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To manipulate the protein concentration at a certain functional state through chemical stabilizers is crucial for protein-related studies. It not only plays a key role in protein structure analysis and protein folding kinetics, but also affects protein functionality to a large extent and thus has wide applications in medicine, food industry, etc. However, due to concerns about side effects or financial costs of stabilizers, identifying optimal strategies for enhancing protein stability with a minimal amount of stabilizers is of great importance. Here, we prove that either for the fixed terminal time (including both finite and infinite cases) or for the free one, the optimal control strategy for stabilizing the folding intermediates with a linear strategy for stabilizer addition belongs to the class of bang–bang controls. The corresponding optimal switching time is derived analytically, whose phase diagram with respect to several key parameters is explored in detail. The bang–bang control will be broken when nonlinear strategies for stabilizer addition are adopted. Moreover, the above theory is applied to the stabilization of erythropoietin by ten different kinds of chemicals, providing theoretical guidance for the selection and rational usage of stabilizers. Our current study on optimal strategies for protein stabilizers not only offers deep insights into the general picture of protein folding kinetics but also provides valuable theoretical guidance on treatments for protein-related diseases in medicine. [ABSTRACT FROM AUTHOR]

Vibrational spectroscopy of protein structure often utilizes 13C18O-labeling of backbone carbonyls to further increase structural resolution. However, sidechains such as arginine, aspartate, and glutamate absorb within the same spectral region, complicating the analysis of isotope-labeled peaks. In this study, we report that the waiting time between pump and probe pulses in two-dimensional infrared spectroscopy can be used to suppress sidechain modes in favor of backbone amide I′ modes based on differences in vibrational lifetimes. Furthermore, differences in the lifetimes of 13C18O-amide I′ modes can aid in the assignment of secondary structure for labeled residues. Using model disordered and β-sheet peptides, it was determined that while β-sheets exhibit a longer lifetime than disordered structures, amide I′ modes in both secondary structures exhibit longer lifetimes than sidechain modes. Overall, this work demonstrates that collecting 2D IR data at delayed waiting times, based on differences in vibrational lifetime between modes, can be used to effectively suppress interfering sidechain modes and further identify secondary structures. [ABSTRACT FROM AUTHOR]

Contactin 2 (CNTN2) is a cell adhesion molecule involved in axon guidance, neuronal migration, and fasciculation. The ectodomains of CNTN1-CNTN6 are composed of six Ig domains (Ig1-Ig6) and four FN domains. Here, we show that CNTN2 forms transient homophilic interactions (KD ∼200 nM). Cryo-EM structures of full-length CNTN2 and CNTN2_Ig1-Ig6 reveal a T-shaped homodimer formed by intertwined, parallel monomers. Unexpectedly, the horseshoe-shaped Ig1-Ig4 headpieces extend their Ig2-Ig3 tips outwards on either side of the homodimer, while Ig4, Ig5, Ig6, and the FN domains form a central stalk. Cross-linking mass spectrometry and cell-based binding assays confirm the 3D assembly of the CNTN2 homodimer. The interface mediating homodimer formation differs between CNTNs, as do the homophilic versus heterophilic interaction mechanisms. The CNTN family thus encodes a versatile molecular platform that supports a very diverse portfolio of protein interactions and that can be leveraged to strategically guide neural circuit development.

We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon–hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements X ijk 2 of collagen's nonlinear susceptibility. Using the three-dimensional atomic positions derived from published crystallographic data of collagen type I, we simulated its X ijk 2 elements for the methylene stretching vibration and compared the predicted response with the experimental results. Our analysis revealed that the carbon–hydrogen stretching range of the SFG spectrum is dominated by symmetric stretching modes of methylene bridge groups on the pyrrolidine rings of the proline and hydroxyproline residues, giving rise to a dominant peak near 2942 cm−1 and a shoulder at 2917 cm−1. Weak asymmetric stretches of the methylene bridge group of glycine are observed in the region near 2870 cm−1, whereas asymmetric CH2-stretching modes on the pyrrolidine rings are found in the 2980 to 3030 cm−1 range. These findings help predict the protein's nonlinear optical properties from its crystal structure, thus establishing a connection between the protein structure and SFG spectroscopic measurements. [ABSTRACT FROM AUTHOR]

Electron spin resonance pulsed dipolar spectroscopy (PDS) has become popular in protein 3D structure analysis. PDS studies yield distance distributions between a pair or multiple pairs of spin probes attached to protein molecules, which can be used directly in structural studies or as constraints in theoretical predictions. Double-quantum coherence (DQC) is a highly sensitive and accurate PDS technique to study protein structures in the solid state and under physiologically relevant conditions. In this work, we have derived analytical expressions for the DQC signal for a system with N-dipolar coupled spin-1/2 particles in the solid state. The expressions are integrated over the relevant spatial parameters to obtain closed form DQC signal expressions. These expressions contain the concentration-dependent "instantaneous diffusion" and the background signal. For micromolar and lower concentrations, these effects are negligible. An approximate analysis is provided for cases of finite pulses. The expressions obtained in this work should improve the analysis of DQC experimental data significantly, and the analytical approach could be extended easily to a wide range of magnetic resonance phenomena. [ABSTRACT FROM AUTHOR]

Cells employ many large macromolecular machines for the execution and regulation of processes that are vital for cell and organismal viability. Interestingly, cells cannot synthesize these machines as functioning units. Instead, cells synthesize the molecular parts that must then assemble into the functional complex. Many important machines, including chaperones such as GroEL and proteases such as the proteasome, comprise protein rings that are stacked on top of one another. While there is some experimental data regarding how stacked-ring complexes such as the proteasome self-assemble, a comprehensive understanding of the dynamics of stacked-ring assembly is currently lacking. Here, we developed a mathematical model of stacked-trimer assembly and performed an analysis of the assembly of the stacked homomeric trimer, which is the simplest stacked-ring architecture. We found that stacked rings are particularly susceptible to a form of kinetic trapping that we term deadlock, in which the system gets stuck in a state where there are many large intermediates that are not the fully assembled structure but that cannot productively react. When interaction affinities are uniformly strong, deadlock severely limits assembly yield. We thus predicted that stacked rings would avoid situations where all interfaces in the structure have high affinity. Analysis of available crystal structures indicated that indeed the majority-if not all-of stacked trimers do not contain uniformly strong interactions. Finally, to better understand the origins of deadlock, we developed a formal pathway analysis and showed that, when all the binding affinities are strong, many of the possible pathways are utilized. In contrast, optimal assembly strategies utilize only a small number of pathways. Our work suggests that deadlock is a critical factor influencing the evolution of macromolecular machines and provides general principles for understanding the self-assembly efficiency of existing machines.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

The application of terahertz radiation has been shown to affect both protein structure and cellular function. As the key to such structural changes lies in the dynamic response of a protein, we focus on the susceptibility of the protein's internal dynamics to mechanical stress induced by acoustic pressure waves. We use the open-boundary molecular dynamics method, which allows us to simulate the protein exposed to acoustic waves. By analyzing the dynamic fluctuations of the protein subunits, we demonstrate that the protein is highly susceptible to acoustic waves with frequencies corresponding to those of the internal protein vibrations. This is confirmed by changes in the compactness of the protein structure. As the amplitude of the pressure wave increases, even larger deviations from average positions and larger changes in protein compactness are observed. Furthermore, performing the mode-projection analysis, we show that the breathing-like character of collective modes is enhanced at frequencies corresponding to those used to excite the protein. [ABSTRACT FROM AUTHOR]

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 μM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of functional affinity in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.

Colloidal delivery systems are widely used in the food industry to enhance the dispersibility, stability, efficacy, or bioavailability. However, when exposed to the high temperature, delivery systems are often prone to degradation, which limits its application in thermal processing. In this paper, the effects of thermal processing on the performance of traditional protein-based or starch-based delivery systems are firstly described, including the molecular structure changes of proteins, starches or lipids, and the degradation of embedded substances. These effects are unfavorable to the application of the delivery system in thermal processing. Then, strategies of improving the heat resistance of food grade colloid delivery system and their use in frying, baking and cooking food are mainly introduced. The heat resistance of the delivery system can be improved by a variety of strategies, including the development of new heat-resistant materials, the addition of heat-resistant coatings to the surface of delivery systems, the cross-linking of proteins or starches using cross-linking agents, the design of particle structures, the use of physical means such as ultrasound, or the optimization of the ingredient formula. These strategies will help to expand the application of heat-resistant delivery systems so that they can be used in real thermal processing. [ABSTRACT FROM AUTHOR]

Ultra-high temperature (UHT) treatments are of high economic relevance for food industries because they contribute to extending the shelf life of food products and facilitating their distribution. In the dairy segment, UHT treatments are applied to a wide range of products containing variable protein amounts. In this sense, the changes in the molecular structure of milk proteins induced by the severity of UHT treatments may lead to fouling in equipment during processing or sedimentation and/or gelation during storage. Nowadays, these concerns are even more relevant due to the increasing demand for UHT-treated high-protein beverages. This review will discuss the two main strategies used by industries to increase the stability of milk proteins during and/or after UHT treatments: (i) addition of chelating agents and (ii) use of polysaccharides. Moreover, the challenges and opportunities associated with promising strategies to improve the stability of milk proteins during and/or after UHT treatments will be covered in this review. The information compiled will be useful to guide researchers and industries in developing more stable UHT dairy products in harmony with consumers' demands. [ABSTRACT FROM AUTHOR]

Food allergens are a major concern for individuals who are susceptible to food allergies and may experience various health issues due to allergens in their food. Most allergenic foods are subjected to heat treatment before being consumed. However, thermal processing and prolonged storage can cause glycation reactions to occur in food. The glycation reaction is a common processing method requiring no special chemicals or equipment. It may affect the allergenicity of proteins by altering the structure of the epitope, revealing hidden epitopes, concealing linear epitopes, or creating new ones. Changes in food allergenicity following glycation processing depend on several factors, including the allergen's characteristics, processing parameters, and matrix, and are therefore hard to predict. This review examines how glycation reactions affect the allergenicity of different allergen groups in allergenic foods. [ABSTRACT FROM AUTHOR]

Plant-based foods are being considered seriously to replace traditional animal-origin foods for various reasons. It is well known that animals release large amounts of greenhouse gases into the environment during feeding, and eating animal-origin foods may also cause some health problems. Moreover, animal resources will likely be in short supply as the world population grows. It is highly likely that serious health problems ascribed to insufficient protein intake in some areas of the world will occur. Studies have shown that environmentally friendly, abundant, and customizable plant-based foods can be an effective alternative to animal-based foods. However, currently, available plant-based foods lack nutrients unique to animal-based foods. Innovative processing technologies are needed to improve the nutritional value and functionality of plant-based foods and make them acceptable to a wider range of consumers. Therefore, protein extraction technologies (e.g., high-pressure extraction, ultrasound extraction, enzyme extraction, etc.), structure improvement and assembly technologies (3D printing, micro-encapsulation, etc.), and technologies to improve digestibility and utilization of bioactive substances (microbial fermentation, physical, etc.) in the field of plant-based foods processing are reviewed. The challenges of plant-based food processing technologies are summarized. The advanced technologies aim to help the food industry solve production problems using efficient, environmentally friendly, and economical processing technologies and to guide the development of plant-based foods in the future. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Disulfide bridge, an important post-translation modification in protein, plays a key role in stabilizing three-dimensional structure of proteins, maintaining correct folded conformation, and thus regulating the biological activities. Disulfide bridge assignment is essential to understand the essence of life process and to develop protein pharmaceutical. In this study, a novel method termed as stepwise reduction and differentiated alkylation (SRDA) was developed analyzing disulfide connectivity for proteins. As a demonstration, three disulfide bridges in insulin aspart were successfully characterized using this SRDA method combined with LC-HRMSn. Firstly, tris (2-carboxyethyl) phosphine (TCEP) was used to partially reduce disulfide bridges with N-ethylmaleimide (NEM) used to block the generated free thiol. Then, dithiothreitol (DTT) was used to reduce the rest disulfide bonds with iodoacetamide (IAM) used to block the newly generated free thiol. After that, an LC-HRMSn method was established to assign disulfide connectivity for an insulin aspart study material, based on the different mass shifts arising from differentiated alkylation. Moreover, this approach allows for the quantitative analysis of various disulfide bond pairings, which can be applied to studies on the consistency and stability among different batches of samples. The results show that SRDA is a valuable tool for reliable quality control and quality assessment of disulfide-rich proteins such as insulin analogues. [ABSTRACT FROM AUTHOR]

The high binding affinity of antibodies toward their cognate targets is key to eliciting effective immune responses, as well as to the use of antibodies as research and therapeutic tools. Here, we propose ANTIPASTI, a convolutional neural network model that achieves state-of-the-art performance in the prediction of antibody binding affinity using as input a representation of antibody-antigen structures in terms of normal mode correlation maps derived from elastic network models. This representation captures not only structural features but energetic patterns of local and global residue fluctuations. The learnt representations are interpretable: they reveal similarities of binding patterns among antibodies targeting the same antigen type, and can be used to quantify the importance of antibody regions contributing to binding affinity. Our results show the importance of the antigen imprint in the normal mode landscape, and the dominance of cooperative effects and long-range correlations between antibody regions to determine binding affinity. [Display omitted] • Antibody-antigen binding affinity prediction using normal modes and deep learning • ANTIPASTI achieves state-of-the-art accuracy and generalization power • ANTIPASTI is interpretable and identifies key antibody regions for binding • Both short and long-range structural correlations are crucial for binding affinity Understanding the drivers of antibody-antigen binding affinity is crucial to design optimized antibodies for research and therapeutic purposes. Michalewicz et al. present ANTIPASTI, a deep learning model that predicts binding affinity from structural data and identifies which antibody regions are important for binding. [ABSTRACT FROM AUTHOR]

Spider silks are renowned for their mechanical properties. The notably tough Argiope trifasciata aciniform silk is spun from a protein predominantly comprising a series of identical 200 residue units (“W units”). In solution, each W unit has a globular 5‐helix domain connected to its neighbors by intrinsically disordered linkers while the fiber contains mixed α‐helical, β‐sheet and disordered structuring. Helix 5 is more dynamic and prone to denaturation than the remainder of the globular domain, implying that it may structurally transform during fiber spinning. Rational cysteine substitutions are introduced at a proximal pair of serines in helix 5 and in helix 1 of the globular domain. Upon disulfide formation, the helix 5 C‐terminal region loses helicity and experiences increased backbone dynamics, while the remainder of the globular structure is effectively unperturbed. Fiber formation by hand‐drawing and wet‐spinning is prevented in the disulfide‐locked state, but readily possible in the reduced (“unlocked”) state. The disulfide‐locked state differs from the unlocked state with much more heterogeneous pre‐fibrillar assembly in spinning dope and through prevention of β‐sheet formation upon cooling following thermal denaturation. This engineered protein thus provides a chemically‐reversible disulfide‐locked state of aciniform silk with modified self‐assembly propensity and fiber formation. [ABSTRACT FROM AUTHOR]

Serratia sp. ATCC 39006 has two tandemly positioned genes, ser4 and ser5, both annotated as sugar aminotransferases, in a putative secondary metabolite biosynthetic gene cluster. Ser5 possesses a complete fold‐type I aminotransferase fold, while Ser4 lacks the N‐ and C‐terminal regions and a catalytically important lysine residue of fold‐type I aminotransferase. We herein revealed that Ser4 and Ser5 formed a heterotetrameric complex (SerTA) with aminotransferase activity and determined the crystal structures. MD simulations and activity assays with SerTA variants indicated that residues from helix α‐8* of inactive Ser4 are important for activity, confirming the importance of heterocomplex formation for activity. Furthermore, the structures suggest that SerTA recognizes a substrate loaded on the carrier protein. [ABSTRACT FROM AUTHOR]

This study investigated the kinetics of ultrasound (US)-assisted hydrolysis of pequi almond protein (PAP) and the conformational and multifunctional characteristics of the hydrolysates. The US-assisted enzymolysis of PAP using Alcalase® was conducted in a US bath (25 kHz, 38 W/L) for 180 min at 60 °C and pH 7.5. The results showed an increase in the rate of proteolysis by 11.3 %, leading to a higher final degree of hydrolysis (25.2 %) and a greater concentration of TCA-soluble proteins after 45 min and 180 min (29 % and 8 %, respectively). Both conventional and US-assisted hydrolysis led to the breakdown of the protein structure, forming low molecular weight peptides (<15 kDa). However, US-assisted hydrolysis demonstrated greater fragmentation of the PAP surface, enhancing enzymatic efficacy. Intrinsic fluorescence and UV spectroscopy indicated increased exposure of hydrophobic groups throughout hydrolysis, and FTIR spectroscopy revealed changes in the amide bands, suggesting structural transitions in the protein. Finally, the hydrolysates obtained from the USassisted reaction showed higher solubility (≤27 % at pH 6.0), foaming capacity (≤82 % at pH 6.0), and antioxidant activity (≤40 %). Therefore, these results indicate the potential of the US to improve the performance of Alcalase® in the hydrolysis of PAP, producing compounds of industrial interest. [Display omitted] • Ultrasound (US)-assisted hydrolysis of Pequi almond protein (PAP) was first studied. • US enhanced PAP hydrolysis, increasing the rate (11.3 %) and peptide production (8 %). • Hydrolysis and US increased the Pequi almond protein solubility (<2.6x at pH 4.0). • US-assisted hydrolysis increased the foaming capacity of PAP (<4.4x at pH 6.0). • US-assisted hydrolysis generated higher antioxidant activity peptides (<40 %). [ABSTRACT FROM AUTHOR]

1. Infectious bursal disease (IBD) is an acute, highly contagious viral disease of chickens caused by a virus (IBDV) which has a bi-segmented, double-stranded RNA genome. It has five viral proteins in its structure; the VP1 gene is encoded in segment B and the other four are in segment A. 2. In this study, bursae of Fabricius and spleen samples taken from chickens suspected of having clinical or subclinical IBD from a total of 50 chicken flocks located in different geographical regions of Turkey were examined. 3. The RT-PCR analysis of the VP2 gene showed that 30 of the 50 samples (60%) tested positive. Eight positive isolates were chosen and RT-PCR was performed to amplify the VP1 gene. 4. The study showed that reassortant field strains that cause clinical or subclinical disease are currently circulating in broiler flocks across Turkey. [ABSTRACT FROM AUTHOR]

The Protein Structure Prediction Problem is one of the most important and challenging open problems in Computer Science and Structural Bioinformatics. Accurately predicting protein conformations would significantly impact several fields, such as understanding proteinopathies and developing smart protein-based drugs. As such, this work has as its primary goal to improve the prediction power of ab initio methods by utilizing a self-adaptive evolutionary algorithm using Monte Carlo based fragment insertion and conformational clustering. A meta-heuristic is used as the core of the conformation sampling process with fragment insertion, feeding domain-specific information into the process. The online parameter control routines allow the method to adapt to a protein's structure specificity and behave dynamically in different stages of the optimization process. The results obtained by the proposed method were compared to results obtained from several other algorithms found in the literature. It is possible to conclude that the proposed method is highly competitive in terms of free-energy and RMSD for the protein set used in the experiments. [ABSTRACT FROM AUTHOR]

Bi-allelic pathogenic variants in GRID2 have been initially associated to an autosomal recessive form of spinocerebellar ataxia, namely SCAR18. Subsequently, few monoallelic cases have been described. Here we present a new subject harboring a novel de novo heterozygous GRID2 missense variant presenting with progressive ataxia together with cerebellar atrophy and, for the first time, alpha-fetoprotein (AFP) elevation. We retrospectively collected data of the patient followed at our clinic. Genetic analysis was performed through clinical exome sequencing with an in-house in-silico ataxia-related genes panel. Variant effect prediction was performed through in silico modeling. The patient had normal psychomotor development except for mild fine and gross motor impairment. In adolescence, he started presenting dysarthria and progressive ataxia. Blood tests showed significant AFP elevation. Brain MRI showed cerebellar atrophy mainly involving the vermis. The novel de novo heterozygous GRID2 (c.1954C>A; p.Leu652Ile) missense variant was disclosed. This variant is located within a highly conserved site with low tolerance to variation and it is predicted to cause protein structure destabilization. GRID2 expression appears to be influenced by other genes related with ataxia and AFP elevation, like ATM and APTX, suggesting a possible shared mechanism. This additional patient increases the scarce literature and genotypic spectrum of the GRID2-related ataxia and evidences a fairly homogeneous phenotype of ataxia with oculomotor abnormalities for the autosomal-dominant form. Alfa-fetoprotein elevation is a novel finding in this condition and this data must be confirmed in larger case-series to definitively state that GRID2-related ataxia can be included among ataxias with AFP increase. [ABSTRACT FROM AUTHOR]

The aminoacyl‐tRNA synthetases (aaRS) are a large group of enzymes that implement the genetic code in all known biological systems. They attach amino acids to their cognate tRNAs, moonlight in various translational and non‐translational activities beyond aminoacylation, and are linked to many genetic disorders. The aaRS have a subtle ontology characterized by structural and functional idiosyncrasies that vary from organism to organism, and protein to protein. Across the tree of life, the 22 coded amino acids are handled by 16 evolutionary families of Class I aaRS and 21 families of Class II aaRS. We introduce AARS Online, an interactive Wikipedia‐like tool curated by an international consortium of field experts. This platform systematizes existing knowledge about the aaRS by showcasing a taxonomically diverse selection of aaRS sequences and structures. Through its graphical user interface, AARS Online facilitates a seamless exploration between protein sequence and structure, providing a friendly introduction to the material for non‐experts and a useful resource for experts. Curated multiple sequence alignments can be extracted for downstream analyses. Accessible at www.aars.online, AARS Online is a free resource to delve into the world of the aaRS. [ABSTRACT FROM AUTHOR]

Pancreatic β-cells in the islets of Langerhans are key to maintaining glucose homeostasis by secreting the peptide hormone insulin. Insulin is packaged within vesicles named insulin secretory granules (ISGs), which recently have been considered to have intrinsic structures and proteins that regulate insulin granule maturation, trafficking, and secretion. Previously, studies have identified a handful of novel ISG-associated proteins, using different separation techniques. The present study combines an optimized ISG isolation technique and mass spectrometry–based proteomics, with an unbiased protein correlation profiling and targeted machine-learning approach to uncover 211 ISG-associated proteins with confidence. Four of these proteins, syntaxin-7, synaptophysin, synaptotagmin-13, and Scamp3 have not been previously associated with ISG. Through colocalization analysis of confocal imaging, we validate the association of these proteins to the ISG in MIN6 and human β-cells. We further validate the role for one (Scamp3) in regulating insulin content and secretion from β-cells for the first time. Scamp3 knockdown INS-1 cells have reduced insulin content and dysfunctional insulin secretion. These data provide the basis for future investigation of Scamp3 in β-cell biology and the regulation of insulin secretion. Article Highlights: This study optimizes insulin granule isolation techniques alongside enhanced proteomics analyses to establish the first murine insulin secretory granule proteome. We investigated what proteins are present on insulin granules from MIN6 cells to further our understanding of insulin granule biogenesis, trafficking, and secretion. We found 211 insulin granule–associated proteins and validated four novel proteins. Through further functional studies, we implicated Scamp3 as a novel protein that regulates insulin content and secretion in β-cells. [ABSTRACT FROM AUTHOR]

For their pungency and tanginess, ginger rhizomes (Zingiber officinale) are often used in cooking. Ginger has long been used in traditional medicine and home remedies to treat pain and inflammation. This research tests Z. officinale (EOZ) essential oils' chemical composition, insecticidal, and antioxidant properties in vitro and in silico. EOZ yielded 0.69% of root mass with 23 chemicals. The main chemicals in EOZ were α-zingiberene (23.850%), Geranial (14.160%), and (E,E)-α-farnesene (9.980%). EOZ demonstrated noteworthy antioxidant activity in all tests, with an IC50 of 9.53 ± 0.41 mg/mL in the DPPH test and an EC50 of 87.46 ± 3.19 mg/mL in the FRAP system. The results indicate that EOZ has strong efficacy against C. maculatus, even at low concentrations (1.00 μL/100 g). Note that EOZ killed 20 ± 0% and 13.33 ± 4.44% of adult C. maculatus in inhalation and contact tests, respectively. High concentration (20.00 μL/100 g) resulted in 100% adult mortality in inhalation tests and 96.67 ± 4.44% mortality in contact testing. We then used molecular docking to identify EO major component binding modes by targeting insecticidal and antioxidant protein structures 1R20 and 1R4U. This research helps create insecticides and antioxidants from common ginger essential oil. [ABSTRACT FROM AUTHOR]

This study aimed to explore the impact of novel method in improving the functionality and nutritional value of lentil-quinoa protein complexes. The method involved manipulating protein interactions through pH shifting and the addition of trehalose, ultimately enhancing water solubility, digestibility, and the functionality. Fluorescence, ultraviolet, and Fourier-transform spectroscopic techniques were applied to characterize the structural and molecular properties of the trehalose-conjugated lentil-quinoa protein complexes at various concentrations. After trehalose conjugation, statistically significant alterations (p < 0.05) in the tertiary and secondary protein structures and protein conformation were observed with trehalose-conjugated lentil-quinoa protein complexes at a trehalose concentration of 5% (w/w). The water solubility and digestibility of the latter conjugate-derived lentil and quinoa complex increased from 75.1 to 80% and 75 to 80.3%, respectively. Modifications in surface properties after conjugation involved significant (p < 0.05) changes in surface charge and hydrophobicity. Overall, combining the protein complexes with trehalose enhanced the digestibility and solubility of the protein complexes and improved their physical properties. The study highlights the potential of this approach to develop more sustainable and efficient plant-based protein sources for various food and nutritional applications in the food industry. [ABSTRACT FROM AUTHOR]

The objective of this research was to investigate and compare the effects of various protein or protein-based powders (WPC, SPI, BSA and hemoglobin) addition on transform of texture properties in egg white (EW)/egg yolk (EY) gels. Results showed that the highest hardness value (38.5 N) was detected in BSA-treated EW gels, while the lowest values (46.6 N) were obtained in hemoglobin-treated EY gels. Additionally, significantly increased value of free –SH groups was obtained in SPI/BSA/hemoglobin-treated EW/EY solutions, and increased value of protein solubility was obtained in EY solutions treated by WPC/SPI/BSA/hemoglobin relative to those of the control. The EW solutions treated by BSA/hemoglobin reached significantly higher H0 values than those of other groups. SDS-PAGE analysis showed that the hemoglobin-treated EW solution had slightly intense bands of ovoinhibitor, ovotransferrin and ovalbumin, and the EY solutions treated by WPC/SPI/BSA had higher level of covalent crosslinking. The EW gels treated by WPC/hemoglobin exhibited relatively more ordered protein secondary structures (α-helix and β-sheet) than those of the SPI group. SEM results suggested that WPC/BSA-treated EW gels developed uniform, porous and fiber-like structure, while SPI/BSA-treated EY gels developed rougher structure. [ABSTRACT FROM AUTHOR]

Introduction: The filamins are cytoskeletal binding proteins that dynamically crosslink actin into orthogonal networks or bundle it into stress fibres. The domain structure of filamin proteins is very well characterised, with an N‐terminal actin‐binding region, followed by 24 immunoglobulin‐like repeat units. The repeat domains are separated into distinct segments by two regions of low‐complexity known as hinge‐1 and hinge‐2. The role of hinge‐1 especially has been proposed to be essential for protein function as it provides flexibility to the otherwise rigid protein, and is a target for cleavage by calpain. Hinge‐1 protects cells from otherwise destructive forces, and the products of calpain cleavage are involved in critical cellular signalling processes, such as survival during hypoxia. Pathogenic variants in FLNA encoding Filamin A, including those that remove the hinge‐1 domain, cause a wide range of survivable developmental disorders. In contrast, complete loss of function of this gene is embryonic lethal in human and mouse. Methods and Results: In this study, we show that removing filamin A hinge‐1 from mouse (FlnaΔH1), while preserving its expression level leads to no obvious developmental phenotype. Detailed characterisation of the skeletons of FlnaΔH1 mice showed no skeletal phenotype reminiscent of that found in the FLNA‐causing skeletal dysplasia. Furthermore, nuclear functions of FLNA are maintained with loss of Filamin A hinge‐1. Conclusion: We conclude that hinge‐1 is dispensable for filamin A protein function during development over the murine lifespan. [ABSTRACT FROM AUTHOR]

Summary: This study aimed to investigate the effects of different oxidation modes on the functional and gel properties of hazelnut proteins. The solubility of hazelnut protein showed a small trend when the oxidation level was low, and it decreased significantly with the increase in concentration. The maximum value of water‐holding capacity (WHC), emulsifying activity index (EAI), and emulsion stability index (ESI) was 343.33%, 56.00 m2 g−1, and 75.85 min at AAPH concentration of 1.0 mmol L−1, respectively. The functional properties of hazelnut proteins, except oil‐holding capacity (OHC), gradually reduced with the increase in malondialdehyde (MDA) concentration. H2O2 modified improved OHC, foaming capacity, foam stability, and EAI of hazelnut proteins, whereas WHC and ESI gradually decreased. The results of the secondary structure of hazelnut protein gels indicated a high ratio of α‐helix and β‐fold of the gels. Also, the increase in oxidation led to an increase in the gels' voids. Also, oxidation led to a rough and loose network structure of hazelnut protein gels. These results suggested that peroxyl radical, hydroxyl radical, and MDA produced during lipid peroxidation affected the interfacial properties of hazelnut protein and disrupted the formation of hazelnut protein gel, thus affecting the quality of hazelnut protein. [ABSTRACT FROM AUTHOR]

Summary: To improve the utilisation of abomasum and explore the material basis of its antioxidant and hyaluronidase inhibitory activities, a homogeneous protein RDA4‐1 was isolated from the abomasum of cultured red deer by two‐step chromatography process for the first time in this study. It has a molecular weight of 15 kDa and comprises 43.56% essential amino acids. RDA4‐1 scavenged 50.97% of DPPH• and 88.37% of ABTS+· at 1.5 mg mL−1, and inhibited hyaluronidase by 79.23% at 1 mg mL−1. The CD and fluorescence spectroscopy was used to initiate explore the hyaluronidase inhibition reaction process. With an increase in the concentration of RDA4‐1 in the inhibition reaction, the wavelength of the negative peak of CD shifted from 208 to 218 nm, and the absorption of the positive peak of CD increased from 0.5 to 8.3 mdeg. Furthermore, fluorescence absorption at a wavelength of 340 nm rose from 248 to 762 au. These findings suggest that the interaction of RDA4‐1 with hyaluronidase induces alterations in the secondary structure of the protein complex, and RDA4‐1 binds reversibly to the enzyme in a non‐covalent manner. The results of this study indicate that RDA4‐1 could serve as a potential hyaluronidase inhibitor in the development of nutraceuticals. [ABSTRACT FROM AUTHOR]

Plant height is one of the most important traits in the breeding of snap bean, which determines the planting density and the resistance to failure, which in turn affects its yield and quality. Therefore, studying the molecular mechanism of plant height in snap bean is an important measure to realize the improvement of snap bean. We obtained a dwarf, thin‐stemmed mutant nts (normal to slim) from a dwarf variety of snap bean A18 via 60CO‐γ‐ray treatment. We conducted phenotypic observations, endogenous hormone content measurements and genetic analyses of the dwarfing thin‐stemmed mutant nts and explored gene localization and candidate gene screening for the mutant trait in snap bean. Compared with wild‐type A18, nts mutant exhibited various abnormal phenotypes such as dwarfing, reduced branching, thinner top stem, reduced plant width, reduced number of leaf area and thousand grain weight and increased content of hormones such as indole‐3‐acetic acid (IAA) in the top stem. Genetic analysis indicated that the mutant trait was controlled by a single recessive nuclear gene, and the mutant gene was named pv‐nts. pv‐nts was localized to a 361.1‐kb segment on chromosome 8 as revealed by bovine serum albumin (BSA) hybrid pool sequencing and simple sequence repeat (SSR) molecular markers. Phvul.008G106300 (encoding LAX2 protein) was identified as a candidate gene corresponding to pv‐nts via gene function annotation. Phvul.008G106300 was significantly upregulated in nts mutant compared with wild‐type A18, which was consistent with the results of endogenous IAA levels. Sequence analysis showed that the Phvul.008G106300 gene in nts had a base mutation that altered the protein structure, thus causing the dwarfed, thin‐stemmed phenotype of cauliflower. Taken together, these results contribute to a better understanding of the genetic basis of dwarfing traits in snap bean. [ABSTRACT FROM AUTHOR]

A natural nano-edible coating based on fish gelatin-rosemary essential oil was developed for the preservation of blunt snout bream (Megalobrama amblycephala) fillets. The results showed that the coating treatment effectively maintained the texture stability of the fish fillets, including appearance and water-holding capacity, significantly delayed lipid and protein oxidation, and inhibited microbial growth. On day 10, compared to the CK group, the TVB-N and TVC of the CT decreased by 41.46% and 31.36%, respectively. Proteomics was used to reveal the biochemical mechanisms affecting fish quality characteristics. Bioinformatics analysis of differential abundant proteins (DAPs) showed that the effects of the coating were related to the cellular process, skeleton structure, and enzymatic activity. Meanwhile, 65 DAPs which related to quality changes were further screened out, and most DAPs showed upregulation in coated fish fillets compared to the control, including myosin heavy chain, myosin-7, T-complex protein 1, isocitrate dehydrogenase (NADP (+)), 40S ribosomal protein, and HSPs, while Prdx-6 showed downregulation. It is believed that the coating delayed and prevented the generation of microbial degradation products and reduced the damage to the protein structure by suppressing microbial growth and protease activity, thereby stabilizing texture properties of refrigerated fish fillets. The shelf life of fillets during storage at 4 °C was significantly extended by at least 30%. The study provides an innovative method for the fish preservation and a theoretical basis for the protective effect of the coating on protein structures at the molecular level. [ABSTRACT FROM AUTHOR]

The ability of laccases to work effectively in properly selected aqueous-organic solutions significantly expands the scope of their application, making them commercially attractive and more competitive. However, it must be considered that molecules of organic solvent are very aggressive towards enzymes and destroy weak non-covalent interactions that maintain the spatial structure of the protein. In this work, we quantitatively described the influence of the nature of water-miscible organic solvents and their concentration on the activity and stability of Trametes versicolor laccase during the oxidation of ABTS (2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) through the maximum rates and the Michaelis constants KM. The obtained kinetic parameters make it possible to compare the effect of various organic solvents on laccase and select the most optimal one, which minimizes the decrease in enzyme activity, maintains its stability and allows repeated use. Among the solvents studied, polyhydroxy compounds have the least negative effect on laccase activity; however, in all cases, a high concentration of organic solvent (≥30% (v/v)) leads to suppression of enzymatic activity. It has been shown that the longer the alkyl chain (hydrophobic fragment of the structure), the stronger the negative effect of alcohols on enzymes. The most toxic solvent for Trametes versicolor laccase is isopropanol. In addition, numerous examples of various uses of Trametes versicolor in aquatic-organic environments are discussed here. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Effect of prawn fed with Catharanthus roseus ethanolic extract incorporated diet in the protein profile approaches in gene expressions related and immune characteristics in giant freshwater prawn (Macrobrachium rosenbergii)." The original article contained mistakes, specifically in the figures and author contributions. The corrected figures include a GC-MS peak level graph, conformational transitions in VP28 protein, and relative mRNA expression levels of certain genes. Additionally, the author contributions were updated to include Velusamy Gayathri. The corrected version of the article has been approved. [Extracted from the article]

The domain‐swapping mechanism involves the exchange of structural elements within a secondary or supersecondary structure between two (or more) proteins. The present paper proposes to interpret the domain‐swapping mechanism using a model that assesses the structure of proteins (and complexes) based on building the structure of a common hydrophobic core in a micelle‐like arrangement (a central hydrophobic core with a polar shell in contact with polar water), which has a considerable impact on the stabilisation of the domain structure built by domain swapping. Domains with a hydrophobicity system that is incompatible with the micelle‐like structure have also been identified. This incompatibility is the form of structural codes related to biological function. [ABSTRACT FROM AUTHOR]

A high protein walnut flour (HPWF) was obtained by defatting walnut flour (WF), which is a by-product of the oil industry. The objective of this study was the chemical and techno-functional characterization of HPWF. Composition, amino acid content, protein secondary structure, protein solubility and thermal transitions were measured. Besides, the techno-functional properties, emulsion activity and stability, and water holding and oil absorption capacities, of HPWF were evaluated. Also, the molecular mass of proteins under denaturing conditions and the microstructure of HPWF were evaluated by electrophoresis and confocal scanning laser microscopy, respectively. HPWF had 55.4% protein content and 21.5% total dietary fibre. In terms of HPWF amino acid composition, the limiting amino acids were the sulphurated cysteine and methionine. By FTIR analysis, the main secondary structures were β-sheet (49%) followed by α-helix (24%); both structures are considered to be ordered. Likewise, HPWF soluble proteins increased at basic pH and HPWF proteins were separated in 11 bands with molecular masses ranging from 97 kDa to 18 kDa by electrophoresis. With respect to techno-functional properties, HPWF presented good emulsion activity (51%) and high thermal emulsion stability (46%). In addition, HPWF retained 571% and 242% of water and oil by weight, respectively. Finally, the micrograph showed the predominance of protein structures and fibre fragments, and the presence of few lipids mostly trapped. These results showed that HPWF is an interesting source of plant-based proteins and walnut flour can be used to obtain high protein ingredients from non-traditional sources. [ABSTRACT FROM AUTHOR]

Recently, a number of message passing neural network (MPNN)‐based methods have been introduced that, based on backbone atom coordinates, efficiently recover native amino acid sequences of proteins and predict modifications that result in better expressing, more soluble, and stable variants. However, usually, X‐ray structures, or artificial structures generated by algorithms trained on X‐ray structures, were employed to define target backbone conformations. Here, we show that commonly used algorithms ProteinMPNN and SolubleMPNN display low sequence recovery on structures determined using NMR. We subsequently propose a computational approach that we successfully apply to re‐engineer AstaP, a protein that natively binds a large hydrophobic ligand astaxanthin (C40H52O4), and for which only a structure determined using NMR is currently available. The engineered variants, designated NeuroAstaP, are 51 amino acid shorter than the 22 kDa parent protein, have 38%–42% sequence identity to it, exhibit good yields, are expressed in a soluble, mostly monomeric form, and demonstrate efficient binding of carotenoids in vitro and in cells. Altogether, our work further tests the limits of using machine learning for protein engineering and paves the way for MPNN‐based modification of proteins based on NMR‐derived structures. [ABSTRACT FROM AUTHOR]

The phosphate‐starvation response transcription‐factor protein family is essential to plant response to low‐levels of phosphate. Proteins in this transcription factor (TF) family act by altering various gene expression levels, such as increasing levels of the acid phosphatase proteins which catalyze the conversion of inorganic phosphates to bio‐available compounds. There are few structural characterizations of proteins in this TF family, none of which address the potent TF activation domains. The phosphate‐starvation response‐like protein‐4 (PHL4) protein from this family has garnered interest due to the unusually high TF activation activity of the N‐terminal domain. Here, we demonstrate using solution nuclear magnetic resonance (NMR) measurements that the PHL4 N‐terminal activating TF effector domain is mainly an intrinsically disordered domain of over 200 residues, and that the C‐terminal region of PHL4 is also disordered. Additionally, we present evidence from size‐exclusion chromatography, diffusion NMR measurements, and a cross‐linking assay suggesting full‐length PHL4 forms a trimeric or tetrameric assembly. Together, the data indicate the N‐ and C‐terminal disordered domains in PHL4 flank a central folded region that likely forms the ordered oligomer of PHL4. This work provides a foundation for future studies detailing how the conformations and molecular motions of PHL4 change as it acts as a potent activator of gene expression in phosphate metabolism. Such a detailed mechanistic understanding of TF function will benefit genetic engineering efforts that take advantage of this activity to boost transcriptional activation of genes across different organisms. [ABSTRACT FROM AUTHOR]

Atherosclerotic cardiovascular diseases (CVDs) are closely linked to factors such as familial hypercholesterolemia (FH), often caused by mutations in low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB). Through a comprehensive bioinformatic analysis, we identified novel LDLR and APOB mutations and their cardiovascular disease (CVD) implications, focusing on unique variants in the Vietnamese population. We used homology modeling to predict protein structures; in addition, through protein-protein molecular docking, we assessed how these mutations affect binding affinities. We identified 10 novel binding residues exclusive to the wild-type and precursor LDLR isoforms, including ASP-47, GLY-48, and GLU-51. Analyses of 154 complexes revealed 5 isoforms with low binding affinities and notable hydrogen-bonding interactions—APOB (Arg3527Trp)-LDLR (Cys318Arg), APOB (His3583Leu)-LDLR (Cys104Tyr), APOB wild-LDLR (Glu228Lys), APOB (Phe2469Cys)-LDLR (Glu288Lys), and APOB wild-LDLR (Ser130Ter). These results suggest strong and potentially detrimental interactions among these proteins. Furthermore, they highlight the molecular mechanisms underlying CVD development, reveal potential therapeutic targets, enhance our understanding of genetic variations, and could guide FH research. [ABSTRACT FROM AUTHOR]

Artificial Intelligence (AI)-based deep learning methods for predicting protein structures are reshaping knowledge development and scientific discovery. Recent large-scale application of AI models for protein structure prediction has changed perceptions about complicated biological problems and empowered a new generation of structure-based hypothesis testing. It is well-recognized that proteins have a modular organization according to archetypal folds. However, it is yet to be determined if predicted structures are tuned to one conformation of flexible proteins or if they represent average conformations. Further, whether or not the answer is protein fold-dependent. Therefore, in this study, we analyzed 2878 proteins with at least ten distinct experimental structures available, from which we can estimate protein topological rigidity verses heterogeneity from experimental measurements. We found that AlphaFold v2 (AF2) predictions consistently return one specific form to high accuracy, with 99.68% of distinct folds (n = 623 out of 628) having an experimental structure within 2.5Å RMSD from a predicted structure. Yet, 27.70% and 10.82% of folds (174 and 68 out of 628 folds) have at least one experimental structure over 2.5Å and 5Å RMSD, respectively, from their AI-predicted structure. This information is important for how researchers apply and interpret the output of AF2 and similar tools. Additionally, it enabled us to score fold types according to how homogeneous versus heterogeneous their conformations are. Importantly, folds with high heterogeneity are enriched among proteins which regulate vital biological processes including immune cell differentiation, immune activation, and metabolism. This result demonstrates that a large amount of protein fold flexibility has already been experimentally measured, is vital for critical cellular processes, and is currently unaccounted for in structure prediction databases. Therefore, the structure-prediction revolution begets the protein dynamics revolution! [ABSTRACT FROM AUTHOR]

Plant-associated microbiota affect pant growth and development by regulating plant hormones homeostasis. Indole-3-acetic acid (IAA), a well-known plant hormone, can be produced by various plant-associated bacteria. However, the prevalence of bacteria with the capacity to degrade IAA in the rhizosphere has not been systematically studied. In this study, we analyzed the IAA degradation capabilities of bacterial isolates from the roots of Arabidopsis and rice. Using genomics analysis and in vitro assays, we found that 21 out of 183 taxonomically diverse bacterial isolates possess the ability to degrade IAA. Through comparative genomics and transcriptomic assays, we identified iac-like or iad-like operon in the genomes of these IAA degraders. Additionally, the putative regulator of the operon was found to be highly conserved among these strains through protein structure similarity analysis. Some of the IAA degraders could utilize IAA as their carbon and energy source. In planta, most of the IAA degrading strains mitigated Arabidopsis and rice seedling root growth inhibition (RGI) triggered by exogenous IAA. Moreover, RGI caused by complex synthetic bacterial community can be alleviated by introducing IAA degraders. Importantly, we observed increased colonization preference of IAA degraders from soil to root according to the frequency of the biomarker genes in metagenome-assembled genomes (MAGs) collected from different habitats, suggesting that there is a close association between IAA degraders and IAA producers. In summary, our findings further the understanding of the functional diversity and potential biological roles of plant-associated bacteria in host plant root morphogenesis. How do plant-associated microbiota regulate plant hormones, affecting their growth and development? This study reveals that bacterial isolates associated with the roots of Arabidopsis and rice can degrade the auxin indole-3-acetic acid (IAA), mitigating the inhibition of root growth induced by IAA-producing bacterial communities. [ABSTRACT FROM AUTHOR]

Studying specific subpopulations of cancer-derived extracellular vesicles (EVs) could help reveal their role in cancer progression. In cancer, an increase in reactive oxygen species (ROS) happens which results in lipid peroxidation with a major product of 4-hydroxynonenal (HNE). Adduction by HNE causes alteration to the structure of proteins, leading to loss of function. Blebbing of EVs carrying these HNE-adducted proteins as a cargo or carrying HNE-adducted on EV membrane are methods for clearing these molecules by the cells. We have referred to these EVs as Redox EVs. Here, we utilize a surface tension-mediated extraction process, termed exclusion-based sample preparation (ESP), for the rapid and efficient isolation of intact Redox EVs, from a mixed population of EVs derived from human glioblastoma cell line LN18. After optimizing different parameters, two populations of EVs were analyzed, those isolated from the sample (Redox EVs) and those remaining in the original sample (Remaining EVs). Electron microscopic imaging was used to confirm the presence of HNE adducts on the outer leaflet of Redox EVs. Moreover, the population of HNE-adducted Redox EVs shows significantly different characteristics to those of Remaining EVs including smaller size EVs and a more negative zeta potential EVs. We further treated glioblastoma cells (LN18), radiation-resistant glioblastoma cells (RR-LN18), and normal human astrocytes (NHA) with both Remaining and Redox EV populations. Our results indicate that Redox EVs promote the growth of glioblastoma cells, likely through the production of H2O2, and cause injury to normal astrocytes. In contrast, Remaining EVs have minimal impact on the viability of both glioblastoma cells and NHA cells. Thus, isolating a subpopulation of EVs employing ESP-based immunoaffinity could pave the way for a deeper mechanistic understanding of how subtypes of EVs, such as those containing HNE-adducted proteins, induce biological changes in the cells that take up these EVs. [ABSTRACT FROM AUTHOR]

Background: Oligoasthenoteratozoospermia (OAT) is a widespread cause of male infertility. One of the usual clinical manifestations of OAT is multiple morphological abnormalities of the sperm flagella (MMAF), which are frequently associated with mutations and defects in the dynein family. However, the relationship between the newly identified Dynein Axonemal Heavy Chain 3 (DNAH3) mutation and oligonasthenospermia in humans has not yet been established. Methods: Whole exome sequencing, pathogenicity analysis, and species conservation analysis of mutation sites were conducted on two patients from different unrelated families with DNAH3 mutations. We identified representative mutation sites and predicted the protein structure following these mutations. The sperm characteristics of the two patients with DNAH3 mutations were verified using Papanicolaou staining, scanning electron microscopy, and transmission electron microscopy. Additionally, mRNA and protein levels were assessed through RT-qPCR and Western blotting. Results: The biallelic mutations in the first progenitor included a heterozygous deletion and insertion, c.6535_6536 delinsAC (to infect mutation (p.Asp2179Thr), and stop codon premutation, c.3249G > A (p.Trp1083Ter). In Family II, the patient (P2) harbored a DNAH3 heterozygous missense mutation, c. 10439G> A(p.Arg3480Gln), along with a stop codon premutation, (c.10260G > A; p.Trp3420Ter). Patients with premature termination of transcription or translation due to DNAH3 mutations exhibit OAT phenotypes, including fibrous sheath dysplasia and multiple tail malformations. We identified the representative sites after mutation, predicted the protein structure, and assessed changes in the protein levels of DNAH3 and related genes following mutations. Notably,a significant reduction in DNAH3 protein expression was validated in these patients. We may explore in the future how DNAH3 affects sperm motility and quality through regulatory mechanisms involving protein structural changes. Conclusion: Novel biallelic mutations in DNAH3, especially those resulting in a premature stop codon, may alter protein expression, structure, and active site, leading to spermatogenic failure and potentially inducing OAT. The discovery of new mutations in DNAH3 may be the key to the diagnosis and treatment of OAT. [ABSTRACT FROM AUTHOR]

A soft set functions as a mathematical instrument crafted to handle uncertainties. Semigraph, a broader form of graph distinct from hypergraph, saw the integration of soft set principles to give rise to soft semigraph. Utilizing parameterization, soft semigraph delineates various representations of a semigraph’s relationships. The pivotal role of graph isomorphism lies in its ability to discern patterns across diverse domains such as image processing, computer systems, social network analysis, chemical bonding exploration, and protein structure analysis. Different types of isomorphisms are present within soft semigraphs, including s-isomorphism, sev-isomorphism, se-isomorphism, and sa-isomorphism. This paper focuses on elucidating certain characteristics associated with these isomorphisms. Additionally, we investigate the complement of a soft semigraph. [ABSTRACT FROM AUTHOR]

Fluorine‐19 NMR spectroscopy has emerged as a powerful tool for studying protein structure, dynamics, and interactions. Of particular interest is the exploitation of trifluoromethyl (tfm) groups, given their high sensitivity and superior transverse relaxation properties, compared to single fluorine atoms. However, biosynthetic incorporation of tfm‐bearing amino acids remains challenging due to cytotoxicity and incompatibility with natural tRNA synthetases. Here, we report on overcoming this challenge using cell‐free synthesis, incorporating trifluoromethyl‐methionine (tfmM) into the protein Cyclophilin A (CypA) with remarkably high efficiency, impossible via biosynthetic means. Importantly, we demonstrate that tfmM CypA binds a native substrate, the N‐terminal domain of HIV‐1 capsid protein (HIV‐1 CA‐NTD), and retains peptidyl prolyl cis/trans isomerase activity. It also binds the peptide inhibitor Cyclosporine A (CsA) with the same affinity as non‐labeled, wild‐type CypA. Furthermore, we show that 19F isotope shifts and 19F solvent paramagnetic relaxation enhancements (PREs) provide valuable structural information on surface exposure. Taken together, our study illustrates that tfmM can be readily incorporated into proteins at very high levels by cell‐free synthesis without disturbing protein structure and function, significantly expanding the scope of 19F NMR spectroscopy for studying protein structure and dynamics. [ABSTRACT FROM AUTHOR]

Background: For decades, KRAS has always been a huge challenge to the field of drug discovery for its significance in cancer progression as well as its difficulties in being targeted as an "undruggable" protein. KRAS regulates downstream signaling pathways through protein–protein interactions, whereas many interaction partners of KRAS remain unknown. Results: We developed a workflow to computationally predict and experimentally validate the potential KRAS-interacting proteins based on the interaction mode of KRAS and its known binding partners. We extracted 17 KRAS-interacting motifs from all experimentally determined KRAS-containing protein complexes as queries to identify proteins containing fragments structurally similar to the queries in the human protein structure database using our in-house protein–protein interaction prediction method, PPI-Miner. Finally, out of the 78 predicted potential interacting proteins of KRAS, 10 were selected for experimental validation, including BRAF, a previously reported interacting protein, which served as the positive control in our validation experiments. Additionally, a known peptide that binds to KRAS, KRpep-2d, was also used as a positive control. The predicted interacting motifs of these 10 proteins were synthesized to perform biolayer interferometry assays, with 4 out of 10 exhibiting binding affinities to KRAS, and the strongest, GRB10, was selected for further validation. Additionally, the interaction between GRB10 (RA-PH domain) and KRAS was confirmed via immunofluorescence and co-immunoprecipitation. Conclusions: These results demonstrate the effectiveness of our workflow in predicting potential interacting proteins for KRAS and deepen the understanding of KRAS-driven tumor mechanisms and the development of therapeutic strategies. [ABSTRACT FROM AUTHOR]

Several recent studies have presented evidence that the human gene catalogue should be expanded to include thousands of short open reading frames (ORFs) appearing upstream or downstream of existing protein-coding genes, each of which might create an additional bicistronic transcript in humans. Here we explore an alternative hypothesis that would explain the translational and evolutionary evidence for these upstream ORFs without the need to create novel genes or bicistronic transcripts. We examined 2,199 upstream ORFs that have been proposed as high-quality candidates for novel genes, to determine if they could instead represent protein-coding exons that can be added to existing genes. We checked for the conservation of these ORFs in four recently sequenced, high-quality human genomes, and found a large majority (87.8%) to be conserved in all four as expected. We then looked for splicing evidence that would connect each upstream ORF to the downstream protein-coding gene at the same locus, thus creating a novel splicing variant using the upstream ORF as its first exon. These protein coding exon candidates were further evaluated using protein structure predictions of the protein sequences that included the proposed new exons. We determined that 541 out of 2,199 upstream ORFs have strong evidence that they can form protein coding exons that are part of an existing gene, and that the resulting protein is predicted to have similar or better structural quality than the currently annotated isoform. Author summary: We analyzed over 2000 human sequences that have been proposed to represent novel protein-coding genes, and that reside just upstream of known genes. These "upstream ORFs" (uORFs) would represent a surprisingly large addition to the human gene catalogue, which after decades of refinement now contains just under 20,000 protein-coding genes. They would also create over 2000 new bicistronic genes, which number only 10 in current human annotation databases. We hypothesized that rather than novel genes, these sequences might instead represent novel exons that can be spliced into existing protein-coding genes, creating new isoforms of those genes. Using a combination of transcriptional evidence and computational predictions, we show that at least 541 of the previously-described uORFs can be used to create novel protein-coding exons, generating new transcripts and new protein isoforms, but not requiring the addition of entirely new genes to the human gene catalogue. We also demonstrate that the predicted three-dimensional structure of some of the new protein isoforms hints at new or improved functions for existing proteins. [ABSTRACT FROM AUTHOR]