (VDAC1) in AD. In addition, we discovered an aberrant interaction between VDAC1 and P-TAU in the brains of AD patients, which led to abnormalities in the structural and functional integrity of the mitochondria. The purpose of our study is to understand the protective effects of reduced VDAC1 against impaired mitochondrial dynamics and defective mitochondrial biogenesis in transgenic TAU mice. Recently, we crossed heterozygote VDAC1 knockout (VDAC1^+/−) mice with transgenic TAU mice to obtain double-mutant VDAC1^+/−/TAU mice. Our goal was to evaluate whether a partial decrease in VDAC1 lessens the amount of mitochondrial toxicity in transgenic Tau (P301L) mice. We found that mitochondrial fission proteins were significantly reduced, and mitochondrial fusion and biogenesis proteins were increased in double-mutant mice compared to TAU mice. On the basis of these discoveries, the current work may have significance for the development of reduced-VDAC1-based treatments for individuals suffering from AD as well as other tauopathies.     

Near-infrared luminescent mesoporous MCM-41 materials covalently bonded with ternary thulium complexes

Two β-diketones 4,4,4-trifluoro-1-2-thenoyl-1,3-butanedione (Htta) and 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione (Htfnb), which contain trifluoroalkyl chain, were selected as the main sensitizer for synthesizing Tm(L)₃phen (L = tta, tfnb) complexes. The two near-infrared (NIR) luminescent thulium complexes have been covalently bonded to the ordered mesoporous material MCM-41 via a functionalized 1,10-phenanthroline (phen) group 5-(N,N-bis-3-(triethoxysilyl)propyl)ureyl-1,10-phenanthroline (phen-Si) [The resultant mesoporous materials are denoted as Tm(L)₃phen–MCM-41 (L = tta, tfnb)]. The Tm(L)₃phen–MCM-41 (L = tta, tfnb) mesoporous materials were characterized by small-angle X-ray diffraction (XRD) and N₂ adsorption/desorption, and they show characteristic mesoporous structure of MCM-41. Luminescence spectra of the Tm(L)₃phen–MCM-41 (L = tta, tfnb) mesoporous materials were recorded and the corresponding luminescence decay curves were obtained. After ligand-mediated excitation, the emission spectra of the Tm(L)₃phen–MCM-41 (L = tta, tfnb) mesoporous materials show the characteristic NIR-luminescence of the Tm³⁺ ion. The full width at half maximum (fwhm) of the 1474-nm emission band are 96 and 100 nm for Tm(tta)₃phen–MCM-41 and Tm(tfnb)₃phen–MCM-41, respectively. The good luminescent performances enable these NIR-luminescent mesoporous materials to have potential applications in optical amplification [broadening amplification band from C band (1530–1560 nm) to S⁺ band (1450–1500 nm)]. Furthermore, the comparison of the luminescence behavior for Tm(tta)₃phen–MCM-41 and Tm(tfnb)₃phen–MCM-41 mesoporous materials was investigated. It shows that Tm(tfnb)₃phen–MCM-41 is somewhat superior to Tm(tta)₃phen–MCM-41 as optical amplifier.     

The dispersants effect on physical properties of long‐lasting luminescent polypropylene

Two series of blends, O‐PP₁₅ and O‐PP₃₅, were prepared by mixing polypropylene (PP), luminescent powders (SrAl₂O₄: Eu²⁺, Dy³⁺) of 15 and 35 μm average particle diameter, and hydrophobic dispersant at about 190°C in the Brabender mixer. The effect of amounts and diameter of luminescent powders on the physical properties of PP material were discussed herein. The luminescence and afterglow time tests indicated that the initial luminescence of all blends increased with the luminescent powders amounts. O‐PP₃₅ blends showed lower afterglow luminance than O‐PP₁₅ blends at low luminescent powder amounts. The melting and crystallization temperatures of the blends appeared at 152–168°C and 87–103°C, respectively. The blends displayed peaks attributable to a α crystal structure at 2θ = 18°–19°. The β crystal structure was only evident from its characteristic 2θ peak at 15°–16° in the WAXD pattern of the O‐PP₃₅ blends with high luminescent powder amounts. All of the blends had lower tensile strengths. However, the improvement in the luminescent powder distribution was evident from the SEM images after adding hydrophobic dispersant. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Regulation of Mitochondrial Respiration by VDAC Is Enhanced by Membrane-Bound Inhibitors with Disordered Polyanionic C-Terminal Domains

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane. When reconstituted into lipid membranes, VDAC responds to sufficiently large transmembrane potentials by transitioning to gated states in which ATP/ADP flux is reduced and calcium flux is increased. Two otherwise unrelated cytosolic proteins, tubulin, and α-synuclein (αSyn), dock with VDAC by a novel mechanism in which the transmembrane potential draws their disordered, polyanionic C-terminal domains into and through the VDAC channel, thus physically blocking the pore. For both tubulin and αSyn, the blocked state is observed at much lower transmembrane potentials than VDAC gated states, such that in the presence of these cytosolic docking proteins, VDAC’s sensitivity to transmembrane potential is dramatically increased. Remarkably, the features of the VDAC gated states relevant for bioenergetics—reduced metabolite flux and increased calcium flux—are preserved in the blocked state induced by either docking protein. The ability of tubulin and αSyn to modulate mitochondrial potential and ATP production in vivo is now supported by many studies. The common physical origin of the interactions of both tubulin and αSyn with VDAC leads to a general model of a VDAC inhibitor, facilitates predictions of the effect of post-translational modifications of known inhibitors, and points the way toward the development of novel therapeutics targeting VDAC.     

Effect of sol maturation on the preparation of luminescent ormosils

An UV‐polymerization approach was proposed to construct luminescence organic‐inorganic hybrid. Methacryloxypropyltrimethoxysilane (MAPTMS) was chosen as sol–gel precursor to prepare the host matrix and acrylic europium Eu(AA)₃ was used as guest dopants. Structural modifications during irradiation were evident in both the inorganic and organic domains of the hybrid material. The influence of condensation degree of the silicate network on the photopolymerization kinetics of organic moieties was investigated. It appeared that the sol maturation time was of vital importance to the phase homogeneity and optical transparency of materials. To get more efficient copolymerization and higher luminescence intensity of the target ormosils, two key processing factors including incorporation time and entrapping concentration are reported. Incorporation time when acrylic europium was introduced into the matrix during sol maturation is found to be helpful to overcome phase separation. The effect of entrapping concentration of europium(III) compound on the luminescence performance was further studied. Material synthesized by this approach has three advantages: optical transparency with little phase separation, organic and inorganic composite with interpenetrating network, covalent grafting of acrylic europium without luminescent species leakage. This “photopolymerization of molecular grafting” strategy is hopeful to offer a promising development for luminescent ormosil glasses. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45146.     

Dysfunction of Mitochondria in Alzheimer’s Disease: ANT and VDAC Interact with Toxic Proteins and Aid to Determine the Fate of Brain Cells

Alzheimer’s disease (AD), certainly the most widespread proteinopathy, has as classical neuropathological hallmarks, two groups of protein aggregates: senile plaques and neurofibrillary tangles. However, the research interest is rapidly gaining ground in a better understanding of other pathological features, first, of all the mitochondrial dysfunctions. Several pieces of evidence support the hypothesis that abnormal mitochondrial function may trigger aberrant processing of amyloid progenitor protein or tau and thus neurodegeneration. Here, our aim is to emphasize the role played by two ‘bioenergetic’ proteins inserted in the mitochondrial membranes, inner and outer, respectively, that is, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), in the progression of AD. To perform this, we will magnify the ANT and VDAC defects, which are measurable hallmarks of mitochondrial dysfunction, and collect all the existing information on their interaction with toxic Alzheimer’s proteins. The pathological convergence of tau and amyloid β-peptide (Aβ) on mitochondria may finally explain why the therapeutic strategies used against the toxic forms of Aβ or tau have not given promising results separately. Furthermore, the crucial role of ANT-1 and VDAC impairment in the onset/progression of AD opens a window for new therapeutic strategies aimed at preserving/improving mitochondrial function, which is suspected to be the driving force leading to plaque and tangle deposition in AD.     

Improvement of self‐activated luminescence from introduced cation disorder in Sr6V2O11

Adjusting the elemental composition of a host is regarded to be an effective strategy to tune its luminescent properties such as peak energy, emission efficiency, and bandwidth. In this work, the cation substitution of (Ba²⁺ → Sr²⁺) in self‐activated Sr₆V₂O₁₁ was conducted to investigate the luminescence modification. All the phosphors of Sr_6‐6xBa_6xV₂O₁₁ (x = 0, 0.1, 0.2, 0.3, 0.4, 0.5) were synthesized by the traditional chemical sol‐gel method. The morphological properties were measured through scanning electron microscope and energy dispersive spectrum measurements. The cation substitution brings out the disorder in the structure, which exerts modifications on the luminescence properties of Sr₆V₂O₁₁. The luminescence intensity and corresponding decay lifetime were enhanced with the cation disorder in the self‐activated phosphor. Cation disorder in a phosphor lattice could be one of the effective approaches to improve the luminescence efficiency.     

Luminescent Conjugated Oligothiophenes for Sensitive Fluorescent Assignment of Protein Inclusion Bodies

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s‐IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co‐localization with proteins reported to accumulate in s‐IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.     

Sol-gel synthesis and luminescent properties of YVO4 : Eu nanoparticles

Experimental results on the synthesis of luminescent YVO₄: Eu nanoparticles with the method of metalopolymer gel decomposition are presented. The sizes of the coherent scattering regions according to the X-ray diffraction data range from 25 to 40 nm and increase with the increase of the annealing temperature from 800 to 1000°C. It was shown that the obtained nanoparticles demonstrate good luminescent properties. The luminescence intensity and lifetime of the excited state increase with the increase of the annealing temperature. Nanoparticles are good candidates to use as luminescent labels.     

Distinct composite structure and properties of Eu(phen)2Cl3(H2O)2 in poly(methyl methacrylate) and polyvinylpyrrolidone

The luminescent europium complex Eu(phen)₂ Cl₃(H₂O)₂ (phen refers to 1,10‐phenanthroline) was doped in poly(methyl methacrylate) (PMMA) and polyvinylpyrrolidone (PVP), respectively. The formed composite systems with different molar ratios of C?O groups in polymers and Eu ions were characterized by X‐ray diffractometry (XRD), FTIR, and photoluminescent (PL) spectroscopy and lifetime measurement. The XRD diffractograms show that the composites of PMMA/Eu(phen)₂Cl₃(H₂O)₂ and PVP/Eu (phen)₂Cl₃(H₂O)₂ have crystalline and amorphous structures, respectively, arising from different interactions between the polymers and the complex, as revealed by FTIR spectra. This leads to distinct luminescent characteristics arising from the ⁵D₀→⁷F_J transitions of Eu(III) ion (J = 0–4). For the composite systems of PMMA/complex, the characteristics of the emission lines change with decreasing molar ratios of C?O/Eu and approach that of the pure complex; whereas the composite systems of PVP/complex have similar spectral features, regardless of the molar ratios, differing from that of the pure complex. The polymer matrices have a substantial influence on the structure and properties of the composites. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3524–3530, 2004     

Isoelectric Focusing of Proteins in Gel Media

Abstract

Separation of proteins according to their isoelectric point can be performed in a pH gradient formed by stationary electrolysis of carrier ampholytes. The pH gradient is stabilized by the use of polyacrylamide, agarose, and Sephadex gels. Separated proteins can be detected by fixation with trichloroacetic acid followed by nonspecific staining, by specific staining, or through immunodiffusion techniques. Isoelectric focusing of proteins in gel media can be carried out in gel columns or on thin-layer plates by using conventional electrophoresis apparatus. Electrofocusing can be followed by electrophoresis in gel media for more complete separation of components.

Multiple samples of microgram quantities can be analyzed simultaneously by simple and rapid procedures. These methods have both analytical and preparative applications in protein fractionation work.     

Downregulation of Mitochondrial TSPO Inhibits Mitophagy and Reduces Enucleation During Human Terminal Erythropoiesis

Translocator protein (TSPO) and voltage dependent anion channels (VDAC) are two proteins forming a macromolecular complex in the outer mitochondrial membrane that is involved in pleiotropic functions. Specifically, these proteins were described to regulate the clearance of damaged mitochondria by selective mitophagy in non-erythroid immortalized cell lines. Although it is well established that erythroblast maturation in mammals depends on organelle clearance, less is known about mechanisms regulating this clearance throughout terminal erythropoiesis. Here, we studied the effect of TSPO1 downregulation and the action of Ro5-4864, a drug ligand known to bind to the TSPO/VDAC complex interface, in ex vivo human terminal erythropoiesis. We found that both treatments delay mitochondrial clearance, a process associated with reduced levels of the PINK1 protein, which is a key protein triggering canonical mitophagy. We also observed that TSPO1 downregulation blocks erythroblast maturation at the orthochromatic stage, decreases the enucleation rate, and increases cell death. Interestingly, TSPO1 downregulation does not modify reactive oxygen species (ROS) production nor intracellular adenosine triphosphate (ATP) levels. Ro5-4864 treatment recapitulates these phenotypes, strongly suggesting an active role of the TSPO/VDAC complex in selective mitophagy throughout human erythropoiesis. The present study links the function of the TSPO/VDAC complex to the PINK1/Parkin-dependent mitophagy induction during terminal erythropoiesis, leading to the proper completion of erythroid maturation.     

Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

Damage induced by oxidative stress is a key driver of the selective motor neuron death in amyotrophic lateral sclerosis (ALS). Mitochondria are among the main producers of ROS, but they also suffer particularly from their harmful effects. Voltage-dependent anion-selective channels (VDACs) are the most represented proteins of the outer mitochondrial membrane where they form pores controlling the permeation of metabolites responsible for mitochondrial functions. For these reasons, VDACs contribute to mitochondrial quality control and the entire energy metabolism of the cell. In this work we assessed in an ALS cell model whether disease-related oxidative stress induces post-translational modifications (PTMs) in VDAC3, a member of the VDAC family of outer mitochondrial membrane channel proteins, known for its role in redox signaling. At this end, protein samples enriched in VDACs were prepared from mitochondria of an ALS model cell line, NSC34 expressing human SOD1G93A, and analyzed by nUHPLC/High-Resolution nESI-MS/MS. Specific over-oxidation, deamidation, succination events were found in VDAC3 from ALS-related NSC34-SOD1G93A but not in non-ALS cell lines. Additionally, we report evidence that some PTMs may affect VDAC3 functionality. In particular, deamidation of Asn215 alone alters single channel behavior in artificial membranes. Overall, our results suggest modifications of VDAC3 that can impact its protective role against ROS, which is particularly important in the ALS context. Data are available via ProteomeXchange with identifier PXD036728.     

Reversible upconversion switching for Ho/Yb codoped (K,Na)NbO3 ceramics with excellent luminescence readout capability

Luminescent readout capability for photochromic materials plays a critical role in 3D optical data storage applications, especially for inorganic photochromic materials in the solid‐state form. In our previous studies, we found that the luminescent readout capability can be improved using two or multiple‐photon excited luminescent mode (upconversion), which can effectively decrease the destruction degree of the excitation energies to the stored information during the luminescent “reading” process. However, the luminescent readout performance is unsatisfactory owing to the absence of nondestructive luminescence readout capability. Herein, we report a new solid‐state photochromic material with excellent upconversion readout capability: Ho³⁺/Yb³⁺ codoped (K,Na)NbO_3. Upon 407 nm light irradiation, the luminescent switching contrast (ΔR_t) is up to 78%. Particularly, the materials almost have no any re‐absorption to 980 nm light, exhibiting extremely low destruction to information recording points. The luminescent readout intensity retains 96% after constant 980 nm irradiation for 4 minutes at a high pumping power of 1W, which is superior to our previously reported results (Er/Yb codoped Bi_2.5Na_0.5Nb₂O₉ materials). This work would help to further develop new inorganic photochromic materials with high performance to satisfy the requirements for optical storage devices.     

Proteomic Identification of Target Proteins of Thiodigalactoside in White Adipose Tissue from Diet-Induced Obese Rats

Previously, galectin-1 (GAL1) was found to be up-regulated in obesity-prone subjects, suggesting that use of a GAL1 inhibitor could be a novel therapeutic approach for treatment of obesity. We evaluated thiodigalactoside (TDG) as a potent inhibitor of GAL1 and identified target proteins of TDG by performing comparative proteome analysis of white adipose tissue (WAT) from control and TDG-treated rats fed a high fat diet (HFD) using two dimensional gel electrophoresis (2-DE) combined with MALDI-TOF-MS. Thirty-two spots from a total of 356 matched spots showed differential expression between control and TDG-treated rats, as identified by peptide mass fingerprinting. These proteins were categorized into groups such as carbohydrate metabolism, tricarboxylic acid (TCA) cycle, signal transduction, cytoskeletal, and mitochondrial proteins based on functional analysis using Protein Annotation Through Evolutionary Relationship (PANTHER) and Database for Annotation, Visualization, Integrated Discovery (DAVID) classification. One of the most striking findings of this study was significant changes in Carbonic anhydrase 3 (CA3), Voltage-dependent anion channel 1 (VDAC1), phosphatidylethanolamine-binding protein 1 (PEBP1), annexin A2 (ANXA2) and lactate dehydrogenase A chain (LDHA) protein levels between WAT from control and TDG-treated groups. In addition, we confirmed increased expression of thermogenic proteins as well as reduced expression of lipogenic proteins in response to TDG treatment. These results suggest that TDG may effectively prevent obesity, and TDG-responsive proteins can be used as novel target proteins for obesity treatment.     

Experimental Characterization of the Binding Affinities between Proapoptotic BH3 Peptides and Antiapoptotic Bcl‐2 Proteins

The Bcl‐2 family proteins are key regulators of the intrinsic apoptotic pathway and are among the validated targets for developing anticancer drugs. Protein–protein interactions between the pro‐ and antiapoptotic members of this family determine mitochondrial outer‐membrane permeabilization. Elucidating such protein–protein interactions in a quantitative way is helpful for network pharmacology studies on the Bcl‐2 family, which, in turn, will provide valuable guidance for developing new anticancer therapies. In this study, the binding affinities of the BH3 peptides derived from eight proapoptotic BH3‐only proteins (i.e., Bid, Bim, Puma, Noxa, Bad, Bmf, Bik, Hrk) against five well‐studied antiapoptotic proteins (i.e., Bcl‐x_L, Bcl‐2, Mcl‐1, Bcl‐w, Bfl‐1) in the Bcl‐2 family have been measured. Three different types of binding assay (i.e., surface plasmon resonance, fluorescence polarization, and homogeneous time‐resolved fluorescence) were employed for cross‐validation. The results confirmed that each proapoptotic BH3 peptide exhibited a distinct binding profile against the five antiapoptotic proteins. The binding data obtained herein serve as a fresh update or correction to existing knowledge. It is expected that such binding data will be helpful for building more accurate mathematical network models for depicting the complex protein–protein interactions within the Bcl‐2 family.