Valacyclovir and Acyclovir Are Substrates of the Guanine Deaminase Cytosolic PSD-95 Interactor (Cypin).

Valacyclovir, enzymatically hydrolyzed in the body to acyclovir, is a guanine-based nucleoside analog commonly prescribed as an antiviral therapy. Previous reports suggest that guanosine analogs bind to guanine deaminase; however, it is unclear whether they act as inhibitors or substrates. Data from our laboratory suggest that inhibition of guanine deaminase by small molecules attenuates spinal cord injury-induced neuropathic pain. Here, we examine whether the guanosine analogs valacyclovir and acyclovir are deaminated by cypin (cytosolic PSD-95 interactor), the major guanine deaminase in the body, or if they act as cypin inhibitors. Using purified Rattus norvegicus cypin, we use NADH-coupled assay to confirm deamination of valacyclovir and determined Michaelis-Menten constants. Subsequently, we use tryptophan fluorescence quenching assay to calculate dissociation constants for valacyclovir and acyclovir and find that inclusion of the valine motif in valacyclovir increases affinity for cypin compared to acyclovir. To our knowledge, neither Km nor KD values for cypin has been previously reported for either compound. We use Amplex Red assay and demonstrate that both valacyclovir and acyclovir are cypin substrates and that their metabolites are further processed by xanthine oxidase and uricase. Using molecular dynamics simulations, we demonstrate that an alpha helix near the active site is displaced when valacyclovir binds to cypin. Furthermore, we used LC-MS-based assay to directly confirm deamination of valacyclovir by cypin. Taken together, our results demonstrate a novel role for cypin in deamination of valacyclovir and acyclovir and suggest that therapeutics based on purine structures may be inactivated by cypin, decreasing inhibitory efficacy.

Study of changes in folding/unfolding properties and stability of Arabidopsis thaliana MYB12 transcription factor following UV-B exposure in vitro.

Flavonoids mostly protect plant cells from the harmful effects of UV-B radiation from the sun. In plants, the R2R3-subfamily of the MYB transcription factor, MYB12, is a key inducer of the biosynthesis of flavonoids. Our study involves the biophysical characterization of Arabidopsis thaliana MYB12 protein (AtMYB12) under UV-B exposure in vitro. Tryptophan fluorescence studies using recombinant full-length AtMYB12 (native) and the N-terminal truncated versions (first N-terminal MYB domain absent in AtMYB12Δ1, and both the first and second N-terminal MYB domains absent in AtMYB12Δ2) have revealed prominent alteration in the tryptophan microenvironment in AtMYB12Δ1 and AtMYB12Δ2 protein as a result of UV-B exposure as compared with the native AtMYB12. Bis-ANS binding assay and urea-mediated denaturation profiling showed an appreciable change in the structural conformation in AtMYB12Δ1 and AtMYB12Δ2 proteins as compared with the native AtMYB12 protein following UV-B irradiation. UV-B-treated AtMYB12Δ2 showed a higher predisposition of aggregate formation in vitro. CD spectral analyses revealed a decrease in α-helix percentage with a concomitant increase in random coiled structure formation in AtMYB12Δ1 and AtMYB12Δ2 as compared to native AtMYB12 following UV-B treatment. Overall, these findings highlight the critical function of the N-terminal MYB domains in maintaining the stability and structural conformation of the AtMYB12 protein under UV-B stress in vitro.

A rapid method to monitor structural perturbations of high-concentrated therapeutic antibody solutions using Intrinsic Tryptophan Fluorescence Emission spectroscopy.

Drug product development of therapeutic antibody formulations is still dictated by the risk of protein particle formation during processing or storage, which can lead to loss of potency and potential immunogenic reactions. Since structural perturbations are the main driver for irreversible protein aggregation, the conformational integrity of antibodies should be closely monitored. The present study evaluated the applicability of a plate reader-based high throughput method for Intrinsic Tryptophan Fluorescence Emission (ITFE) spectroscopy to detect protein aggregation due to protein unfolding in high-concentrated therapeutic antibody samples. The impact of fluorophore concentration on the ITFE signal in microplate readers was investigated by analysis of dilution series of two therapeutic antibodies and pure tryptophan. At low antibody concentrations (< 5 mg/mL, equivalent to 0.8 mM tryptophan), the low inner filter effect suggests a quasi-linear relationship between antibody concentration and ITFE intensity. In contrast, the constant ITFE intensity at high protein concentrations (> 40 mg/mL, equivalent to 6.1 mM tryptophan) indicate that ITFE spectroscopy measurements of IgG1 antibodies are feasible in therapeutically relevant concentrations (up to 223 mg/mL). Furthermore, the capability of the method to detect low levels of unfolding (around 1 %) was confirmed by limit of detection (LOD) determination with temperature-stressed antibody samples as degradation standards. Change of fluorescence intensity at the maximum (ΔIaM) was identified as sensitive descriptor for protein degradation, providing the lowest LOD values. The results demonstrate that ITFE spectroscopy performed in a microplate reader is a valuable tool for high-throughput monitoring of protein degradation in therapeutic antibody formulations.

The molecular determinants of calcium ATPase inhibition by curcuminoids.

The natural product curcumin and some of its analogs are known inhibitors of the transmembrane enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). Despite their widespread use, the curcuminoids' binding site in SERCA and their relevant interactions with the enzyme remain elusive. This lack of knowledge has prevented the development of curcuminoids into valuable experimental tools or into agents of therapeutic value. We used the crystal structures of SERCA in its E1 conformation in conjunction with computational tools such as docking and surface screens to determine the most likely curcumin binding site, along with key enzyme/inhibitor interactions. Additionally, we determined the inhibitory potencies and binding affinities for a small set of curcumin analogs. The predicted curcumin binding site is a narrow cleft in the transmembrane section of SERCA, close to the transmembrane/cytosol interface. In addition to pronounced complementarity in shape and hydrophobicity profiles between curcumin and the binding pocket, several hydrogen bonds were observed that were spread over the entire curcumin scaffold, involving residues on several transmembrane helices. Docking-predicted interactions were compatible with experimental observations for inhibitory potencies and binding affinities. Based on these findings, we propose an inhibition mechanism that assumes that the presence of a curcuminoid in the binding site arrests the catalytic cycle of SERCA by preventing it from converting from the E1 to the E2 conformation. This blockage of conformational change is accomplished by a combination of steric hinderance and hydrogen-bond-based cross-linking of transmembrane helices that require flexibility throughout the catalytic cycle.

Transfer of ANS-Like Drugs from Micellar Drug Delivery Systems to Albumin Is Highly Favorable and Protected from Competition with Surfactant by "Reserved" Binding Sites.

Micellar drug delivery systems (MDDS) for the intravenous administration of poorly soluble drugs have great advantages over alternative formulations in terms of the safety of their excipients, storage stability, and straightforward production. A classic example is mixed micelles of glycocholate (GC) and lecithin, both endogenous substances in human blood. What limits the use of MDDS is the complexity of the transitions after injection. In particular, as the MDDS disintegrate partially or completely after injection, the drug has to be transferred safely to endogenous carriers in the blood, such as human serum albumin (HSA). If this transfer is compromised, the drug might precipitate─a process that needs to be excluded under all circumstances. The key question of this paper is whether the high local concentration of GC at the moment and site of MDDS dissolution might transiently saturate HSA binding sites and, hence, endanger quick drug transfer. To address this question, we have used a new approach, which is time-resolved fluorescence spectroscopy of the single tryptophan in HSA, Trp-214, to characterize the competitive binding of GC and the drug substitute anilinonaphthalenesulfonate (ANS) to HSA. Time-resolved fluorescence of Trp-214 showed important advantages over established methods for tackling this problem. ANS has been the standard "model drug" to study albumin binding for decades, given its structural similarity to the class of naphthalene-containing acidic drugs and the fact that it is displaced from HSA by numerous drugs (which presumably bind to the same sites). Our complex global fit uses the critical approximation that the average lifetimes behave similarly to a single lifetime, but the resulting errors are found to be moderate and the results provide a convincing explanation of the, at first glance, counterintuitive behavior. Accordingly, and largely in line with the literature, we observed two types of sites binding ANS at HSA: 3 type A, rather peripheral, and 2 type B, likely more central sites. The latter quench Trp-214 by Förster Resonance Energy Transfer (FRET) with a rate constant of ≈0.4 ns-1 per ANS. Adding millimolar concentrations of GC displaces ANS from the A sites but not from B sites. At incomplete ANS saturation, this causes a GC-induced translocation of ANS from A to the more FRET-active B sites. This leads to the apparent paradox that the partial displacement of ANS from HSA increases its quenching effect on Trp-214. The most important conclusion is that (ANS-like) drugs cannot be displaced from the type-B sites, and consequently, drug transfer to these sites is not impaired by competitive binding of GC in the vicinity of a dissolving micelle. The second conclusion is that for unbound GC above the CMC (9 mM), ANS equilibrates between HSA and GC micelles but with a strong preference for free sites on HSA. That means that even persisting micelles would lose their cargo readily once exposed to HSA. For all MDDS sharing this property, targeted drug delivery approaches involving them as the nanocarrier would be pointless.

Spectroscopic analysis of the bacterially expressed head domain of rotavirus VP6.

The rotavirus capsid protein VP6 forms the middle of three protein layers and is responsible for many critical steps in the viral life cycle. VP6 as a structural protein can be used in various applications including as a subunit vaccine component. The head domain of VP6 (VP6H) contains key sequences that allow the protein to trimerize and that represent epitopes that are recognized by human antibodies in the viral particle. The domain is rich in ß-sheet secondary structures. Here, VP6H was solubilised from bacterial inclusion bodies and purified using a single affinity chromatography step. Spectral (far-UV circular dichroism and intrinsic tryptophan fluorescence) analysis revealed that the purified domain had native-like secondary and tertiary structures. The domain could maintain structure up to 44°C during thermal denaturation following which structural changes result in an intermediate forming and finally irreversible aggregation and denaturation. The chemical denaturation with urea and guanidinium hydrochloride produces intermediates that represent a loss in the cooperativity. The VP6H domain is stable and can fold to produce its native structure in the absence of the VP6 base domain but cannot be defined as an independent folding unit.

A conserved but structurally divergent loop in acyl protein thioesterase 1 regulates its catalytic activity, ligand binding, and folded stability.

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding ß-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/ß hydrolase structure and a potential allosteric binding site within human APTs.

The four subunits of rabbit skeletal muscle lactate dehydrogenase do not exert their catalytic action additively.

Oligomeric enzymes containing multiple active sites are usually considered to perform their catalytic action at higher rates when compared with their monomeric counterparts. This implies, in turn, that the activity performed by different holoenzyme subunits features additivity. Nevertheless, the extent of this additivity occurring in holoenzymes is far from being adequately understood. To tackle this point, we used tetrameric rabbit lactate dehydrogenase (rbLDH) as a model system to assay the reduction of pyruvate catalysed by this enzyme at the expense of ß-NADH under pre-steady-state conditions. In particular, we observed the kinetics of reactions triggered by concentrations of ß-NADH equimolar to 1, 2, 3, or all 4 subunits of the rbLDH holoenzyme, in the presence of an excess of pyruvate. Surprisingly, when the concentration of the limiting reactant exceeded that of a single holoenzyme subunit, we observed a sharp slowdown of the enzyme conformational rearrangements associated to the generation and the release of l-lactate. Furthermore, using a model to interpret the complex kinetics observed under the highest concentration of the limiting reactant, we estimated the diversity of the rates describing the action of the different rbLDH subunits.

In vitro binding analysis of legacy-linear and new generation-cyclic perfluoro-alkyl substances on sex hormone binding globulin and albumin, suggests low impact on serum hormone kinetics of testosterone.

In humans, serum testosterone (T) is largely bound to the sex hormone binding globulin (SHBG) and human serum albumin (hSA), resulting in a 2-3 % of unbound or "free" active quote (FT). Endocrine-disrupting chemicals, including perfluoro-alkyl substances (PFAS), are recognized to interfere with the hormonal axes, but the possible impact on the FT quote has not been addressed so far. Here we investigated the possible competition of two acknowledged PFAS molecules on T binding to SHBG and hSA. In particular, perfluoro-octanoic acid (PFOA) and acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)-1,3-dioxolan-4-yl)oxy)-ammonium salt (1:1) (C6O4) were used as, respectively, legacy-linear and new-generation-cyclic PFASs. Human recombinant SHBG 30-234 domain (SHBG30-234), produced in HEK293-F cells, and delipidated recombinant hSA were used as in vitro protein models. Isothermal Titration Calorimetry (ITC) and tryptophan fluorescence quencing (TFQ) were used to evaluate the binding modes of T and PFAS to SHBG30-234 and hSA. ITC revealed the binding of T to SHBG30-234 with a Kd of 44 ± 2 nM whilst both PFOA and C6O4 showed no binding activity. Results were confirmed by TFQ, since only T modified the fluorescence profile of SHBG30-234. In hSA, TFQ confirmed the binding of T on FA6 site of the protein. A similar binding mode was observed for PFOA but not for C6O4, as further verified by displacement experiments with T. Although both PFASs were previously shown to bind hSA, only PFOA is predicted to possibly compete with T for the binding to hSA. However, on the base of the binding stoichiometry and affinity of PFOA for hSA, this appears unlikely at the blood concentrations of the chemical documented to date.

Red edge excitation shift spectroscopy is highly sensitive to tryptophan composition.

Red edge excitation shift (REES) spectroscopy relies on the unique emission profiles of fluorophore-solvent interactions to profile protein molecular dynamics. Recently, we reported the use of REES to compare the stability of 32 polymorphic IgG antibodies natively containing tryptophan reporter fluorophores. Here, we expand on this work to investigate the sensitivity of REES to variations in tryptophan content using a subset of IgG3 antibodies containing arginine to tryptophan polymorphisms. Structural analysis revealed that the additional tryptophan residues were situated in highly solvated environments. Subsequently, REES showed clear differences in fluorescence emission profiles when compared with the unmutated variants, thereby limiting direct comparison of their structural dynamics. These findings highlight the exquisite sensitivity of REES to minor variations in protein structure and tryptophan composition.

Avidin cooperative allosterism upon binding biotin observed by differential changes in intrinsic fluorescence.

Similar to streptavidin, the binding of biotin by avidin does not appear to be cooperative in the traditional sense of altered binding strength, though it appears to be cooperative in terms of ligand induced structural communication across subunits in the protein as previously shown for streptavidin. In this work we provide data from intrinsic tryptophan fluorescence as evidence of a cooperative structural change. The technique involves examination of the changes in fluorescence emission corresponding to the various tryptophan populations accompanying avidin-biotin binding. We note that the 335 nm emission population (i.e. more hydrophobic local environment) saturates prior to full ligation and the saturation of the 350 nm emission population commonly used in standard binding activity assays. We also note that total integrated fluorescence emission and peak height during the titration of ligand into streptavidin also reach saturation prior to the 4:1 stoichiometric end point. Unique to avidin and distinct from the behavior of streptavidin described in our prior work, the wavelength of maximum emission and full width at half maximum (FWHM) data do not saturate prior to the 4:1 stoichiometric end point. Avidin also exhibited larger FWHM for both apo and holo forms suggesting greater heterogeneity in local tryptophan environments, as compared to streptavidin. Taken together, the data suggests that the binding of the first 3 biotins effect greater structural changes in the protein than the final ligand in a similar way for avidin and streptavidin.

Steady-State and Time-Resolved Fluorescence Study of Selected Tryptophan-Containing Peptides in an AOT Reverse Micelle Environment.

The aim of this study was to demonstrate the utility of time-resolved fluorescence spectroscopy in the detection of subtle changes in the local microenvironment of a tryptophan chromophore in a confined and crowded medium of AOT reverse micelles, which mimic biological membranes and cell compartmentalization. For this purpose, fluorescence properties of L-tryptophan and several newly synthesized tryptophan-containing peptides in buffer and in an AOT reverse micelle medium were determined. It was shown that insertion of tryptophan and its short di- and tripeptides inside micelles led to evident changes in both the steady-state emission spectra and in fluorescence decay kinetics. The observed differences in spectral characteristics, such as a blue shift in the emission maxima, changes in the average fluorescence lifetime, and the appearance of environmental-dependent fluorescent species, showed the utility of time-resolved fluorescence spectroscopy as a sensitive tool for detecting subtle conformational modifications in tryptophan and its peptides induced by changes in polarity, viscosity, and specific interactions between chromophores and water molecules/polar groups/ions that occur inside reverse micelles.

Changes in Hemoglobin Properties in Complex with Glutathione and after Glutathionylation.

Hemoglobin is the main protein of red blood cells that provides oxygen transport to all cells of the human body. The ability of hemoglobin to bind the main low-molecular-weight thiol of the cell glutathione, both covalently and noncovalently, is not only an important part of the antioxidant protection of red blood cells, but also affects its affinity for oxygen in both cases. In this study, the properties of oxyhemoglobin in complex with reduced glutathione (GSH) and properties of glutathionylated hemoglobin bound to glutathione via an SS bond were characterized. For this purpose, the methods of circular dichroism, Raman spectroscopy, infrared spectroscopy, tryptophan fluorescence, differential scanning fluorimetry, and molecular modeling were used. It was found that the glutathionylation of oxyhemoglobin caused changes in the secondary structure of the protein, reducing the alpha helicity, but did not affect the heme environment, tryptophan fluorescence, and the thermostability of the protein. In the noncovalent complex of oxyhemoglobin with reduced glutathione, the secondary structure of hemoglobin remained almost unchanged; however, changes in the heme environment and the microenvironment of tryptophans, as well as a decrease in the protein's thermal stability, were observed. Thus, the formation of a noncovalent complex of hemoglobin with glutathione makes a more significant effect on the tertiary and quaternary structure of hemoglobin than glutathionylation, which mainly affects the secondary structure of the protein. The obtained data are important for understanding the functioning of glutathionylated hemoglobin, which is a marker of oxidative stress, and hemoglobin in complex with GSH, which appears to deposit GSH and release it during deoxygenation to increase the antioxidant protection of cells.

Structural Insights into the Ligand-LsrK Kinase Binding Mode: A Step Forward in the Discovery of Novel Antimicrobial Agents.

LsrK is a bacterial kinase that triggers the quorum sensing, and it represents a druggable target for the identification of new agents for fighting antimicrobial resistance. Herein, we exploited tryptophan fluorescence spectroscopy (TFS) as a suitable technique for the identification of potential LsrK ligands from an in-house library of chemicals comprising synthetic compounds as well as secondary metabolites. Three secondary metabolites (Hib-ester, Hib-carbaldehyde and (R)-ASME) showed effective binding to LsrK, with KD values in the sub-micromolar range. The conformational changes were confirmed via circular dichroism and molecular docking results further validated the findings and displayed the specific mode of interaction. The activity of the identified compounds on the biofilm formation by some Staphylococcus spp. was investigated. Hib-carbaldehyde and (R)-ASME were able to reduce the production of biofilm, with (R)-ASME resulting in the most effective compound with an EC50 of 14 mg/well. The successful application of TFS highlights its usefulness in searching for promising LsrK inhibitor candidates with inhibitor efficacy against biofilm formation.

Peptide Models of the Cytoplasmic Tail of Influenza A/H1N1 Virus Hemagglutinin Expand Understanding its pH-Dependent Modes of Interaction with Matrix Protein M1.

Influenza A virus hemagglutinin (HA) is a major virus antigen. No cryo-electron microscopy or X-ray data can be obtained for the HA intraviral (cytoplasmic) domain (CT) post-translationally modified with long fatty acid residues bound to three highly conserved cysteines. We recently proposed a model of HA CT of Influenza A/H1N1 virus possessing an antiparallel beta structure based on the experimental secondary structure analysis of four 14-15 amino acid long synthetic peptides, corresponding to the HA CT sequence, with free or acetaminomethylated cysteines. To dispel doubts about possible non-specific "amyloid-like" aggregation of those synthetic peptides in phosphate buffer solution, we have determined the order of oligomers based on blue native gel electrophoresis, membrane filtration, fluorescence spectroscopy and molecular modeling approaches. We have found that unmodified peptides form only low molecular weight oligomers, while modified peptides form both oligomers of low order similar to those found for unmodified peptides and high order conglomerates, which however are not of beta-amyloid-like fold. This study confirms that the beta structure previously detected by circular dichroism spectroscopy analysis is more likely the result of intrinsic propensity of the HA CT amino acid sequence than the consequence of aggregation. The structures of low order oligomers of the synthetic peptides were used for in silico experiments on modeling of HA CT interactions with matrix protein M1 at physiological and acidic pH levels and revealed two different areas of binding. Finally, tripeptides capable of blocking interactions between HA CT and M1 were proposed.

Early Stage UV-B Induced Molecular Modifications of Human Eye Lens γD-Crystallin.

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, ß-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains. Early UV-B induced damage of hγD with molecular resolution is studied by solution NMR and fluorescence spectroscopy. hγD modifications are restricted to Tyr 17 and Tyr 29 in the N-terminal domain, where a local unfolding of the hydrophobic core is observed. None of the tryptophan residues assisting fluorescence energy transfer is modified and hγD is remained soluble over month. Investigating isotope-labeled hγD surrounded by eye lens extracts from cataract patients reveals very week interactions of solvent-exposed side chains in the C-terminal hγD domain and some remaining photoprotective properties of the extracts. Hereditary E107A hγD found in the eye lens core of infants developing cataract shows under the here used conditions a thermodynamic stability comparable to the wild type but an increased sensitivity toward UV-B irradiation.

A Neuroprotective Action of Quercetin and Apigenin through Inhibiting Aggregation of Aß and Activation of TRKB Signaling in a Cellular Experiment.

Alzheimer's disease (AD) is a neurodegenerative disease with progressive memory loss and the cognitive decline. AD is mainly caused by abnormal accumulation of misfolded amyloid ß (Aß), which leads to neurodegeneration via a number of possible mechanisms such as down-regulation of brain-derived neurotrophic factor-tropomyosin-related kinase B (BDNF-TRKB) signaling pathway. 7 ,8-Dihydroxyflavone (7,8-DHF), a TRKB agonist, has demonstrated potential to enhance BDNF-TRKB pathway in various neurodegenerative diseases. To expand the capacity of flavones as TRKB agonists, two natural flavones quercetin and apigenin, were evaluated. With tryptophan fluorescence quenching assay, we illustrated the direct interaction between quercetin/apigenin and TRKB extracellular domain. Employing Aß folding reporter SH-SY5Y cells, we showed that quercetin and apigenin reduced Aß-aggregation, oxidative stress, caspase-1 and acetylcholinesterase activities, as well as improved the neurite outgrowth. Treatments with quercetin and apigenin increased TRKB Tyr516 and Tyr817 and downstream cAMP-response-element binding protein (CREB) Ser133 to activate transcription of BDNF and BCL2 apoptosis regulator (BCL2), as well as reduced the expression of pro-apoptotic BCL2 associated X protein (BAX). Knockdown of TRKB counteracted the improvement of neurite outgrowth by quercetin and apigenin. Our results demonstrate that quercetin and apigenin are to work likely as a direct agonist on TRKB for their neuroprotective action, strengthening the therapeutic potential of quercetin and apigenin in treating AD.

UBQLN2 undergoes a reversible temperature-induced conformational switch that regulates binding with HSPA1B: ALS/FTD mutations cripple the switch but do not destroy HSPA1B binding.

Here we present evidence, based on alterations of its intrinsic tryptophan fluorescence, that UBQLN2 protein undergoes a conformational switch when the temperature is raised from 37 °C to 42 °C. The switch is reset on restoration of the temperature. We speculate that the switch regulates UBQLN2 function in the heat shock response because elevation of the temperature from 37 °C to 42 °C dramatically increased in vitro binding between UBQLN2 and HSPA1B. Furthermore, restoration of the temperature to 37 °C decreased HSPA1B binding. By comparison to wild type (WT) UBQLN2, we found that all five ALS/FTD mutant UBQLN2 proteins we examined had attenuated alterations in tryptophan fluorescence when shifted to 42 °C, suggesting that the conformational switch is crippled in the mutants. Paradoxically, all five mutants bound similar amounts of HSPA1B compared to WT UBQLN2 protein at 42 °C, suggesting that either the conformational switch is not instrumental for HSPA1B binding, or that, although damaged, it is still functional. Comparison of the poly-ubiquitin chain binding revealed that WT UBQLN2 binds more avidly with K63 than with K48 chains. The avidity may explain the involvement of UBQLN2 in autophagy and cell signaling. Consistent with its function in autophagy, we found UBQLN2 binds directly with LC3, the autophagosomal-specific membrane-tethered protein. Finally, we provide evidence that WT UBQLN2 can homodimerize, and heterodimerize with WT UBQLN1. We show that ALS mutant P497S-UBQLN2 protein can oligomerize with either WT UBQLN1 or 2, providing a possible mechanism for how mutant UBQLN2 proteins could bind and inactivate UBQLN proteins, causing loss of function.

Analysis of Biologics Molecular Descriptors towards Predictive Modelling for Protein Drug Development Using Time-Gated Raman Spectroscopy.

Pharmaceutical proteins, compared to small molecular weight drugs, are relatively fragile molecules, thus necessitating monitoring protein unfolding and aggregation during production and post-marketing. Currently, many analytical techniques take offline measurements, which cannot directly assess protein folding during production and unfolding during processing and storage. In addition, several orthogonal techniques are needed during production and market surveillance. In this study, we introduce the use of time-gated Raman spectroscopy to identify molecular descriptors of protein unfolding. Raman spectroscopy can measure the unfolding of proteins in-line and in real-time without labels. Using K-means clustering and PCA analysis, we could correlate local unfolding events with traditional analytical methods. This is the first step toward predictive modeling of unfolding events of proteins during production and storage.

Modeling of Proteolysis of ß-Lactoglobulin and ß-Casein by Trypsin with Consideration of Secondary Masking of Intermediate Polypeptides.

The opening of protein substrates during degradation by proteases and the corresponding exposure of their internal peptide bonds for a successful enzymatic attack, the so-called demasking effect, was studied for ß-lactoglobulin (ß-LG) and ß-casein (ß-CN) hydrolyzed by trypsin. Demasking was estimated by monitoring the redshift in intrinsic tryptophan fluorescence, characterizing the accessibility of polypeptide chains to aqueous medium. The secondary masking of intermediate polypeptides, giving an inverse effect to demasking, caused a restriction of the substrate opening. This led to the limitations in the red shift of fluorescence and the degree of hydrolysis with a long time of hydrolysis of ß-LG and ß-CN at a constant substrate concentration and reduced trypsin concentrations. The proposed proteolysis model included demasking of initially masked bonds in the protein globule or micelle, secondary masking of intermediate polypeptides, and their subsequent slow demasking. The hydrolysis of peptide bonds was modeled taking into account different hydrolysis rate constants for different peptide bonds. It was demonstrated that demasking competes with secondary masking, which is less noticeable at high trypsin concentrations. Modeling of proteolysis taking into account two demasking processes and secondary masking made it possible to simulate kinetic curves consistent with the experimental data.