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Abstract Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among their complex clinical features, including musculoskeletal, neurological, and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early-onset Alzheimer’s disease (AD). This dementia is attributed to the increased gene dosage of the amyloid-β (Aβ) precursor protein gene, the formation of self-propagating Aβ and tau prion conformers, and the deposition of neurotoxic Aβ plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here, we report the characterization of brain samples from four DS cases spanning 36–63 years of age by spectral confocal imaging with conformation-specific dyes and cryo–electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures revealed paired helical filament (PHF) and straight filament (SF) conformations of tau that were identical to those determined from AD cases. The PHFs and SFs are made of two C-shaped protofilaments, each containing a cross-β/β-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (approximately 20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene oxide surface derivatized with anti-tau antibodies. This method improved isolation and revealed that primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II–like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.

Orthopedic disorders are increasing in our society due to population aging. Numerous biomaterials have been developed to support bone regeneration, however showing a strong discrepancy between in vitro and in vivo results. This has been attributed to a lack of knowledge about protein adsorption, an early step occurring after biomaterial implantation. Bioactive glass S53P4 is clinically accepted for orthopedic applications pertaining to its osteoconductive and osteogenic behavior. However, its interactions with proteins are still unclear. To better understand the impact of surface chemistry on the glass-protein interactions, bare and silanized S53P4 were placed in contact with fibronectin (fn), in static and dynamic conditions. The surfaces were characterized by zeta potential, confocal microscopy and FTIR-ATR spectroscopy.The impact of fn on the cell response was assessed by live-dead, proliferation and morphology tests, using human adipose stem cells (hASCs). Both S53P4 and silanized-S53P4 showed good cell viability. Fn was found to affect cell alignment on both bare and silanized substrates. The impact of the surface treatments on osteogenesis was evaluated studying the expression of relevant osteogenic markers (hDLX5, hRUNX2A, hOSTERIX), which was particularly promoted by the concomitant action of silanization and fn coating.

The effects of glutamine transaminase (TGase), hydroxypropyl distarch phosphate (HDP), gellan gum and their complex (THG) on the water distribution and protein conformation of low-salt tilapia surimi gel prepared with microwave and ultrasound were analyzed. In addition, the effects of different exogenous substances on the characteristics of low-salt tilapia fish cake were explored through in vitro digestion experiment. The results showed that compared with the control group, THG increased the bound water and immovable water of surimi to 98.71% and 14.75%, respectively, and significantly decreased the free water content (P

The present study investigated the impact of plasma-activated water (PAW), slightly acidic electrolytic water (SAEW) and plasma-activated slightly acidic electrolytic water (PASW) treatment on myofibrillar protein (MP) in salmon fillets. Additionally, the interaction mechanism between myosin and reactive oxygen species was explored by molecular docking. Compared with the control group (719.26 nm), PASW treatment group exhibited the smallest particle size (408.97 nm). The PASW treatment exhibited efficacy in reducing MP aggregation and inhibiting protein oxidation. In comparison with other treatments, PASW treatment demonstrated a greater ability to mitigate damage to the secondary and tertiary structures of MP. O3 and H2O2 interact with myosin through hydrogen bonding. Specifically, O3 interacts with Lys676, Gly677, and Met678 of myosin while H2O2 binds to Thr681, Asp626, Arg680, and Met678. This study offers novel insights into the impact of PASW on MP, and provides a theoretical foundation for its application in aquatic product processing.

AapoA-I, the main protein of high-density lipoprotein, plays a key role in the biogenesis and atheroprotective properties of high-density lipoprotein. We showed previously that a naturally occurring apoA-I mutation, L178P, induces major defects in protein’s structural integrity and functions that may underlie the increased cardiovascular risk observed in carriers of the mutation. Here, a library of marketed drugs (956 compounds) was screened against apoA-I[L178P] to identify molecules that can stabilize the normal conformation of apoA-I. Screening was performed by the thermal shift assay in the presence of fluorescent dye SYPRO Orange. As an orthogonal assay, we monitored the change in fluorescence intensity of 8-anilinonaphthalene-1-sulfonic acid upon binding on hydrophobic sites on apoA-I. Screening identified four potential structure correctors. Subsequent analysis of the concentration-dependent effect of these compounds on secondary structure and thermodynamic stability of WT apoA-I and apoA-I[L178P] (assessed by thermal shift assay and circular dichroism spectroscopy), as well as on macrophage viability, narrowed the potential structure correctors to two, the drugs atorvastatin and bexarotene. Functional analysis showed that these two compounds can restore the defective capacity of apoA-I[L178P] to promote cholesterol removal from macrophages, an important step for atheroprotection. Computational docking suggested that both drugs target a positively charged cavity in apoA-I, formed between helix 1/2 and helix 5, and make extensive interactions that could underlie thermodynamic stabilization. Overall, our findings indicate that small molecules can correct defective apoA-I structure and function and may lead to novel therapeutic approaches for apoA-I–related dyslipidemias and increased cardiovascular risk.

High-moisture extrusion technology has the characteristics of low energy consumption, green processing and high-efficiency production. Extruded products produced using plant proteins as the major raw material can be processed directly without rehydration for consumption, but have some problems such as poor texture, weak mouthfeel and flavor, so they require quality improvement. The current research on physicochemical changes during high-moisture extrusion has provided evidence that exogenous additives are useful for product quality improvement and innovations and can endow the product with more functional properties. This article reviews the research progress on modifiers for the quality improvement of high-moisture extruded plant protein products, outlines the classification and characteristics of plant proteins and modifiers, and summarizes the methods used to evaluate the product quality, with a focus on the role of modifiers in regulating the conformation, functional properties and quality of high-moisture extruded plant protein products, aiming to provide a reference for the quality control of high-moisture extruded products.

To improve the gel properties of squid surimi products, we optimized the addition of modified corn starch acetylated distarch phosphate (ADSP) and the non-muscle proteins egg white protein (EWP) and soy protein isolate (SPI), and we evaluated the effects of the addition of ADSP, EWP and SPI on the texture, intermolecular force, protein secondary structure and microstructure of squid surimi products. Furthermore, we explored the mechanism by which modified starch and non-muscle protein improve the gel quality of squid surimi products. The results showed that the gel properties of squid surimi products with 10% ADSP, 7% EWP and 6% SPI were the best. Compared with the control group, the addition of modified starch or non-muscle protein decrease the relative contents of random coil and α-helix in proteins, and increased the contents of β-sheet and β-turn. Their combined addition was beneficial for the retention of β-sheet and β-turn structures during the gelation process, and promoted the hydrophobic interaction between proteins, resulting in the formation of a denser gel network structure, which in turn improved the gel strength and water holding capacity of squid surimi products. Correlation analysis showed that the gel strength and water holding capacity, hydrophobic interaction, and relative contents of β-turn and immobilized water of squid surimi products were significantly positively correlated with each other (P < 0.01), and significantly negatively correlated with the contents of free water and random coil (P < 0.01). Therefore, the addition of 10% ADSP, 7% EWP and 6% SPI improved the gel quality of squid surimi products by affecting protein secondary structure and water distribution.

The Hofmeister series categorizes ions based on their effects on protein stability, yet the microscopic mechanism remains a mystery. In this series, NaCl is neutral, Na2SO4 and Na2HPO4 are kosmotropic, while GdmCl and NaSCN are chaotropic. This study employs CD and NMR to investigate the effects of NaCl, Na2SO4, and Na2HPO4 on the conformation, stability, binding, and backbone dynamics (ps-ns and µs-ms time scales) of the WW4 domain with a high stability and accessible side chains at concentrations ≤ 200 mM. The results indicated that none of the three salts altered the conformation of WW4 or showed significant binding to the four aliphatic hydrophobic side chains. NaCl had no effect on its thermal stability, while Na2SO4 and Na2HPO4 enhanced the stability by ~5 °C. Interestingly, NaCl only weakly interacted with the Arg27 amide proton, whereas Na2SO4 bound to Arg27 and Phe31 amide protons with Kd of 32.7 and 41.6 mM, respectively. Na2HPO4, however, bound in a non-saturable manner to Trp9, His24, and Asn36 amide protons. While the three salts had negligible effects on ps-ns backbone dynamics, NaCl and Na2SO4 displayed no effect while Na2HPO4 significantly increased the µs-ms backbone dynamics. These findings, combined with our recent results with GdmCl and NaSCN, suggest a microscopic mechanism for the Hofmeister series. Additionally, the data revealed a lack of simple correlation between thermodynamic stability and backbone dynamics, most likely due to enthalpy–entropy compensation. Our study rationalizes the selection of chloride and phosphate as the primary anions in extracellular and intracellular spaces, as well as polyphosphate as a primitive chaperone in certain single-cell organisms.

The formation of amyloid fibrils is a common feature of many protein systems. It has implications in both health, as amyloid fibrils are implicated in over 30 degenerative diseases, and in the biological functions of proteins. Surfaces have long been known to affect the formation of fibrils but the specific effect depends on the details of both the surface and protein. Fully understanding the role of surfaces in fibrillization requires microscopic information on protein conformation on surfaces. In this paper replica exchange molecular dynamics simulation is used to investigate the model fibril forming protein, Aβ(10–40) (a 31-residue segment of the amyloid-beta protein) on surfaces of different hydrophobicity. Similar to other proteins Aβ(10–40) is found to adsorb strongly onto hydrophobic surfaces. It also adopts significantly different sets of conformations on hydrophobic and polar surfaces, as well as in bulk solution. On hydrophobic surfaces, it adopts partially helical structures, with the helices overlapping with beta-strand regions in the mature fibril. These may be helical intermediates on the fibril formation pathway, suggesting a mechanism for the enhanced fibril formation seen on hydrophobic surfaces.

GdmCl and NaSCN are two strong chaotropic salts commonly used in protein folding and stability studies, but their microscopic mechanisms remain enigmatic. Here, by CD and NMR, we investigated their effects on conformations, stability, binding and backbone dynamics on ps-ns and µs-ms time scales of a 39-residue but well-folded WW4 domain at salt concentrations ≤200 mM. Up to 200 mM, both denaturants did not alter the tertiary packing of WW4, but GdmCl exerted more severe destabilization than NaSCN. Intriguingly, GdmCl had only weak binding to amide protons, while NaSCN showed extensive binding to both hydrophobic side chains and amide protons. Neither denaturant significantly affected the overall ps-ns backbone dynamics, but they distinctively altered µs-ms backbone dynamics. This study unveils that GdmCl and NaSCN destabilize a protein before the global unfolding occurs with differential binding properties and µs-ms backbone dynamics, implying the absence of a simple correlation between thermodynamic stability and backbone dynamics of WW4 at both ps-ns and µs-ms time scales.

ABSTRACTCongenital thrombotic thrombocytopenic purpura (TTP) is a rare autosomal recessive genetic disorder caused by mutations in the ADAMTS13 gene. Approximately 200 mutations of the ADAMTS-13 gene have been identified, although only a few have been characterized through in vitro expression studies. We conducted an investigation on a male congenital TTP patient with reduced plasma levels of ADAMTS13 activity. DNA sequence analysis revealed two mutations on chromosome 9 (1.9q34.2) in the patient’s ADAMTS13 gene. One mutation was a non-synonymous mutation (exon 5: c.A530G: p.Y177C), while the other was a nonsense mutation (exon 21: c.G2651A: p.W884X). Both mutations were found to be heterozygous. The patient’s parents had no history of thrombocytopenia or neurological symptoms. DNA sequence analysis showed the patient’s father was a heterozygote for the nonsense mutation of the ADAMTS13 gene (exon 21: c.G2651A: p.W884X), while the mother was a heterozygote for the non-synonymous mutation of the ADAMTS13 gene (exon 5: c.A530G: p.Y177C). To investigate the mechanism behind ADAMTS13 deficiency in this patient, wild type (WT), ADAMTS13 p.Y177C, and ADAMTS13 p.W884X were transiently expressed in 293-6E cells. Expression studies revealed a significant reduction in enzyme activity and secretion, although the protease was detected within the cells. The 3D structures of the natural and mutated ADAMTS-13 proteins were partially reconstructed using the Phyre2 web server. The mutation that replaces the tyrosine residue at amino acid position 177 with cysteine may result in decreased steric hindrance and a looser structure. This mutation affects the binding of calcium ions and the secretion of the enzyme from intracellular to extracellular compartments.

Water filling occurs during the rapid salting of separated egg yolks. The purpose of this study was to investigate the changes in water migration, protein aggregation and structures of the separated egg yolks salted with 20% salt solution over 24 hours to understand the principles of this phenomenon. The results showed that water filling occurred within 12 h. The hardness of the salted egg yolk increased significantly within approximately 0−6 h and then decreased significantly. The springiness and cohesiveness of the egg yolk did not change significantly during salting. With the appearance of water filling, the T23, A23, soluble protein content, free sulfhydryl content, β-turns, α-helices and maximum fluorescence intensity (FImax) increased; the T22, A22, disulfide bonds, β-sheets, irregular curls, maximum emission wavelength (λmax) and average particle sizes decreased. This work showed that water filling enhanced the hydrogen bonding between proteins and water and inhibited protein aggregation.

In this study, the effect of Zanthoxylum bungeanum aqueous extract (ZBAE) with the addition of 0, 0.125%, 0.25%, 0.5% and 1.0% on the characteristics of mandarin fish surimi gel, including water-holding capacity, gel strength, texture, whiteness, secondary structure and microstructure were performed. The results showed that the fish surimi gel's water-holding capacity, gel strength, hardness, and adhesion presented a trend of first increasing and then decreasing with the increasing of ZBAE, which had a maximum value of 0.25%. Also, FTIR analysis showed that α-helix, β-fold, and random coil structure exerted a trend of first increasing and then decreasing, and the content of β-fold and α-helix both reached the highest value at 0.125% and 0.25%, respectively. SEM results showed that when adding 0.25% ZBAE, the network structure was more ordered and denser than that of the non-addition group, and the fractal dimension, average diameter and water holes were also perfect. In addition, low addition (

The distance geometry problem asks to find a realization of a given simple edge-weighted graph in a Euclidean space of given dimension $K$, where the edges are realized as straight segments of lengths equal (or as close as possible) to the edge weights. The problem is often modelled as a mathematical programming formulation involving decision variables that determine the position of the vertices in the given Euclidean space. Solution algorithms are generally constructed using local or global nonlinear optimization techniques. We present a new modelling technique for this problem where, instead of deciding vertex positions, the formulations decide the length of the segments representing the edges in each cycle in the graph, projected in every dimension. We propose an exact formulation and a relaxation based on a Eulerian cycle. We then compare computational results from protein conformation instances obtained with stochastic global optimization techniques on the new cycle-based formulation and on the existing edge-based formulation. While edge-based formulations take less time to reach termination, cycle-based formulations are generally better on solution quality measures.

Cerium oxide (CeO2) nanoparticles are expected to have applications in the biomedical field because of their antioxidative properties. Inorganic nanoparticles interact with proteins at the nanoparticle surface and change their conformation when administered; however, the principle underlying this interaction is still unclear. This study aimed to investigate the secondary structural changes occurring in bovine serum albumin (BSA) mixed with CeO2 nanoparticles having different surface modifications using Fourier transform infrared spectroscopy. CeO2 nanoparticles (diameter: 240 nm) were synthesized from an aqueous cerium (III) nitrate solution using a homogeneous precipitation method. The surfaces of the nanoparticles were modified by the catechol compounds dopamine and 3,4-dihydroxyhydrocinnamic acid (DHCA). In the presence of these CeO2 nanoparticles (0.11–0.43 mg/mL), β-sheet formation of BSA (30 mg/mL) was promoted especially on the amine-modified (positively charged) nanoparticles. The local concentration of BSA on the surface of the positively charged nanoparticles may have resulted in structural changes due to electrostatic and other interactions with BSA. Further investigations of the interaction mechanism between nanoparticles and proteins are expected to lead to the safe biomedical applications of inorganic nanoparticles.

The protein conformation of soymilk is the key to affecting the instant solubility of soymilk flour. This study aimed to evaluate the effect of cavitation jet treatment time (0, 2, 4, 6, and 8 min) on the instant solubility of soymilk flour based on the conformational changes of protein in soymilk. The results showed that the cavitation jet treatment for 0–4 min significantly unfolded the protein structure of soymilk and increased the content of soluble protein, which reduced the particle size and increased the electrostatic repulsion and the viscosity of soymilk. This was beneficial for soymilk droplets fully atomized and repolymerized in the spray drying tower, forming soymilk flour particles with large size, smooth surface, and uniform distribution. When the cavitation jet treatment time was 4 min, the wettability (from 127.3 ± 2.5 s to 84.7 ± 2.1 s), dispersibility (from 70.0 ± 2.0 s to 55.7 ± 2.1 s), and solubility (from 56.54% to 78.10%) of soymilk flour were significantly improved. However, when the time of the cavitation jet treatment was extended to 8 min, the protein of soymilk aggregated and the stability of soymilk decreased, which reduced the particle size and hurt the surface characteristics of soymilk flour after spray drying. It resulted in a decrease in the instant solubility of soymilk flour. Therefore, the cavitation jet treatment with proper time increases the instant solubility of soymilk flour by improving the protein conformation of soymilk.

Intrinsically disordered regions (IDRs) of transcription factors play an important biological role in liquid condensate formation and gene regulation. It is thus desirable to investigate the druggability of IDRs and how small-molecule binders can alter their conformational stability. For the androgen receptor (AR), certain covalent ligands induce important changes, such as the neutralization of the condensate. To understand the specificity of ligand–IDR interaction and potential implications for the mechanism of neutralizing liquid–liquid phase separation (LLPS), we modeled and performed computer simulations of ligand-bound peptide segments obtained from the human AR. We analyzed how different covalent ligands affect local secondary structure, protein contact map, and protein–ligand contacts for these protein systems. We find that effective neutralizers make specific interactions (such as those between cyanopyrazole and tryptophan) that alter the helical propensity of the peptide segments. These findings on the mechanism of action can be useful for designing molecules that influence IDR structure and condensate of the AR in the future.

Abstract Understanding material‐protein interactions is the basis for regulating material‐blood interactions, which is a common topic of interest for medical material developers. In recent years, researchers have conducted extensive studies on (1) the structural characteristics of the plasma protein adsorption layer on the material surface, including the evolution of the protein adsorption layer and its typical binary structure. (2) Influence factors of the protein adsorption layer formation include protein factors (e.g., isoelectric point, structural stability), material factors (e.g., wettability, surface charge, morphology, size), and environmental factors. (3) Effects of some common plasma proteins in the protein adsorption layer on material‐blood interactions. Here, we review the important research results in this field, hoping to provide a reference for future development of advanced blood contact materials.

Protein conformation and cell compartmentalization are fundamental concepts and subjects of vast scientific endeavors. In the last two decades, we have witnessed exciting advances that unveiled the conjunction of these concepts. An avalanche of studies highlighted the central role of biomolecular condensates in membraneless subcellular compartmentalization that permits the spatiotemporal organization and regulation of myriads of simultaneous biochemical reactions and macromolecular interactions. These studies have also shown that biomolecular condensation, driven by multivalent intermolecular interactions, is mediated by order-disorder transitions of protein conformation and by protein domain architecture. Conceptually, protein condensation is a distinct level in protein conformational landscape in which collective folding of large collections of molecules takes place. Biomolecular condensates arise by the physical process of phase separation and comprise a variety of bodies ranging from membraneless organelles to liquid condensates to solid-like conglomerates, spanning lengths from mesoscopic clusters (nanometers) to micrometer-sized objects. In this review, we summarize and discuss recent work on the assembly, composition, conformation, material properties, thermodynamics, regulation, and functions of these bodies. We also review the conceptual framework for future studies on the conformational dynamics of condensed proteins in the regulation of cellular processes.

Clostridium difficile toxins are the primary causative agents for hospital-acquired diarrhea and pseudomembranous colitis. Numerous monoclonal antibodies (mAbs) targeting different domains of Clostridium difficile toxin have been reported. Here we report the crystal structures of two mAbs, B1 and B2, in complex with the glycosyltransferase domain (GTD) of the Clostridium difficile toxin B (TcdB). B2 bound to the N-terminal 4 helix bundle of the GTD, a conserved membrane localization domain (MLD) found in the large clostridial glycosylating toxin family implicated in targeting plasma membrane. B1 bound to a distinct epitope at the hinge region between the MLD and the catalytic subdomain of the GTD. Functional studies revealed the potency of these mAbs in vitro and in vivo to be synergistic when given in combination.

The development and improvement of methods for comparing and searching for three-dimensional protein structures remain urgent tasks in modern structural biology. To solve this problem, we developed a new tool, SAFoldNet, which allows for searching, aligning, superimposing, and determining the exact coordinates of fragments of protein structures. The proposed search and alignment tool was built using neural networking. Specifically, we implemented the integrative synergy of neural network predictions and the well-known BLAST algorithm for searching and aligning sequences. The proposed method involves multistage processing, comprising a stage for converting the geometry of protein structures into sequences of a structural alphabet using a neural network, a search stage for forming a set of candidate structures, and a refinement stage for calculating the structural alignment and overlap and evaluating the similarity with the starting structure of the search. The effectiveness and practical applicability of the proposed tool were compared with those of several widely used services for searching and aligning protein structures. The results of the comparisons confirmed that the proposed method is effective and competitive relative to the available modern services. Furthermore, using the proposed approach, a service with a user-friendly web interface was developed, which allows for searching, aligning, and superimposing protein structures; determining the location of protein fragments; mapping onto a protein molecule chain; and providing structural similarity metrices (expected value and root mean square deviation).

The quality deterioration that is induced by freezing treatment limits the development of frozen dough technology for standardized and delayed baking. In this study, laccase (LAC) and ferulic acid (FA) were employed to improve the rheological properties and microstructure of frozen unfermented dough. The results showed that the dough with LAC + FA had a lower softening degree than the dough with FA alone. Correspondingly, LAC + FA incorporation enhanced the viscoelastic behavior of frozen unfermented dough with better stability. Furthermore, a more uniform and homogeneous gluten network was observed in the LAC + FA-supplemented dough after 21 d of storage. The structural stability of the frozen gluten sample increased after LAC + FA treatment, possibly owing to an increase in the oxidation degree of FA. Moreover, LAC + FA treatment promoted the oxidation of the sulfhydryl groups to some extent, resulting in more extensive cross-linking. LAC + FA treatment hindered the protein conformational changes typically induced by frozen storage compared with LAC alone. Overall, LAC + FA treatment has a synergistic effect on enhancing the viscoelastic behaviors of frozen unfermented dough and inhibiting the conformational variation in frozen gluten; thus, it shows promise for improving frozen dough.

The effect of high-pressure processing (100–4 00 MPa) on conformation, water distribution, and gel characteristics of reduced-sodium (0.3 M NaCl) myofibrillar protein containing 0.15% Artemisia sphaerocephala Krasch gum (AG) was investigated. The addition of AG resulted in the increase of WHC, proportion of immobilized water, and gel strength. Then, the WHC, proportion of immobilized water, and gel strength peaked after 200 MPa treatment, attributed to increased solubilization and zeta potential of MP, decreased particle size of MP, exposure of intrinsic tryptophan residues and the partial transformation of α-helix into β-sheet in MP. Moreover, 300 and 400 MPa induced decreases in surface hydrophobicity, solubility and storage modulus, resulting in the formation of loose and disordered gel structures with attenuated WHC. These results suggest that application of moderate HPP (200 MPa) combined with AG could provide a novel approach to improve the WHC and gelation properties of reduced-sodium meat products.

The hotspot mutations of SF3B1, the most frequently mutated splicing gene in cancers, contribute to oncogenesis by corrupting the mRNA splicing. Further SF3B1 mutations have been reported in cancers but their consequences remain unclear. Here, we screened for SF3B1 mutations in the vicinity of the hotspot region in tumors. We then performed in-silico prediction of the functional outcome followed by in-cellulo modelling of different SF3B1 mutants. We show that cancer-associated SF3B1 mutations present varying functional consequences that are loosely predicted by the in-silico algorithms. Analysis of the tertiary structure of SF3B1 mutants revealed that the resulting splicing errors may be due to a conformational change in SF3B1 N-terminal region, which mediates binding with other splicing factors. Our study demonstrates a varying functional impact of SF3B1 mutations according to the mutated codon and the amino acid substitution, implying unequal pathogenic and prognostic potentials of SF3B1 mutations in cancers.

This study assessed the gel properties and conformational changes of duck myofibrillar protein (DMP) affected by plasma-activated water (PAW) generated at various discharge times (0 s, 10 s, 20 s, 30 s, and 40 s). With the treatment of PAW-20 s, the gel strength and water-holding capacity (WHC) of DMP gels were significantly increased when compared to the control group. Throughout the heating process, dynamic rheology revealed that the PAW-treated DMP had a higher storage modulus than the control. The hydrophobic interactions between protein molecules were significantly improved by PAW, resulting in a more ordered and homogeneous gel microstructure. The increased sulfhydryl and carbonyl content in DMP indicated a higher degree of protein oxidation with PAW treatment. Additionally, the circular dichroism spectroscopy demonstrated that PAW induced α-helix and β-turn transformed to β-sheet in DMP. Surface hydrophobicity, fluorescence spectroscopy, and UV absorption spectroscopy suggested that PAW altered DMP’s tertiary structure, although the electrophoretic pattern indicated that the primary structure of DMP was mostly unaffected. These results suggest that PAW can improve the gel properties of DMP through mild alteration in its conformation.

Striated muscle contraction and relaxation is regulated by Ca2+ at the myofilament level via conformational modulations of the troponin complex. To understand the structure–function relationship of troponin in normal muscle and in myopathies, it is necessary to study the functional effects of troponin isoforms and mutations at the level of allosteric conformations of troponin subunits. Traditional methodologies assessing such conformational studies are laborious and require significant amounts of purified protein, while many current methodologies require non-physiological conditions or labeling of the protein, which may affect their physiological conformation and function. To address these issues, we developed a novel approach using site-specific monoclonal antibodies (mAb) as molecular probes to detect and monitor conformational changes of proteins. Here, we present examples for its application in studies of two subunits of troponin: the Ca2+-binding subunit, TnC, and the tropomyosin-binding/thin filament-anchoring subunit, TnT. Studies using a high-throughput microplate assay are compared with that using localized surface plasmon resonance (LSPR) to demonstrate the effectiveness of using mAb probes to assess ligand-induced conformations of troponin subunits in physiological conditions. The assays utilize relatively small amounts of protein and are free of protein modification, which may bias results. Detailed methodologies using various monoclonal antibodies (mAbs) are discussed with considerations for the optimization of assay conditions and the broader application in studies of other proteins as well as in screening of therapeutic reagents that bind a specific target site with conformational and functional effects.

Abstract Integrins have crucial roles in BM homing, survival, proliferation, or drug resistance of multiple myeloma (MM) cells. Especially, integrin α4β1 (VLA-4) and α4β7 has been reported to have important functions in MM cells, and therefore are potential therapeutic targets. We have recently shown that integrin β7 constitutively adopts the active conformation specifically in MM cells, and found that chimeric antigen receptor (CAR) T cells targeting the activated conformation of integrin β7 is promising for MM. Although the mechanism for the constitutive activation is still being investigated, our results indicate that integrin conformation is different between MM and normal cells and suggest that it may be associated with the pathology of MM.

One of the most common drivers in human cancer is the mutant KRAS protein. Not so long ago KRAS was considered as an undruggable oncoprotein. After a long struggle, however, we finally see some light at the end of the tunnel as promising KRAS targeted therapies are in or approaching clinical trials. In recent years, together with the promising progress in RAS drug discovery, our understanding of KRAS has increased tremendously. This progress has been accompanied with a resurgence of publicly available KRAS structures, which were limited to nine structures less than ten years ago. Furthermore, the ever-increasing computational capacity has made biologically relevant timescales accessible, enabling molecular dynamics (MD) simulations to study the dynamics of KRAS protein in more detail at the atomistic level. In this minireview, my aim is to provide the reader an overview of the publicly available KRAS structural data, insights to conformational dynamics revealed by experiments and what we have learned from MD simulations. Also, I will discuss limitations of the current data and provide suggestions for future research related to KRAS, which would fill out the existing gaps in our knowledge and provide guidance in deciphering this enigmatic oncoprotein.

Protein kinases are crucial components of the cell-signalling machinery that orchestrate and convey messages to their downstream targets. Most often, kinases are activated upon a phosphorylation to their activation loop, which will shift the kinase into the active conformation. The Dual specificity mitogen-activated protein kinase kinase 4 (MKK4) exists in a unique conformation in its inactive unphosphorylated state, where its activation segment appears in a stable α-helical conformation. However, the precise role of this unique conformational state of MKK4 is unknown. Here, by all-atom molecular dynamics simulations (MD simulations), we show that this inactive state is unstable as monomer even when unphosphorylated and that the phosphorylation of the activation segment further destabilizes the autoinhibited α-helix. The specific phosphorylation pattern of the activation segment has also a unique influence on MKK4 dynamics. Furthermore, we observed that this specific inactive state is stable as a dimer, which becomes destabilized upon phosphorylation. Finally, we noticed that the most frequent MKK4 mutation observed in cancer, R134W, which role has not been disclosed to date, contributes to the dimer stability. Based on these data we postulate that MKK4 occurs as a dimer in its inactive autoinhibited state, providing an additional layer for its activity regulation.

The effect of bamboo shoot dietary fibre (BSDF) (0%-4%) on the gel quality, protein thermal stability and secondary structure of pork salt-soluble proteins (SSP) was investigated. The water holding capacity (WHC), breaking force and particle size were increased with the addition of 2% BSDF. Differential scanning calorimetry (DSC) analysis indicated that the addition BSDF changed the thermal denaturation temperature of myosin tail and actin, and led to the disappearance of myosin head as the increase of BSDF up to 4%. SDS-PAGE showed that the band intensity of actin was obviously enhanced by the addition of BSDF. Fourier transform infrared (FTIR) spectroscopy showed that BSDF could affect the protein secondary structure. The α-helix content was significantly decreased and the β-sheet content was significantly increased (P

As a member of the melanocortin receptor family, melanocortin 4 receptor (MC4R) plays a critical role in regulating energy homeostasis and feeding behavior, and has been proven as a promising therapeutic target for treating severe obesity syndrome. Numerous studies have demonstrated that central MC4R signaling is significantly affected by melanocortin receptor accessory protein 2 (MRAP2) in humans, mice and zebrafish. MRAP2 proteins exist as parallel or antiparallel dimers on the plasma membrane, but the structural insight of dual orientations with the pharmacological profiles has not yet been fully studied. Investigation and optimization of the conformational topology of MRAP2 are critical for the development of transmembrane allosteric modulators to treat MC4R-associated disorders. In this study, we synthesized a brand new single transmembrane protein by reversing wild-type mouse and zebrafish MRAP2 sequences and examined their dimerization, interaction and pharmacological activities on mouse and zebrafish MC4R signaling. We showed that the reversed zebrafish MRAPa exhibited an opposite function on modulating zMC4R signaling and the reversed mouse MRAP2 lost the capability for regulating MC4R trafficking but exhibited a novel function for cAMP cascades, despite proper expression and folding. Taken together, our results provided new biochemical insights on the oligomeric states and membrane orientations of MRAP2 proteins, as well as its pharmacological assistance for modulating MC4R signaling.

Plasma glutathione peroxidase (GPx3) belongs to the GPx superfamily, and it is the only known secreted selenocysteine (Sec)−containing GPx in humans. It exists as a glycosylated homotetramer and catalyzes the reduction of hydrogen peroxide and lipid peroxides, depending on the Sec in its active center. In this study, a previously reported chimeric tRNAUTuT6 was used for the incorporation of Sec at the UAG amber codon, and the mature form of human GPx3 (hGPx3) without the signal peptide was expressed in amber−less E. coli C321.ΔA.exp. Reactive Sec−hGPx3, able to reduce H2O2 and tert−butyl hydroperoxide (t−BuOOH), was produced with high purity and yield. Study of the quaternary structure suggested that the recombinant Sec−hGPx3 contained an intra−molecular disulfide bridge but failed to form tetramer. Mutational and structural analysis of the mutants with three Cys residues, individually or jointly replaced with Ser, indicated that the formation of intra−molecular disulfide bridges involved structure conformational changes. The secondary structure containing Cys77 and Cys132 was flexible and could form a disulfide bond, or form a sulfhydryl–selenyl bond with Sec49 in relative mutants. Mutation of Cys8 and Cys132 to Sec8 and Sec132 could fix the oligomerization loop through the formation of diselenide bond, which, in turn, facilitated tetramer formation and noticeably improved the GPx activity. This research provides an important foundation for the further catalysis and functional study of hGPx3.

So as to investigate the effect of using sodium bicarbonate (SB) to replace sodium tripolyphosphate (ST) in the chicken meat batters with ST or SB (0.30% and 0.50%), the changes of gel properties and protein conformation were studied. The pH, salt-soluble protein solubility, cooking yield, b* value and texture properties were increased significantly (p

Insights into the conformational organization and dynamics of proteins complexes at membranes is essential for our mechanistic understanding of numerous key biological processes. Here, we introduce graphene-induced energy transfer (GIET) to probe axial orientation of arrested macromolecules at lipid monolayers. Based on a calibrated distance-dependent efficiency within a dynamic range of 25 nm, we analyzed the conformational organization of proteins and complexes involved in tethering and fusion at the lysosome-like yeast vacuole. We observed that the membrane-anchored Rab7-like GTPase Ypt7 shows conformational reorganization upon interactions with effector proteins. Ensemble and time-resolved single-molecule GIET experiments revealed that the HOPS tethering complex, when recruited via Ypt7 to membranes, is dynamically alternating between a ‘closed’ and an ‘open’ conformation, with the latter possibly interacting with incoming vesicles. Our work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub-nanometer resolution.

Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (αSyn), linking it to the pathophysiology of Parkinson’s disease. Our earlier results have revealed that the potent small molecular PREP inhibitor KYP-2047 is able to increase autophagy and decrease dimerization of αSyn but other PREP inhibitors have not been systematically studied for these two protein-protein interaction mediated biological functions of PREP. In this study, we characterized these effects for 12 known PREP inhibitors with IC50-values ranging from 0.2 nM to 1010 nM. We used protein-fragment complementation assay (PCA) to assess αSyn dimerization and Western Blot of microtubule-associated protein light chain 3B II (LC3B-II) and a GFP-LC3-RFP expressing cell line to study autophagy. In addition, we tested selected compounds in a cell-free αSyn aggregation assay, native gel electrophoresis, and determined the compound concentration inside the cell by LC-MS. We found that inhibition of the proteolytic activity of PREP did not predict decreased αSyn dimerization or increased autophagy, and we also confirmed that this result did not simply reflect concentration differences of the compounds inside the cell. Thus, PREP ligands regulate the effect of PREP on autophagy and αSyn aggregation through a conformational stabilization of the enzyme that is not equivalent to inhibiting its proteolytic activity.

Owing to the wide application of miniature pigs in biomedicine, the formation mechanism of its short stature must be elucidated. The insulin-like growth factor 1 receptor (IGF-1R), which receives signals through the extracellular domain (ECD) binding with ligands, is crucial in regulating cell growth and bone matrix mineralization. In this study, two haplotypes of Igf1r with four synonymous mutations in the coding sequences of IGF-1R ECD between large pigs (LP) and Bama pigs (BM) were stably expressed in the Igf1r-knockout MC3T3-E1 cells and named as MC3T3-LP cells (LP group) and MC3T3-BM cells (BM group), respectively. IGF-1R expression was lower in the BM group than in the LP group both in terms of transcription and translation levels, and IGF-1R expression inhibited cell proliferation. In addition, IGF-1R expression in the BM group promoted early-stage differentiation but delayed late-stage differentiation, which not only suppressed the expression of bone-related factors but also reduced alkaline phosphatase activity and calcium deposition. Moreover, different haplotypes of Igf1r changed the stability and conformation of the protein, further affecting the binding with IGF-1. Our data indicated that the four synonymous mutations of IGF1R ECD encoded by affect gene transcription and translation, thereby further leading to differences in the downstream pathways and functional changes of osteoblasts.

Phosphorylation of the C-terminal tail of the heavy neurofilament subunit (NF-H) impacts neurofilament (NF) axonal transport and residence within axons by fostering NF-NF associations that compete with transport. We tested the role of phosphorylation of a GSK-3β consensus site (S493) located in the proximal portion of the NF-H tail in NF dynamics by transfection of NB2a/d1 cells with NF-H, where S493 was mutated to aspartic acid (S493D) or to alanine (S493A) to mimic constitutive phosphorylation and non-phosphorylation. S493D underwent increased transport into axonal neurites, while S493A displayed increased perikaryal NF aggregates that were decorated by anti-kinesin. Increased levels of S493A co-precipitated with anti-kinesin indicating that reduced transport of S493A was not due to reduced kinesin association but due to premature NF-NF interactions within perikarya. S493D displayed increased phospho-immunoreactivity within axonal neurites at downstream C-terminal sites attributable to mitogen-activated protein kinase and cyclin-dependent kinase 5. However, S493D was more prone to proteolysis following kinase inhibition, suggesting that S493 phosphorylation is an early event that alters sidearm configuration in a manner that promotes appropriate NF distribution. We propose a novel model for sidearm configuration.

Amyloid structures assemble through a repeating type of bonding called “cross-β”, in which identical sequences in many protein molecules form β-sheets that interdigitate through side chain interactions. We review the structural characteristics of such bonds. Single cell force microscopy (SCFM) shows that yeast expressing Als5 adhesin from Candida albicans demonstrate the empirical characteristics of cross-β interactions. These properties include affinity for amyloid-binding dyes, birefringence, critical concentration dependence, repeating structure, and inhibition by anti-amyloid agents. We present a model for how cross-β bonds form in trans between two adhering cells. These characteristics also apply to other fungal adhesins, so the mechanism appears to be an example of a new type of cell–cell adhesion.

Rare germline pathogenic TP53 missense variants often predispose to a wide spectrum of tumors characterized by Li-Fraumeni syndrome (LFS) but a subset of variants is also seen in families with exclusively hereditary breast cancer (HBC) outcomes. We have developed a logistic regression model with the aim of predicting LFS and HBC outcomes, based on the predicted effects of individual TP53 variants on aspects of protein conformation. A total of 48 missense variants either unique for LFS (n = 24) or exclusively reported in HBC (n = 24) were included. LFS-variants were over-represented in residues tending to be buried in the core of the tertiary structure of TP53 (p = 0.0014). The favored logistic regression model describes disease outcome in terms of explanatory variables related to the surface or buried status of residues as well as their propensity to contribute to protein compactness or protein-protein interactions. Reduced, internally validated models discriminated well between LFS and HBC (C-statistic = 0.78−0.84; equivalent to the area under the ROC (receiver operating characteristic) curve), had a low risk for over-fitting and were well calibrated in relation to the known outcome risk. In conclusion, this study presents a phenotypic prediction model of LFS and HBC risk for germline TP53 missense variants, in an attempt to provide a complementary tool for future decision making and clinical handling.

Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are substantially reduced. To better understand the structural basis for these functional differences, the 2.15 Å crystal structure of CYP3A7 has been solved. Comparative analysis of CYP3A enzymes shows that decreased structural plasticity rather than the active site microenvironment defines the ligand binding ability of CYP3A7. In particular, a rotameric switch in the gatekeeping amino acid F304 triggers local and long-range rearrangements that transmit to the F-G fragment and alter its interactions with the I-E-D-helical core, resulting in a more rigid structure. Elongation of the β3-β4 strands, H-bond linkage in the substrate channel, and steric constraints in the C-terminal loop further increase the active site rigidity and limit conformational ensemble. Collectively, these structural distinctions lower protein plasticity and change the heme environment, which, in turn, could impede the spin-state transition essential for optimal reactivity and oxidation of substrates.

Noncovalent interactions play a pivotal role in regulating protein conformation, stability and dynamics. Among the quantum mechanical (QM) overlap-based noncovalent interactions, n→π* is the best understood with studies ranging from small molecules to β-turns of model proteins such as GB1. However, these investigations do not explore the interplay between multiple overlap interactions in contributing to local structure and stability. In this work, we identify and characterize all noncovalent overlap interactions in the β-turn, an important secondary structural element that facilitates the folding of a polypeptide chain. Invoking a QM framework of natural bond orbitals, we demonstrate the role of several additional interactions such as n→σ* and π→π* that are energetically comparable to or larger than n→π*. We find that these interactions are sensitive to changes in the side chain of the residues in the β-turn of GB1, suggesting that the n→π* may not be the only component in dictating β-turn conformation and stability. Furthermore, a database search of n→σ* and π→π* in the PDB reveals that they are prevalent in most proteins and have significant interaction energies (∼1 kcal/mol). This indicates that all overlap interactions must be taken into account to obtain a comprehensive picture of their contributions to protein structure and energetics. Lastly, based on the extent of QM overlaps and interaction energies, we propose geometric criteria using which these additional interactions can be efficiently tracked in broad database searches.

This study aimed to evaluate the effect of riboflavin (RF) and Rose Bengal (RB) photosensitizer modified adhesive resin on the degree of conversion (DC), and antimicrobial capacity after bonded to tooth surface. Different concentrations of RB and RF were prepared by homogenization method. An ultraviolet light source A (UVA) (375 nm wavelength, 3 mW/cm2 power) was used for 30 min irradiation. FTIR was performed for control and test adhesives to analyze the DC. Antibacterial testing was performed using the MTT assay. Metal brackets were bonded using the modified adhesives and subjected for SEM examination. The surfaces of teeth and metal brackets were examined at ×10 magnification for assessing adhesive remnant index (ARI) after PDT, 24 h and thermocycling. For DC, control group, 0.1% RB and RF after PDT showed the highest value. SEM imaging indicated lowest growth of Streptococcus mutans over 0.5% of RB-PDT and RF-PDT as compared to the control group. The MTT assay outcomes reported that the activity of S. mutans substantially decreased with the addition of a high amount of either RB or RF (p < 0.01). Mean ARI scores showed a significant difference between all groups. This study concluded that 0.1% of either RB or RF after PDT can be used for bonding orthodontic brackets to the tooth surface with substantial antibacterial properties.

Detailed study of conformational rearrangements and dynamics of proteins is central to our understanding of their physiological functions and the loss of function. This review outlines the applications of the electron paramagnetic resonance (EPR) technique to study the structural aspects of proteins transitioning from a solution environment to the states in which they are associated with the surfaces of biological membranes or engineered nanoobjects. In the former case these structural transitions generally underlie functional protein states. The latter case is mostly relevant to the application of protein immobilization in biotechnological industries, developing methods for protein purification, etc. Therefore, evaluating the stability of the protein functional state is particularly important. EPR spectroscopy in the form of continuous-wave EPR or pulse EPR distance measurements in conjunction with protein spin labeling provides highly versatile and sensitive tools to characterize the changes in protein local dynamics as well as large conformational rearrangements. The technique can be widely utilized in studies of both protein-membrane and engineered nanoobject-protein complexes.

Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

The normal cellular isoform of prion protein, designated PrPC, is constitutively converted to the abnormally folded, amyloidogenic isoform, PrPSc, in prion diseases, which include Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. PrPC is a membrane glycoprotein consisting of the non-structural N-terminal domain and the globular C-terminal domain. During conversion of PrPC to PrPSc, its 2/3 C-terminal region undergoes marked structural changes, forming a protease-resistant structure. In contrast, the N-terminal region remains protease-sensitive in PrPSc. Reverse genetic studies using reconstituted PrPC-knockout mice with various mutant PrP molecules have revealed that the N-terminal domain has an important role in the normal function of PrPC and the conversion of PrPC to PrPSc. The N-terminal domain includes various characteristic regions, such as the positively charged residue-rich polybasic region, the octapeptide repeat (OR) region consisting of five repeats of an octapeptide sequence, and the post-OR region with another positively charged residue-rich polybasic region followed by a stretch of hydrophobic residues. We discuss the normal functions of PrPC, the conversion of PrPC to PrPSc, and the neurotoxicity of PrPSc by focusing on the roles of the N-terminal regions in these topics.

How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics is poorly understood. Here, we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but a considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11–29) emerge as “correlated segments”, identified by their frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11–14 and 23–28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent; segments where internal interactions are more prevalent manifest elevated “collective” motions on the 5–10 ns timescale and suppressed local motions on the sub-ns timescale. Amide proton exchange rates provides corroboration, with residues in the most correlated segment exhibiting the highest protection factors. We propose the correlated segment as a defining feature for the conformations and dynamics of IDPs.

A protein is a kind of major biomacromolecule of life. Its sequence, structure, and content in organisms contains quite important information for normal or pathological physiological process. However, research of proteomics is facing certain obstacles. Only a few technologies are available for protein analysis, and their application is limited by chemical modification or the need for a large amount of sample. Solid-state nanopore overcomes some shortcomings of the existing technology, and has the ability to detect proteins at a single-molecule level, with its high sensitivity and robustness of device. Many works on detection of protein molecules and discriminating structure have been carried out in recent years. Single-molecule protein sequencing techniques based on solid-state nanopore are also been proposed and developed. Here, we categorize and describe these efforts and progress, as well as discuss their advantages and drawbacks.

Polyphenols such as epigallocatechin gallate (EGCg) may have roles in preventing some chronic diseases when they are ingested as components of plant-based foods and beverages. Human serum albumin (HSA) is a multi-domain protein that binds various ligands and aids in their transport, distribution, and metabolism in the circulatory system. In the present study, the HSA-EGCg interaction in the absence or presence of fatty acid has been investigated. Förster resonance energy transfer (FRET) was used to determine inter- and intra-domain distances in the protein with and without EGCg and palmitic acid (PA). By labeling Cys-34 with 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CPM), the distance between Trp-214 at domain IIA and CPM-Cys-34 at domain IA could be established. A small amount of PA decreased the distance, while a large amount increased the distance up to 5.4 Å. EGCg increased the inter-domain distance in HSA and HSA-PA up to 2.8 and 7.6 Å, respectively. We concluded that PA affects protein conformation more significantly compared to EGCg. Circular dichroism (CD) established that EGCg affects protein secondary structure more significantly than PA. PA had little effect on the α-helix content of HSA, while EGCg decreased the α-helix content in a dose-dependent fashion. Moreover, EGCg decreased α-helix content in HSA and HSA-PA to the same level. Dynamic light scattering (DLS) data revealed that both PA and EGCg increased HSA aggregation. EGCg increased HSA aggregation more significantly and promoted formation of aggregates that were more heterogenous. Any of these effects could impact the ability of serum albumin to transport and stabilize ligands including EGCg and other polyphenols.

The stereoselective flavoenzyme D-amino acid oxidase (DAAO) catalyzes the oxidative deamination of neutral and polar D-amino acids producing the corresponding α-keto acids, ammonia, and hydrogen peroxide. Despite its peculiar and atypical substrates, DAAO is widespread expressed in most eukaryotic organisms. In mammals (and humans in particular), DAAO is involved in relevant physiological processes ranging from D-amino acid detoxification in kidney to neurotransmission in the central nervous system, where DAAO is responsible of the catabolism of D-serine, a key endogenous co-agonist of N-methyl-D-aspartate receptors. Recently, structural and functional studies have brought to the fore the distinctive biochemical properties of human DAAO (hDAAO). It appears to have evolved to allow a strict regulation of its activity, so that the enzyme can finely control the concentration of substrates (such as D-serine in the brain) without yielding to an excessive production of hydrogen peroxide, a potentially toxic reactive oxygen species (ROS). Indeed, dysregulation in D-serine metabolism, likely resulting from altered levels of hDAAO expression and activity, has been implicated in several pathologies, ranging from renal disease to neurological, neurodegenerative, and psychiatric disorders. Only one mutation in DAO gene was unequivocally associated to a human disease. However, several single nucleotide polymorphisms (SNPs) are reported in the database and the biochemical characterization of the corresponding recombinant hDAAO variants is of great interest for investigating the effect of mutations. Here we reviewed recently published data focusing on the modifications of the structural and functional properties induced by amino acid substitutions encoded by confirmed SNPs and on their effect on D-serine cellular levels. The potential significance of the different hDAAO variants in human pathologies will be also discussed.

The mitotic checkpoint monitors kinetochore-microtubule attachment, delays anaphase onset and prevents aneuploidy when unattached or tensionless kinetochores are present in cells. Mitotic arrest deficiency 1 (MAD1) is one of the evolutionarily conserved core mitotic checkpoint proteins. MAD1 forms a cell cycle independent complex with MAD2 through its MAD2 interaction motif (MIM) in the middle region. Such a complex is enriched at unattached kinetochores and functions as an unusual catalyst to promote conformational change of additional MAD2 molecules, constituting a crucial signal amplifying mechanism for the mitotic checkpoint. Only MAD2 in its active conformation can be assembled with BUBR1 and CDC20 to form the Mitotic Checkpoint Complex (MCC), which is a potent inhibitor of anaphase onset. Recent research has shed light on how MAD1 is recruited to unattached kinetochores, and how it carries out its catalytic activity. Here we review these advances and discuss their implications for future research.