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Hydrogen sulfide (H₂S) has emerged as a significant signaling molecule involved in various physiological processes, including vasodilation, neurotransmission, and cytoprotection. Its interactions with biomolecules are critical to understand its roles in health and disease. Recent advances in biophysical characterization techniques have shed light on the complex interactions of H₂S with proteins, nucleic acids, and lipids. Proteins are primary targets for H₂S, which can modify cysteine residues through S-sulfhydration, impacting protein function and signaling pathways. Advanced spectroscopic techniques, such as mass spectrometry and NMR, have enabled the identification of specific sulfhydrated sites and provided insights into the structural and functional consequences of these modifications. Nucleic acids also interact with H₂S, although this area is less explored compared to proteins. Recent studies have demonstrated that H₂S can induce modifications in nucleic acids, affecting gene expression and stability. Techniques like gel electrophoresis and fluorescence spectroscopy have been utilized to investigate these interactions, revealing that H₂S can protect DNA from oxidative damage and modulate RNA stability and function. Lipids, being integral components of cell membranes, interact with H₂S, influencing membrane fluidity and signaling. Biophysical techniques such as electron paramagnetic resonance (EPR) and fluorescence microscopy have elucidated the effects of H₂S on lipid membranes. These studies have shown that H₂S can alter lipid packing and dynamics, which may impact membrane-associated signaling pathways and cellular responses to stress. In the current work we have integrated this with key scientific explainations to provide a comprehensive review. [Display omitted] • Hydrogen sulfide (H₂S) plays a pivotal role in cell signaling, influencing vasodilation, neurotransmission, and cytoprotection. • Mass spectrometry (MS) identifies and quantifies S-sulfhydrated proteins, revealing persulfide modifications and their specificity. • Nuclear magnetic resonance (NMR) spectroscopy elucidates structural changes and interaction dynamics of H₂S-modified proteins. • Fluorescence resonance energy transfer (FRET)-based techniques offer real-time spatial and temporal resolution of H₂S interactions in living cells. • Electron paramagnetic resonance (EPR) spectroscopy explores the redox properties of H₂S and its interaction with metal-containing enzymes. [ABSTRACT FROM AUTHOR]

Using a combination of pea protein isolates (PPI) and β-lactoglobulin (βlg) presents an interesting approach to improve the gelling properties of PPI compared to the use of PPI alone. While controlling the 7S/11S ratio has proven effective in improving the gelling properties of PPI, more complex models need to be studied. Specifically, the effects of diverse 7S/11S ratios and the inclusion of βlg and 2S albumin on gel formation remain unexplored. Therefore, this study aimed to 1) determine the optimal 7S/11S ratios and 2) identify the interactions involved in the formation of model βlg—pea protein gels generated from globulin and albumin-enriched fractions. Results revealed that pea protein gels with a 7S/11S ratio of ∼ 1.89 achieved the highest firmness (131 Pa). Gelation was primarily driven by hydrophobic interactions (∼ 41%) involving 7S vicilin and 2S albumin, while most of the 11S legumin did not contribute to the protein gel. Conversely, a 7S/11S ratio of ≤ 1 facilitated the formation of the firmest gels (300–657 Pa) in the presence of βlg. These gels were primarily formed via disulfide bonds (36–65%) with βlg, 11S legumin, and 2S albumin as the main proteins involved, while 7S vicilin gelled independently through hydrophobic interactions. Based on these results, we propose a mechanism illustrating the interactions in mixed βlg—pea protein gels. This study provides new insights into how 7S/11S ratios, gel firmness, and protein interactions interplay during the gelation of mixed βlg–pea protein systems, paving the way for innovation in developing a new food category. [Display omitted] • Without βlg, gel firmness peaks at a 7S/11S ratio of 1.89 • Without βlg, hydrophobic interactions dominate pea protein gel formation. • Gel firmness peaks at a 7S/11S ratio ≤1 with βlg present. • With βlg, 7S independently gels through hydrophobic interactions. • With βlg, βlg, 11S, and 2S gel via disulfide bonds. [ABSTRACT FROM AUTHOR]

[Display omitted] • Improved thiolysis and LC-HRMS analysis were applied to grape seed extract proanthocyanidins (PACs). • GSE PACs with DPs up to 16 were identified by improved HRMS data processing. • DP affected interactions of PAC with cream proteins and the physical property of cream. • Low-DP PACs primarily engaged in hydrogen bonding with cream proteins. • High-DP PACs mainly utilized multiple hydrophobic interaction to form aggregates. This study aimed to comprehensively characterize chemical profiles of proanthocyanidins (PACs) from grape seed extract (GSE), examine their interactions with proteins in a cream system, and define the mechanisms mediating PAC-protein interactions. GSE PACs were fractionated and characterized by thiolysis followed by liquid chromatography–high resolution mass spectrometry (LC-HRMS) analysis. New PACs with a degree of polymerization (DP) up to 16 were identified by improved HRMS data processing methods. In the model cream system, high-DP PACs exhibited greater precipitation capacity and protein binding than low-DP PACs. Low-DP PACs primarily engaged in hydrogen bonding, while high-DP PACs predominantly utilized multiple hydrophobic interaction sites to form cream protein aggregates. Furthermore, particle size and viscosity measurement of cream revealed a progressively DP-dependent increase in aggregated fat globules and cream viscosity. These findings enhanced our understanding of PACs' structural intricacies and highlighted their functional role as PAC-rich natural ingredients in creating structured cream systems. [ABSTRACT FROM AUTHOR]

Across all eukaryotic kingdoms, ubiquitin regulatory X (UBX) domain-containing adaptor proteins control the segregase cell division control protein 48 (CDC48), and thereby also control cellular proteostasis and adaptation. The structures and biological roles of UBX proteins in animals and fungi have garnered considerable attention. However, their counterparts in plants remain markedly understudied. Since 2021, the artificial intelligence (AI)-based algorithm AlphaFold has provided predictions of protein structural features that can be highly accurate. Predictions of the proteomes of all major model organisms are now freely accessible to the entire research community through user-friendly web interfaces. We propose that the combination of cross-kingdom comparison with AF analysis produces a wealth of testable hypotheses to inspire and guide experimental research on plant UBX domain-containing (PUX) proteins. Ubiquitin regulatory X (UBX) domain-containing adaptor proteins guide and control the AAA-ATPase cell division control protein 48 (CDC48) to mediate an astonishing variety of fast-response mechanisms. Recent structural and functional studies have clarified the biological roles of many UBX proteins from animals and fungi, but most plant UBX domain-containing (PUX) proteins remain poorly understood. Despite their evolutionary distance, many domains and their combinations are conserved across eukaryotic UBX proteins, suggesting that they form functionally coherent 'work packages'. The artificial intelligence (AI)-based program AlphaFold is now able to accurately predict protein 3D structures and connections between structural units. The combination of a cross-kingdom comparison and AlphaFold analysis reveals conserved motifs and higher-order features for PUX proteins that collectively provide testable hypotheses to efficiently guide future experimental research. [ABSTRACT FROM AUTHOR]

Whey proteins in milk are globular proteins that can be converted into fibrils to enhance functional properties such gelation, emulsification, and foaming. A model fibrillated milk protein concentrate (MPC) was developed by mixing micellar casein concentrate (MCC) with fibrillated milk whey proteins. Similarly, a control model MPC was obtained by mixing MCC with milk whey proteins. The resulting fibrillated model MPC and control model MPC contained 5% protein and a ratio of casein to whey proteins similar to milk. The objective of the current study was to understand the rheological characteristics of fibrillated and control model MPC during acid gelation, using Förster resonance energy transfer (FRET) to assess small amplitude oscillation and casein–whey protein interaction. The results from the FRET index images showed greater interactions between caseins and whey proteins in fibrillated model MPC compared with the moderate and uniform interactions in control model MPC gels. Rheological study showed that the maximum storage modulus of acid gel of fibrillated model MPC was 546.9 ± 15.5 Pa, which was significantly higher than acid gel made from control model MPC (336.9 ± 11.3 Pa), indicating that fibrillated model MPC produced a firmer gel. Therefore, it can be concluded that acid gel produced from fibrillated model MPC was stronger than control model MPC. Selective fibrillation of the whey protein fraction in MPC can be used to improve gelation characteristics of acid gel type products. [ABSTRACT FROM AUTHOR]

Protein cross-linking has assumed an irreplaceable role in structural proteomics. Recently, significant efforts have been made to develop novel mass spectrometry (MS)-cleavable reagents. At present, only water-insoluble MS-cleavable cross-linkers are commercially available. However, to comprehensively analyse the various chemical and structural motifs making up proteins, it is necessary to target different protein sites with varying degrees of hydrophilicity. Here we introduce the new MS-cleavable cross-linker disulfodisuccinimidyl dibutyric urea (DSSBU), which we have developed in-house for this purpose. DSSBU contains an N -hydroxysulfosuccinimide (sulfo-NHS) reactive group, so it can serve as a water-soluble counterpart to the widely used cross-linker disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers: bis[sulfosuccinimidyl] suberate (BS3), disuccinimidyl suberate (DSS), DSBU and DSSBU with bovine serum albumin. In addition, we compared the efficacy of DSBU and DSSBU with human haemoglobin. Our results demonstrate that the sulfo-NHS group ensures the superior water solubility of DSSBU and thus negates the need for organic solvents such as dimethyl sulfoxide while preserving the effectivity of urea-based MS-cleavable crosslinkers such as DSBU. Additionally, it makes it possible to target polar regions in proteins. The data gathered are available via ProteomeXchange under identifier PXD055284. We have synthesized the novel protein cross-linker DSSBU, which combines sulfo-NHS ester chemistry with a mass spectrometry- cleavable urea group. This makes DSSBU a water-soluble, MS-cleavable cross-linker that reacts with amino groups. To our knowledge, it is the first cross-linker which combines all three of these characteristics. We have tested the performance of our novel cross-linker on bovine serum albumin, a model widely used by the cross-linking mass spectrometry community, and on human haemoglobin. We have comprehensively assessed the performance of DSSBU and compared its efficacy with that of three other cross-linkers in current use (BS3, DSS and DSBU). We conclude that our novel cross-linker surpasses its MS-non-cleavable analogue BS3 in performance and that its water solubility eliminates the need for organic solvents while its hydrophilicity allows for the targetting of polar regions in proteins. Therefore, it will likely become a significant addition to the portfolio of N -hydroxysuccinimide ester cross-linkers. [Display omitted] • We have synthesized and tested the novel water-soluble and MS-cleavable amino-specific protein cross-linker DSSBU. • The effectivity of DSSBU is comparable to that of its water-insoluble analogue DSBU and it performs better than its MS-non-cleavable analogue BS3. • DSSBU performs well in water, eliminating the need for an organic solvent. • DMSO containing 2 % water is an effective solvent for DSSBU and DSBU alike. • DSSBU in combination with DSBU improves the yield of BSA linkages compared to the separate application of these cross-linkers. [ABSTRACT FROM AUTHOR]

Gibberellic acids (GAs) are a group of endogenous phytohormones that play important roles in plant growth and development. SLENDER RICE (SLR) serves as a vital component of the DELLA gene family, which plays an irreplaceable role in regulating plant flowering and height, as well as stress responses. SLR gene has not been reported in mango, and its function is unknown. In present study, two DELLA subfamily genes MiSLR1 and MiSLR2 were identified from mango. MiSLR1 and MiSLR2 were highly expressed in the stems of the juvenile stage, but were expressed at a low level in flower buds and flowers. Gibberellin treatment could up-regulate the expression of MiSLR1 and MiSLR2 genes, but gibberellin biosynthesis inhibitor prohexadione-calcium (Pro-Ca) and paclobutrazol (PAC) treatments significantly down-regulated the expression of MiSLR1 , while MiSLR2 was up-regulated. The expression levels of MiSLR1 and MiSLR2 were up-regulated under both salt and drought treatments. Overexpression of MiSLR1 and MiSLR2 genes significantly resulted early flowering in transgenic Arabidopsis and significantly up-regulated the expression levels of endogenous flower-related genes, such as SUPPRESSOR OF CONSTANS1 (SOC1), APETALA1 (AP1), and FRUITFULL (FUL). Interestingly, MiSLR1 significantly reduced the height of transgenic plants, while MiSLR2 gene increased. Overexpression of MiSLR1 and MiSLR2 increased seed germination rate, root length and survival rate of transgenic plants under salt and drought stress. Physiological and biochemical detection showed that the contents of proline (Pro) and superoxide dismutase (SOD) were significantly increased, while the contents of malondialdehyde (MDA) and H 2 O 2 were significantly decreased. Additionally, protein interaction analysis revealed that MiSLR1 and MiSLR2 interacted with several flowering-related and GA-related proteins. The interaction between MiSLR with MiGF14 and MiSOC1 proteins was found for the first time. Taken together, the data showed that MiSLR1 and MiSLR2 in transgenic Arabidopsis both regulated the flowering time and plant height, while also acting as positive regulators of abiotic stress responses. • Two DELLA genes, MiSLR1 and MiSLR2 were identified and characterized. • MiSLR1 and MiSLR2 confers early flowering and different phenotypes in plant height. • MiSLR1 and MiSLR2 enhances abiotic stress tolerance to abiotic stress tolerance in Arabidopsis. • Discovered for the first time the interaction of MiSLR with MiGF14 and MiSOC1 proteins. [ABSTRACT FROM AUTHOR]

Extended-spectrumβ-lactam producing Escherichia coli (ESBL-EC) readily colonizes live poultry and serves as a major source of contamination in retail chicken meat, posing significant threats to public health. This study aims to investigate the impact of inappropriate antibiotic use on the dissemination and exacerbation of antibiotic resistance in ESBL-EC and explore the underlying molecular mechanisms. Through experimental analysis, we propose a hypothesis that inappropriate antibiotic use may exacerbate resistance by affecting vesicle formation and protein secretion. Experimental results demonstrate that under the influence of amoxicillin, the concentration of proteins secreted in outer membrane vehicles (OMVs) by ESBL-EC significantly increases, along with a significant upregulation in the expression of the CTX-M-55-type Extended-spectrum beta-lactamase (CTX-M-55). Proteomic analysis and differential gene knockout experiments identified the key protein YdcZ, associated with OMVs formation and protein transportation in ESBL-EC under amoxicillin treatment. Further investigations reveal direct interactions between YdcZ and other proteins (YdiH and BssR). Upon ydcz gene knockout, a significant decrease in protein concentration within OMVs is observed, accompanied by a noticeable reduction in protection against sensitive bacteria. These findings suggest a critical role of YdcZ in regulating the process of protein transportation to OMVs in ESBL-EC under the influence of amoxicillin. In summary, our research uncovers the significant role of inappropriate antibiotic use in promoting the secretion of OMVs by ESBL-EC, aiding the survival of antibiotic-sensitive bacteria in the vicinity of infection sites. These findings provide new insights into the mechanisms underlying antibiotic-induced bacterial resistance dissemination and offer novel avenues for exploring prevention and control strategies against bacterial resistance propagation. • Unveiled antibiotic-vesicle dynamics, vital for food safety. • Revealed intricate dynamics, notably increased OMVs secretion in response to subinhibitory amoxicillin. • Emphasizes OMVs' potential role in antibiotic resistance transmission, paving the way for innovative strategies. [ABSTRACT FROM AUTHOR]

Auxin response factors (ARFs) are central components of the growth hormone signaling pathway, responsible for regulating the expression of corresponding growth hormone genes. To date, ARFs have demonstrated diverse functions and roles, with identification in various species, including Arabidopsis thaliana and Oryza sativa. However, their specific functions in Torreya grandis , an evergreen and economically significant tree species primarily found in China, remain elusive. In this study, we identified 15 ARF genes (TgARF1 - TgARF15) in T. grandis and observed their distribution across 11 chromosomes. We analyzed the expression patterns of TgARFs in various tissues and their responses to drought stress. Our findings revealed a significant reduction of TgARF15 expression in leaf tissues under drought conditions, implying a potential key role for TgARF15 in drought stress resistance. Additionally, we determined that TgARF15 was localized in the nucleus. Furthermore, overexpression of TgARF15 in Arabidopsis resulted in enhanced drought resistance, attributed to increased enzymatic activity of antioxidant enzymes. Moreover, we identified a direct interaction between TgARF15 and TgIAA1, both in vivo and in yeast. These findings will facilitate future efforts in establishing a foundation for in-depth exploration of their specific mechanisms related to drought resistance, and addressing challenges in the T. grandis industry. • 15A RF genes was identified in Torreya grandis. • Overexpression of TgARF15 enhanced the tolerance of tomato to drought stress. • TgARF15 directly interacted with TgIAA1 in vivo and in yeast. [ABSTRACT FROM AUTHOR]

• The interaction between AtTFIIS and AtLeo1 of AtPaf1C was identified • AtTFIIS LW domain interacts with an LFG motif of AtLeo1 through a hydrophobic pocket formed by several critical and conserved residues • Our studies revealed the structural basis of the interaction between AtTFIIS and AtLeo1 [ABSTRACT FROM AUTHOR]

The purpose of this investigation was to highlight the utility of nuclear magnetic resonance (NMR) as a multi-attribute method for the characterization of therapeutic antibodies. In this case study, we compared results from isothermal chemical denaturation (ICD) and NMR with standard methods to relate conformational states of a model monoclonal antibody (mAb1) with protein-protein interactions (PPI) that lead to self – association in concentrated solutions. The increase in aggregation rate and relative viscosity for mAb1 was found to be both concentration and pH dependent. The free energy of unfolding (∆G⁰) from ICD and thermal analysis in dilute solutions indicated that although the native state predominated between pH 4 – pH 7, it was disrupted at the CH 2 and unfolded noncooperatively under acidic conditions. One-dimensional (1D) 1H NMR and two-dimensional (2D) 13C-1H NMR performed, in concentrated solutions, confirmed that PPI between pH 4–7 occurred while mAb1 was in the native state. NMR corroborated that mAb1 maintained a dominant native state at formulation-relevant conditions at the tested pH range, had increased global molecular tumbling dynamics at lower pH and confirmed increased PPI at higher pH conditions. This report aligns and compares typical characterization of an IgG1 with assessment of structure by NMR and provided a more precise assessment and deeper insight into the conformation of an IgG1 in concentrated solutions. [ABSTRACT FROM AUTHOR]

• Fast Field-Cycling can assess proteoglycan content in vitro. • The T 1 vs field strength profile of cartilage varies in osteoarthritis. • These biomarkers can potentially be accessed in vivo using Field-Cycling Imaging. • Potential application in clinical trials of new treatments for early osteoarthritis. Osteoarthritis in synovial joints remains a major cause of long-term disability worldwide, with symptoms produced by the progressive deterioration of the articular cartilage. The earliest cartilage changes are thought to be alteration in its main protein components, namely proteoglycan and collagen. Loss of proteoglycans bound in the collagen matrix which maintain hydration and stiffness of the structure is followed by collagen degradation and loss. The development of new treatments for early osteoarthritis is limited by the lack of accurate biomarkers to assess the loss of proteoglycan. One potential biomarker is magnetic resonance imaging (MRI). We present the results of a novel MRI methodology, Fast Field-Cycling (FFC), to assess changes in critical proteins by demonstrating clear quantifiable differences in signal from normal and osteoarthritic human cartilage for in vitro measurements. We further tested proteoglycan extracted cartilage and the key components individually. Three clear signals were identified, two of which are related predominantly to the collagen component of cartilage and the third, a unique very short-lived signal, is directly related to proteoglycan content; we have not seen this in any other tissue type. In addition, we present the first volunteer human scan from our whole-body FFC scanner where articular cartilage measurements are in keeping with those we have shown in tissue samples. This new clinical imaging modality offers the prospect of non-invasive monitoring of human cartilage in vivo and hence the assessment of potential treatments for osteoarthritis. Keywords: Fast Field-Cycling NMR; human hyaline cartilage; Osteoarthritis; T1 dispersion; quadrupolar peaks; protein interactions [ABSTRACT FROM AUTHOR]

[Display omitted] It is well known that protein corona affects the "biological identity" of nanoparticles (NPs), which has been seen as both a challenge and an opportunity. Approaches have moved from avoiding protein adsorption to trying to direct it, taking advantage of the formation of a protein corona to favorably modify the pharmacokinetic parameters of NPs. Although promising, the results obtained with engineered NPs still need to be completely understood. While much effort has been put into understanding how the surface of nanomaterials affects protein absorption, less is known about how proteins can affect corona formation due to their specific physicochemical properties. This review addresses this knowledge gap, examining key protein factors influencing corona formation, highlighting current challenges in studying protein–protein interactions, and discussing future perspectives in the field. [ABSTRACT FROM AUTHOR]

Interactions between food and saliva govern complex mouthfeel perceptions such as astringency. Herein, we present a study of the interactions of salivary proteins with the main pea protein fractions that are obtained by isoelectric and salt precipitation (legumin-rich, vicilin-rich and albumin-rich fractions). The sensory evaluations performed on protein solutions by trained panelists evidenced that all three protein fractions exhibit a basal level of astringency, but that the albumin fraction was perceived as the most astringent one. All three fractions induced significant but comparable loss of salivary lubrication. Yet, when compared to the other fractions, the albumin fraction showed the formation of a thicker and more rigid film on salivary conditioning film-coated sensors as measured using a quartz crystal microbalance with dissipation monitoring (QCM-D). We also present proteomics studies on the precipitates obtained from the mixtures of saliva and pea protein fractions. Protein identification finds a pool of salivary proteins involved in non-specific interactions with all the three pea protein fractions. Yet, 13 pea proteins specific to the albumin fraction were identified as being involved in specific interactions with salivary proteins. Several of these proteins are part of the plant defense mechanisms and are likely to interact with many salivary proteins. This could explain the higher number of salivary proteins found in the precipitate induced by the albumin fraction when compared to the other two. These quantitative results increase the understanding of the complex links between plant protein-salivary protein interactions and astringency. [Display omitted] • The albumin pea protein fraction was the most astringent pea protein fraction. • Globulin and albumin pea proteins induced a similar salivary lubrication breakdown. • Albumin pea proteins formed a rigid and thick film on a salivary pellicle. • 13 pea proteins of the albumin fraction are likely to trigger interactions with saliva. [ABSTRACT FROM AUTHOR]

SQUAMOSA promoter-binding protein-like (SPL) family genes play an important role in regulating plant flowering and resistance to stress. However, understanding the function of the SPL family in mango is still limited. In a previous study, two MiSPL3 genes, MiSPL3a and MiSPL3b (MiSPL3a/b), were identified in 'SiJiMi' mango and exhibited the highest expression in flowers at the initial flowering stage [ 24 ]. Therefore, in this study, we further investigated the expression pattern and gene function of MiSPL3a/b. The results showed that the expression of MiSPL3a was greatest at the end of floral bud differentiation, and MiSPL3b was expressed mainly during the flowering induction and vegetative growth stages. Subcellular localization showed that MiSPL3a/b localized to the nucleus. In addition, ectopic expression of MiSPL3a/b promoted earlier flowering in Arabidopsis thaliana by 3 d-6 d than in wild-type (WT) plants, which increased the expression of SUPPRESSOR OF CONSTANS1 (AtSOC1), FRUITFULL (AtFUL), and APETALA1 (AtAP1). MiSPL3a/b transgenic lines exhibited increased tolerance to drought, GA 3 , and abscisic acid (ABA) treatments but were sensitive to Pro-Ca treatment. Furthermore, protein interaction analysis revealed that MiSPL3a/b could interact with several stress-related proteins, flowering-related proteins, and the bridge protein 14–3-3. Taken together, MiSPL3a and MiSPL3b acted as positive regulators of flowering time and stress tolerance in transgenic Arabidopsis. [ABSTRACT FROM AUTHOR]

This study investigated casein-whey protein interactions in high-protein milk dispersions (5% protein wt/wt) during heating at 90°C for 1.5 to 7.5 min at 3 different pH of 6.5, 6.8, and 7.0, using both conventional methods (gel electrophoresis, physicochemical properties) and fluorescence spectroscopy. Conventional methods confirmed the presence of milk protein aggregates during heating, similar to skim milk. These methods were able to help in understanding the denaturation and aggregation of milk proteins as a function of heat treatment. However, the results from the conventional methods were greatly affected by batch-to-batch variations and, therefore, differentiation could be drawn only in nonheated samples and samples heated for a longer duration. The front-face fluorescence spectroscopy was found to be a useful tool that provided additional information to conventional methods and helped in understanding differences between nonheated, low-, and high-heated samples, along with the type of sample used (derived from liquid or powder milk protein concentrates). At all pH values, tryptophan maxima in nonheated samples derived from powdered milk protein concentrates presented a blue shift in comparison to samples derived from liquid milk protein concentrates, and tryptophan maxima in heated samples presented a red shift. With the heating of the sample, Maillard emission and excitation spectra also showed increases in the peak intensities from 408 to 432 and 260 to 290 nm, respectively. As the level of denaturation increased with heating, a marked differentiation can be seen in the principal component analysis plots of tryptophan, Maillard emission, and excitation spectra, indicating that the front-face fluorescence technique has a potential to monitor and classify samples according to milk protein interactions as a function of pH and heat exposure. Overall, it can be said that the pattern of protein-protein interactions in high-protein dispersions was similar to the observation reported in skim milk systems, and fluorescence spectroscopy with chemometrics can be used as a rapid, nondestructive, and complementary method to conventional methods for following heat-induced changes. [ABSTRACT FROM AUTHOR]

Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine. [ABSTRACT FROM AUTHOR]

Salt stress is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple (Malus × domestica). In this study, we demonstrate that a salt-responsive MYB transcription factor (TF), designated as MdMYB63, promotes survival under salt stress. Overexpression of MdMYB63 in apple calli significantly enhanced salt tolerance. Screening of the AP2/ERF family of TFs identified MdERF106 as an interaction partner of MdMYB63. Further analyses showed that the MdMYB63–MdERF106 complex significantly promotes the expression of downstream MdSOS1 , thereby improving the Na+ expulsion and salt tolerance of apple. These functional analyses of MdMYB63 have provided valuable insights into the regulatory network of salt tolerance, and lay a theoretical foundation for the cultivation of new salt-tolerant apple varieties. • Functional analyses of MdMYB63 provide valuable insights into the salt tolerance. • Overexpression of MdMYB63 in apple calli promotes its survival under salt stress. • MdMYB63 binds to MBS on the promoter of MdSOS1 to enhance its transcription. • MdERF106 can significantly promote the effect of MdMYB63 on the expression of downstream MdSOS1 by forming a protein complex. • MYBs can directly regulate SOS1 to participate in the salt tolerance of plants. [ABSTRACT FROM AUTHOR]

Circulating CRP dissociates into monomeric CRP (mCRP) and deposits in inflamed loci. mCRP interacts with FN and enhances monocytes adhesion, upregulates adhesion molecules, and mediates the inflammatory process. • mCRP exhibits an enhanced binding capacity to FN independent of Ca2+ and pH. • mCRP binds to FN at residues 35–47. • In vivo , mCRP mainly interacted with FN in the tissue-bound form. • mCRP enhances adhesion of monocytes to FN and upregulates endothelial expression of adhesion molecules. The acute phase reactant C-reactive protein (CRP) binds with high affinity to fibronectin (FN), but this binding occurs only at pH 6.5 or lower, and the binding is inhibited by calcium ions at physiological pH. Since CRP in the circulating blood exists in a calcium-binding form, the interaction between CRP and FN in vivo has been uncertain. CRP can undergo a conformational rearrangement in the absence of calcium or in the local microenvironment (e.g. , acidic pH) of inflamed tissue to dissociate into monomeric CRP (mCRP). Therefore, we tested whether these discrepancies can be explained by the different isoforms and locations of CRP. Surface plasmon resonance and ELISA assays showed that mCRP binds with high affinity to FN, and the binding of mCRP to FN was unaffected by calcium or pH. Peptide competition assay, deletion mutant binding assay and protein docking analyse verified that the binding site of mCRP to FN is residues a.a.35–47. Furthermore, mCRP can significantly enhance the adhesion of monocytes to FN as well as upregulate the adhesion molecules expression on endothelial cell. Colocalization of mCRP with FN was observed in mice with DSS-induced colitis, whereas there was very little signal orcolocalization of CRP. These results provide in vitro and in vivo evidence that mCRP formed by local dissociation from circulating CRP is the major isoform that interacts with FN and regulates FN-mediated monocyte adhesion, which is involved in the pro-inflammatory process. [ABSTRACT FROM AUTHOR]

Genetic screens in cancer cell lines inform gene function and drug discovery. More comprehensive screen datasets with multi-omics data are needed to enhance opportunities to functionally map genetic vulnerabilities. Here, we construct a second-generation map of cancer dependencies by annotating 930 cancer cell lines with multi-omic data and analyze relationships between molecular markers and cancer dependencies derived from CRISPR-Cas9 screens. We identify dependency-associated gene expression markers beyond driver genes, and observe many gene addiction relationships driven by gain of function rather than synthetic lethal effects. By combining clinically informed dependency-marker associations with protein-protein interaction networks, we identify 370 anti-cancer priority targets for 27 cancer types, many of which have network-based evidence of a functional link with a marker in a cancer type. Mapping these targets to sequenced tumor cohorts identifies tractable targets in different cancer types. This target prioritization map enhances understanding of gene dependencies and identifies candidate anti-cancer targets for drug development. [Display omitted] • 500 gene dependencies are identified from CRISPR screens in 930 cancer cell lines • Clinically derived multi-omics markers are linked with gene dependencies • Protein-protein networks provide functional links between markers and dependencies • 370 candidate targets are identified and their prevalence in tumors vary widely Pacini et al. identify selective gene dependencies from CRISPR screens in 930 cell lines. Using clinically curated multi-omics data and protein-protein interaction networks, they curate 370 candidate targets and estimate their prevalence in patient tumor cohorts. This enhances understanding of gene dependencies and comprehensively maps targetable cancer dependencies. [ABSTRACT FROM AUTHOR]

Over the last 40 years nuclear magnetic resonance (NMR) spectroscopy has established itself as one of the most versatile techniques for the characterization of biomolecules, especially proteins. Given the molecular size limitations of NMR together with recent advances in cryo-electron microscopy and artificial intelligence-assisted protein structure prediction, the bright future of NMR in structural biology has been put into question. In this mini review we argue the contrary. We discuss the unique opportunities solution NMR offers to the protein chemist that distinguish it from all other experimental or computational methods, and how it can benefit from machine learning. • Future of solution protein NMR in the machine learning era of structural biology. • NMR spectroscopy provides unique insights into protein function that are not possible by other methods. • NMR spectroscopy as a versatile tool for understanding the complex dynamic nature of biomolecules. • Biomolecular NMR itself can benefit from the developments in the AI field. [ABSTRACT FROM AUTHOR]

Salt stress is one of the major environmental stressors that remarkably hinders the processes of plant growth and development, thereby limiting crop productivity. An understanding of the molecular mechanisms underlying plant responses against salinity stimulus will help guide the rational design of crop plants to counter these challenges. Nitric oxide (NO) is a redox-related signaling molecule regulating diverse biological processes in plant. Accumulating evidences indicated NO exert its biological functions through posttranslational modification of proteins, notably via S -nitrosylation. During the past decade, the roles of S -nitrosylation as a regulator of plant and S -nitrosylated candidates have also been established and detected. Emerging evidence indicated that protein S -nitrosylation is ubiquitously involved in the regulation of plant response to salt stress. However, little is known about this pivotal molecular amendment in the regulation of salt stress response. Here, we describe current understanding on the regulatory mechanisms of protein S -nitrosylation in response to salt stress in plants and highlight key challenges in this field. • S -nitrosylation is involved in the regulation of plant response to salt stress. • S -nitrosylation-induced conformational change is an important mechanism in response to salt stress. • S -nitrosylated protein regulates phytohormones signaling under salt stress. • NO-mediated S -nitrosylation activated carbohydrate metabolism, protecting plants from salinity. [ABSTRACT FROM AUTHOR]

• Transgenic soybean lines overexpressing GmHsp90A2 and with GmHsp90A2 knockout by the CRISPR/Cas9 system were obtained. • Heat shock protein 90A2 (GmHsp90A2) was identified as a positive regulator under heat stress in soybean. • GmHsp90A2 can interact with GmHsp90A1, and GmHsp90A1 may play a significant role in promoting GmHsp90A2 entry into nucleus. • Overexpression of GmHsp90A2 can reduce chlorophyll degradation and lipid peroxidation during heat stress. Heat shock protein 90 s (Hsp90s), one of the most conserved and abundant molecular chaperones, is an essential component of the protective stress response. A previous study reported at least 12 genes in the GmHs p90 s family in soybean and that GmHs p90 A2 overexpression enhanced thermotolerance in Arabidopsis thaliana. Here, we investigate the roles of GmHsp90A2 in soybean by utilizing stable transgenic soybean lines overexpressing GmHs p90 A2 and mutant lines generated by the CRISPR/Cas9 system. The results showed that compared with wild-type plants (WT) and empty vector control plants (VC), T 3 transgenic soybean plants overexpressing GmHs p90 A2 exhibited increased tolerance to heat stress through higher chlorophyll and lower malondialdehyde (MDA) contents in plants. Conversely, reduced chlorophyll and increased MDA contents in T 2 homozygous GmHs p90 A2- knockout mutants indicated decreased tolerance to heat stress. GmHsp90A2 was found to interact with GmHsp90A1 in yeast two-hybrid assays. Furthermore, subcellular localization analyses revealed that GmHsp90A2 was localized to the cytoplasm and cell membrane; as shown by bimolecular fluorescence complementation (BiFC) assays, GmHsp90A2 interacted with GmHsp90A1 in the nucleus and cytoplasm and cell membrane. Hence, we conclude that GmHsp90A1 is able to bind to GmHsp90A2 to form a complex and that this complex enters the nucleus. In summary, GmHsp90A2 might respond to heat stress and positively regulate thermotolerance by interacting with GmHsp90A1. [ABSTRACT FROM AUTHOR]

The behavior of a cell depends on how its adhesion molecules interact with the cellular microenvironment. Hemidesmosomal collagen XVII essentially contributes to cell adhesion and modulates keratinocyte directionality and proliferation during skin regeneration, however only little is known about the involved interactions. Here, we used keratinocytes from patients with junctional epidermolysis bullosa with late onset, which exclusively produce a collagen XVII mutant with the p.R1303Q mutation within its extracellular C-terminus. Although this mutant was normally expressed and targeted to the membrane and the expression of integrins α3β1, α6β4 and of laminin-332 was unchanged, the keratinocytes were less adhesive, showed migratory defects and decreased clonogenic growth. Since the p.R1303Q substitution is located within the predicted laminin-332 binding site of collagen XVII, we anticipated an altered collagen XVII-laminin-332 interaction. Indeed, the pR1303Q collagen XVII ectodomain showed decreased binding capability to laminin-332 and was less co-localized with pericellular laminin-332 molecules in cell culture. Thus, aberrant collagen XVII-laminin-332 interaction results in reduced cell adhesion, destabilized cell motility and decreased clonogenicity, which in turn lead to blister formation, delayed wound healing and skin atrophy. • Here, we investigated the missense mutation p.R1303Q of collagen XVII resulting in mild JEB. • Analysis of p.R1303Q protein revealed structural changes in the C-terminus causing reduced interactions with laminin 332. • p.R1303Q collagen XVII was normal expressed and membrane targeted. • JEBR1303Q keratinocytes were less adhesive, showed migratory defects and decreased clonogenic growth. • Reduction of collagen XVII-laminin 332 interaction leads to mild skin blistering, delayed wound healing and skin atrophy. [ABSTRACT FROM AUTHOR]

Abstract Di- and poly-nuclear Manganese-Cobalt complexes (dnc and pnc) were selected to be investigated in their anticancer activities against MCF-7 breast cancer cell lines. The results show that the pnc can reduce the cell viability to about 12% of the control after 72 h of exposure, in the event that dnc don't have a significant cellular cytotoxic impact on cell viability in MCF-7 cell line. The interaction of complexes with βLG and HSA were investigated and showed that (i) both complexes strongly quenched the fluorescence of proteins through the static quenching (ii) in the dnc -protein systems, HSA had stronger binding affinity toward complex than βLG, (iii) the association affinity of pnc bound to HSA or βLG were close to each other; (iv) the interaction of pnc with two proteins was greater than that of dnc (v) hydrogen bonding and hydrophobic interactions (especially in the pnc) are predominant forces in the binding process (vi) the binding of pnc and dnc to HSA and βLG can induce conformational changes of these proteins. These results can confirm that in addition to the type of ligands, the nuclear numbers and surface of the metal complex has a major impact on its biological activity. Graphical abstract Image 1 Highlights • Effects of pnc and dnc were studied against MCF-7 breast cancer lines, HSA and βLG. • pnc had a stronger anticancer activity than dnc. • The binding ability of HSA with both complexes was stronger than that of βLG. • Hydrogen bonding and hydrophobic interactions are main forces in the binding. • The complexes are located within subdomain IB of HSA and second site of βLG. [ABSTRACT FROM AUTHOR]

Highlights • Nanoapertures in metal films allow for optical trapping of proteins, DNA fragments and other biomolecules, as well as small viruses. • They provide a label free approach to identify proteins based on their trapping dynamics, including in unpurified solutions. • Protein interactions with DNA and small molecules have been measured with this approach. • The vibrational modes of the trapped nanoparticles can be measured using a modification of nanoaperture trapping called extraordinary acoustic Raman (EAR). Abstract Nanoaperture optical tweezers extend the range of optical tweezers to dielectric particles below 50 nm in size. This allows for optical trapping of proteins, DNA fragments and other biomolecules, as well as small viruses. With this label-free, tether-free approach proteins have been trapped, sized and their conformational changes observed in real-time. The molecular weight of proteins in the nanoaperture trap was determined from their Brownian motion statistics. This is useful for analysis of heterogeneous solutions: since this is a single molecule technique, it can be operated in “dirty” solutions with minimal sample preparation. The acoustic modes of proteins, DNA fragments and other trapped nanoparticles can be measured using a nanoaperture optical tweezer with two lasers creating a GHz to THz beat frequency. Interactions between proteins and DNA, small molecules (i.e., binding) and other proteins have also been demonstrated. This single molecule technique allows for measuring the dissociation constants of small molecules binding to proteins, both at equilibrium and at the single molecule level. For DNA fragments in the trap, it has been shown that the protein p53 can suppress unzipping and mutant p53 is ineffective to do so. This is promising for the discovery of drugs that effectively restore the function to p53. Integration of nanoapertures on the ends of fibers allows for translocation of the trapped object and may function as an optical “nanopipette” for microwell single molecule protein sampling. There is also potential to combine nanoapertures with nanopore translocation studies as well as fluorescence correlation microscopy studies and several researchers are already pursuing these areas of research. [ABSTRACT FROM AUTHOR]

Abstract Protein-protein interactions (PPI) and solution viscosities were measured at low and high protein concentrations under a range of formulation conditions for 4 different monoclonal antibodies. Static light scattering was used to quantify the osmotic second virial coefficient (B 22) and the zero-q limit static structure factor (S q=0), versus protein concentration (c 2) from low to high c 2. Dynamic light scattering was used to measure the collective diffusion coefficient as a function of c 2 and to determine the protein interaction parameter (k D). Static light scattering and dynamic light scattering were combined to determine the hydrodynamic factor (H q=0), which accounts for changes in hydrodynamic PPI as a function of c 2. The net PPI ranged from strongly repulsive to attractive interactions, via changes in buffer pH, ionic strength, and choice of monoclonal antibodies. Multiple-particle tracking microrheology and capillary viscometery were used to measure monoclonal antibodies solution viscosities under the same solution conditions. In most cases, even large and qualitative changes in PPI did not result in significant changes in protein solution viscosity. This highlights the complex nature of PPI and how they influence protein solution viscosity and raises questions as to the validity of using experimental PPI metrics such as k D or B 22 as predictors of high viscosity. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Chondroitin sulfate (CS) derivatives sulfation-dependently bind growth factors. • The disposition of the sulfates modulates the surface charge of the helical structure. • O-6 sulfation favours the arrangement of sulfates on the surface. • High-sulfated CS variants on the surface enhance the binding capacity. Abstract Chondroitin sulfate (CS) is a relevant family of polysaccharides that participates in a large variety of biological events that are related to neural processes by regulating various growth factors through the pattern and degree of sulfation of the polysaccharide. However, their own complexity makes their optimization for biomedical applications a difficult undertaking. Thus, a different perspective has to be taken. Herein, we show that the particular sulfate distribution within the disaccharide repeating-unit plays a key role in the binding of growth factors (GFs). In particular, this disposition modulates the surface charge of the helical structure that, interestingly, has a significant influence on the binding capacity of CSs with several GFs. This fact should be carefully considered in the design of new ligands with improved activity as GFs ligands. [ABSTRACT FROM AUTHOR]

Flavonoids, such as anthocyanins and proanthocyanidins (PAs), play essential roles in plant growth, development, and stress response. Red-fleshed apples represent a valuable germplasm resource with high flavonoid content. Understanding and enriching the regulatory network controlling flavonoid synthesis in red-fleshed apples holds significant importance for cultivating high-quality fruits. In this study, we successfully isolated an NAC transcription factor, MdNAC14-Like, which exhibited a significant negative correlation with the content of anthocyanin. Transient injection of apple fruit and stable expression of callus confirmed that MdNAC14-Like acts as an inhibitor of anthocyanin synthesis. Through yeast monohybrid, electrophoretic mobility shift, and luciferase reporter assays, we demonstrated the ability of MdNAC14-Like to bind to the promoters of MdMYB9 , MdMYB10 , and MdUFGT , thus inhibiting their transcriptional activity and subsequently suppressing anthocyanin synthesis. Furthermore, our investigation revealed that MdNAC14-Like interacts with MdMYB12, enhancing the transcriptional activation of MdMYB12 on the downstream structural gene MdLAR , thereby promoting PA synthesis. This comprehensive functional characterization of MdNAC14-Like provides valuable insights into the intricate regulatory network governing anthocyanin and PA synthesis in apple. • The expression level of MdNAC14-Like was negatively correlated with the content of anthocyanin. • MdNAC14-Like inhibits transcriptional activity of MdMYB9 , MdMYB10 and MdUFGT , thereby inhibiting anthocyanin synthesis. • MdNAC14-Like interacts with MdMYB12 to enhance the transcriptional activation of MdLAR , which promotes PAs synthesis. [ABSTRACT FROM AUTHOR]

• Mechanism of HAX1 regulates ASFV replication through interactions with MGF360–9 L. • Key regions of interaction between ASFV MGF360–9 L & HAX1 were predicted & validated. • The biological function of HAX1 in inhibiting ASFV replication was demonstrated. • Mechanism of MGF360–9L-linked K48 ubiquitin chain. African swine fever virus (ASFV) infection causes African swine fever (ASF), a virulent infectious disease that threatens the safety of livestock worldwide. Studies have shown that MGF360–9 L is important for the virulence of ASFV and the host protein HS1-associated protein X-1 (HAX1) plays an important role in viral pathogenesis. This study aimed to clarify the mechanism by which HAX1 mediates ASFV replication through interactions with MGF360–9 L. The regions of interaction between MGF360–9 L and HAX1 were predicted and validated. HAX1 overexpression and RNA interference studies revealed that HAX1 is a host restriction factor that suppresses ASFV replication. Moreover, HAX1 expression was inhibited in ASFV-infected mature bone marrow–derived macrophages, and infection with the virulent MGF360–9 L gene deletion strain (∆MGF360–9 L) attenuated the inhibitory effect of the wild-type strain (WT) on HAX1 expression, suggesting a complex regulatory relationship between MGF360–9 L and HAX1. Furthermore, the E3 ubiquitin ligase RNF114 interacted with MGF360–9 L and HAX1, MGF360–9 L degraded HAX1 through the ubiquitin–proteasome pathway, and RNF114 facilitated the degradation of HAX1 by MGF360–9L-linked K48 ubiquitin chains through the ubiquitin–proteasome pathway, thereby facilitating ASFV replication. In conclusion, this study has enriched our understanding of the regulatory networks between ASFV proteins and host proteins and provided a reference for investigation into the pathogenesis and immune escape mechanism of ASFV. [ABSTRACT FROM AUTHOR]

The viscoelasticity of concentrated protein suspensions associates closely with the mixing efficiency and cleaning frequency of facility during high-protein food development. This study investigated the effects of thermal-mechanical treatment on the viscoelasticity of milk protein isolate (MPI) suspensions and their underlying mechanisms to develop protein ingredient with low viscoelasticity. MPI suspensions (20%) were treated at 25, 50 and 85 °C for 10–60 min under constant shear (100 s−1), followed by storage at 4 °C. The viscosity (η) of MPI suspension treated at 50 °C and 85 °C was ∼1–10% as those treated at 25 °C. After four days of storage at 4 °C, η showed the least value in 50°C-treated samples compared to those at 25 °C and 85 °C. The η and storage modulus (G') was decreased with prolonged treatment at 25 and 50 °C, whereas opposite trend was found in 85 °C treated samples. Differential scanning calorimetry found proteins in 50 °C treated samples had smaller enthalpy than those in the control and 25 °C treated samples. Protein surface hydrophobicity was increased slightly from 25 °C to 50 °C, but remarkably in 85 °C treated samples. Ultra-small angle x-ray scattering showed the radius of gyration (R g) of casein micelle was ∼38 nm at 25 °C and 50 °C treated samples but increased to ∼44 nm 85 °C treated samples with reduced compactness. A new sphere-like structure with R g of 18 nm was generated in 85 °C treated samples. These findings suggested modulating temperature during thermal-mechanical treatment is essential to alter protein structures and morphology for desirable rheological properties. [Display omitted] • 50 °C treated MPI suspensions had the least viscosity and elasticity. • Proteins in 50 °C treated MPI suspensions had lower enthalpy than those at 25 °C. • 85 °C increased the size of casein micelle but reduced its compactness. • Duration of treatment has milder effect on viscoelasticity compared to temperature. [ABSTRACT FROM AUTHOR]

[Display omitted] • The specific interactions of fullerene nanomaterials with proteins were reported in literature over last 10 years. • More detailed target-related approach to interactions is necessary. • Buckyballs can be incorporated with proteins (e.g. lysozymes, albumin) to improve their water-solubility. • Fullerenes can interact with serum proteins so that a protein corona is formed around the nanoparticles, but the detailed composition of corona is still unknown. Fullerenes have numerous properties that fill the gap between small molecules and nanomaterials. Several types of chemical reaction allow their surface to be ornamented with functional groups designed to change them into 'ideal' nanodelivery systems. Improved stability, and bioavailability are important, but chemical modifications can render them practically soluble in water. 'Buckyball' fullerene scaffolds can interact with many biological targets and inhibit several proteins essential for tumorigeneses. Herein, we focus on the inhibitory properties of fullerene nanomaterials against essential proteins in cancer nanotechnology, as well as the use of dedicated proteins to improve the bioavailability of these promising nanomaterials. [ABSTRACT FROM AUTHOR]

Advanced techniques in nanofabrication are essential in order to manifest theoretical works in establishing nanobio-sensor devices. Metal-insulator-metal (MIM) geometry has been effectively used for tuning plasmonic effect that dramatically altered biosensor sensitivity. Normally, adhesive metal at metal-insulator interface; sub-nm chromium is required to stabilize the metallic nano-layer structure. This deteriorates the performances of the MIM sensors, by severely damping the plasmon modes. We replaced the commonly used sub-nm Cr with an organic molecular layer of (3‑Mercaptopropyl) trimethoxysilane; MPTMS, as an adhesive molecule to strengthen the structure of the MIM-based biosensor. This successfully demonstrated a non-deterioration plasmon effect due to no imaginary part exist in the refractive index of the MPTMS. The new structure of metal/(MPTMS)/insulator/(MPTMS)/metal, instead of metal/(Cr)/insulator/(Cr)/metal or metal/poly(methyl methacrylate) or PMMA/metal was used as biosensing platform to detect molecular interactions. We simulated the biosensing concept using transfer matrix methods and construct the theoretically optimized MIM structure. The metal (Au) thin-layer was prepared by means of thermal evaporation method on a pre-cleaned glass slide. MPTMS is then deposited using solution deposition technique. This enables larger usable area on the MIM platform that can be utilized as the sensing area compared to an MIM using PMMA as insulator layer. [ABSTRACT FROM AUTHOR]

Abstract Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has within the last 25 years evolved to become a well-established technique for analysing the solution-phase conformational dynamics and molecular interactions of proteins, attributes that can be difficult to access by other methods for structural analysis. This review presents the latest development within HDX-MS with a special focus on how improvements in instrumentation, methodology and data analysis have expanded the capability of HDX-MS to analyse complex and hitherto elusive protein states. Such protein states include large or multi-protein complexes, lipid-associated or integral membrane proteins, proteins with highly dynamic regions, disulfide-bonded proteins, heavily glycosylated proteins and proteins in formulations. Highlights • HDX-MS has become an established method to analyse the conformational dynamics and interactions of proteins. • HDX-MS is often used to detect and map conformational differences between protein states in solution. • Improvements in instrumentation, methodology and data analysis is enabling HDX-MS of protein states of unprecedented complexity and size. [ABSTRACT FROM AUTHOR]

SNAP-25 is a protein involved in regulated membrane fusion and part of the SNARE complex. It exists as two splicing variants, SNAP-25a and SNAP-25b, which differ in 9 out of 206 amino acids. SNAP-25 together with Syntaxin 1 and VAMP-2 forms the ternary SNARE complex essential for mediating activity-dependent release of hormones and neurotransmitters. The functional difference between SNAP-25a and SNAP-25b is poorly understood as both can participate in SNARE complexes and mediate membrane fusion. However, we recently demonstrated that SNAP-25b-deficiency results in metabolic disease and increased insulin secretion. Here we investigated if SNAP-25a and SNAP-25b differently affect interactions with other SNAREs and SNARE-interacting proteins in mouse hippocampus. Adult mice almost exclusively express the SNAP-25b protein in hippocampus whereas SNAP-25b-deficient mice only express SNAP-25a. Immunoprecipitation studies showed no significant differences in amount of Syntaxin 1 and VAMP-2 co-precipitated with the different SNAP-25 isoforms. In contrast, Munc18-1, that preferentially interacts with SNAP-25 via Syntaxin 1 and/or the trimeric SNARE complex, demonstrated an increased ability to bind protein-complexes containing SNAP-25b. Moreover, we found that both SNAP-25 isoforms co-precipitated the Gβγ subunits of the heterotrimeric G proteins, an interaction known to play a role in presynaptic inhibition. We have identified Gβ 1 and Gβ 2 as the interacting partners of both SNAP-25 isoforms in mouse hippocampus, but Gβ 2 was less efficiently captured by SNAP-25a. These results implicate that the two SNAP-25 isoforms could differently mediate protein interactions outside the ternary SNARE core complex and thereby contribute to modulate neurotransmission. [ABSTRACT FROM AUTHOR]

The present work attempts to clarify the influence of montmorillonite nanoclay content and pH on the mechanical properties of extruded soy protein nanocomposites. The mechanical behaviour is dominated by the formation of positive synergies between protein and nanoclay above a nanoclay concentration threshold. Moreover, the presence of nanoclay can improve water uptake. The pH also exerts a strong influence on mechanical and water absorption properties, although montmorillonite tend to reduce this effect. Eventually, this study put forward the feasibility of using a combination of soy protein and montmorillonite to obtain potentially attractive biodegradable nanocomposite materials, processed by means of a simple and easily scalable twin-screw extruder. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is the most common reversible post-translational modification in eukaryotes. Humans have over 500 protein kinases, of which more than a dozen are established targets for anticancer drugs. All kinases share a structurally similar catalytic domain, yet each one is uniquely positioned within signaling networks controlling essentially all aspects of cell behavior. Kinases are distinguished from one another based on their modes of regulation and their substrate repertoires. Coupling specific inputs to the proper signaling outputs requires that kinases phosphorylate a limited number of sites to the exclusion of hundreds of thousands of off-target phosphorylation sites. Here, we review recent progress in understanding mechanisms of kinase substrate specificity and how they function to shape cellular signaling networks. [ABSTRACT FROM AUTHOR]

The tetranuclear Pt complex (PtL) 4 (where L 2− is the anion derived from para -isopropyl thiosemicarbazone) was first described in A.G. Quiroga et al., J. Med. Chem. 41, 1998, 1399–1408. (PtL) 4 manifests antiproliferative properties toward various cancer cell lines being a promising anticancer drug candidate. Yet, details of its reactivity with biomolecules have not been elucidated. To this end, we investigated the reactions of (PtL) 4 with a few model proteins, i.e. bovine pancreatic ribonuclease (RNase A), cytochrome c (Cyt c ) and hen egg white lysozyme (Lysozyme), through electrospray ionization mass spectrometry and other biophysical methods. A rich reactivity of (PtL) 4 with the above-mentioned model proteins is observed, leading to the formation of numerous metallodrug-protein adducts. The tetranuclear complex breaks down and various fragments bind proteins up to high metal/protein ratios; this typically results into very complicated mass spectral patterns. However, some of the main mass peaks could be assigned in the case of the Lysozyme adduct. In addition, crystallographic data were obtained for the (PtL) 4 /Lysozyme and (PtL) 4 /RNase A adducts pointing at His side chains as the primary binding sites for monometallic Pt fragments. Notably, a few selected features of the interactions observed in the (PtL) 4 /protein adducts were reproduced by reacting (PtL) 4 with a small molecule, i.e. N -methylimidazole. In conclusion, the present study confirms the prodrug nature of the tetraplatinum complex, clarifies one possible pathway for its activation through cluster disassembly and allows initial identification of adducts formed with a representative protein. [ABSTRACT FROM AUTHOR]

Wnt signaling is an important pathway that regulates several aspects of embryogenesis, stem cell maintenance, and neural connectivity. We have recently determined that opioids decrease Wnt secretion, presumably by inhibiting the recycling of the Wnt trafficking protein Wntless (Wls). This effect appears to be mediated by protein-protein interaction between Wls and the mu-opioid receptor (MOR), the primary cellular target of opioid drugs. The goal of this study was to identify novel protein interactors of Wls that are expressed in the brain and may also play a role in reward or addiction. Using genetic and candidate gene approaches, we show that among a variety of protein, Wls interacts with the dopamine transporter (target of cocaine), cannabinoid receptors (target of THC), Adenosine A2A receptor (target of caffeine), and SGIP1 (endocytic regulator of cannabinoid receptors). Our study shows that aside from opioid receptors, Wntless interacts with additional proteins involved in reward and/or addiction. Future studies will determine whether Wntless and WNT signaling play a more universal role in these processes. [ABSTRACT FROM AUTHOR]

A novel Schiff base Zn(II) complex using a tetradentate Schiff base ligand was synthesized and characterized. The results of cytotoxicity assay revealed that the prepared complex induced cytotoxicity in a breast cancer cell line. Thus, the binding of the Zn(II) complex to human serum albumin (HSA) was investigated using spectroscopic and molecular docking methods. The fluorescence data showed that the Zn(II) complex quenched protein fluorescence by a static quenching mechanism. The binding constant (K b ), number of binding sites ( n ), and thermodynamic parameters were calculated and showed that the Zn(II) complex binds to HSA through hydrogen bond and Vander Waals interactions with one binding site. Protein-ligand docking analysis confirmed that the Zn(II) complex binds to residues located in the subdomain IIA of HSA. This Zn(II) complex was used for the preparation of ZnO nanoparticles, and their photocatalytic activities were examined via photocatalytic degradation of ethidium bromide (EBr) in the absence of UV–vis irradiation. The results showed that these nanoparticles are promising materials for photocatalytic degradation. [ABSTRACT FROM AUTHOR]

The phenomenon of protein phase separation, which underlies the formation of biomolecular condensates, has been associated with numerous cellular functions. Recent studies indicate that the amino acid sequences of most proteins may harbour not only the code for folding into the native state but also for condensing into the liquid-like droplet state and the solid-like amyloid state. Here we review the current understanding of the principles for sequence-based methods for predicting the propensity of proteins for phase separation. A guiding concept is that entropic contributions are generally more important to stabilise the droplet state than they are for the native and amyloid states. Although estimating these entropic contributions has proven difficult, we describe some progress that has been recently made in this direction. To conclude, we discuss the challenges ahead to extend sequence-based prediction methods of protein phase separation to include quantitative in vivo characterisations of this process. [ABSTRACT FROM AUTHOR]

Over the last two decades, awareness of the (patho)physiological roles of thromboxane A 2 signaling has been greatly extended. From humble beginnings as a short-lived stimulus that activates platelets and causes vasoconstriction to a dichotomous receptor system involving multiple endogenous ligands capable of modifying tissue homeostasis and disease generation in almost every tissue of the body. Thromboxane A 2 receptor (TP) signal transduction is associated with the pathogenesis of cancer, atherosclerosis, heart disease, asthma, and host response to parasitic infection amongst others. The two receptors mediating these cellular responses (TPα and TPβ) are derived from a single gene (TBXA2R) through alternative splicing. Recently, knowledge about the mechanism(s) of signal propagation by the two receptors has undergone a revolution in understanding. Not only have the structural relationships associated with G-protein coupling been established but the modulation of that signaling by post-translational modification to the receptor has come sharply into focus. Moreover, the signaling of the receptor unrelated to G-protein coupling has become a burgeoning field of endeavor with over 70 interacting proteins currently identified. These data are reshaping the concept of TP signaling from a mere guanine nucleotide exchange factors for Gα activation to a nexus for the convergence of diverse and poorly characterized signaling pathways. This review summarizes the advances in understanding in TP signaling, and the potential for new growth in a field that after almost 50 years is finally coming of age. [ABSTRACT FROM AUTHOR]

Bacillus subtilis spores, representatives of an exceptionally resistant dormant cell type, are encircled by a thick proteinaceous layer called the spore coat. More than 80 proteins assemble into four distinct coat layers: a basement layer, an inner coat, an outer coat and a crust. As the spore develops inside the mother cell, spore coat proteins synthesized in the cytoplasm are gradually deposited onto the prespore surface. A small set of morphogenetic proteins necessary for spore coat morphogenesis are thought to form a scaffold to which the rest of the coat proteins are attached. Extensive localization and proteomic studies using wild type and mutant spores have revealed the arrangement of individual proteins within the spore coat layers. In this study we examined the interactions between the proteins localized to the outer coat and crust using a bacterial two hybrid system. These two layers are composed of at least 25 components. Self-interactions were observed for most proteins and numerous novel interactions were identified. The most interesting contacts are those made with the morphogenetic proteins CotE, CotY and CotZ; these could serve as a basis for understanding the specific roles of particular proteins in spore coat morphogenesis. [ABSTRACT FROM AUTHOR]

Recognizing of binding mechanisms between drugs and carrier proteins is basic for us to understand the pharmacokinetics and pharmacodynamics of them. In this research, the anticancer activities of a binuclear complex [Co(dipic) 2 Ni(OH 2 ) 5 ]·2H 2 O (dipic = dipicolinate) against MDA-MB-231 cell lines were studied. Results of MTT assay and flow cytometry analysis revealed that above complex can induce the cytotoxicity and the apoptosis in breast cancer cell lines. So, this complex was selected to investigate its binding to human serum albumin (HSA) and bovine β-lactoglobulin (βLG) by spectroscopic methods (UV–visible, fluorescence and FT-IR) along with molecular docking technique. The fluorescence data showed Co-Ni complex quench the fluorescence of both proteins by a static quenching mechanism and HSA has stronger binding affinity toward Co-Ni complex than βLG. The binding constant (K b ), number of binding sites (n) and thermodynamic parameters were calculated and showed that the Co-Ni complex binds to protein (HSA and βLG) through hydrogen bonding and van der Waals forces with one binding site. The results of UV–visible measurements indicated that the binding of above complex to HSA and βLG may induce conformational and micro-environmental changes of studied proteins. Protein–ligand docking analysis confirmed that the Co-Ni complex binds to residues located in the subdomain IIA of HSA and site II of βLG. [ABSTRACT FROM AUTHOR]

Two complexes of Ru(II) possessing the general formula [Ru(ssz) 2 L](PF 6 ) were prepared, in which ssz is sulfasalazine and L is either 2,2′-bipyridine ( 1 ) or 1,10-phenanthroline ( 2 ). The complexes were characterized by elemental and conductivity analyses, UV–Vis, vibrational, NMR and electrospray ionization mass spectroscopies. The geometry around ruthenium(II) is distorted octahedral. The metal ion is bound to the carboxylate of ssz in a bidentate way and to two heterocyclic nitrogens of bpy or phen. Spectrofluorimetric measurements indicate that complexes bind to BSA with affinity constants ranging from 10 4 to 10 5 M −1 . Both compounds are significantly cytotoxic in a chronic myelogenous leukemia cell line in a concentration dependent manner. UV-light exposure for 5 min increases the cytotoxicity of 1 by three times and that of 2 by ten times. In addition, upon UV light irradiation, the complexes are able to damage the bovine serum albumin molecule, at concentrations that do not affect BSA in dark conditions. It is worth to note that the complexes also bind to Abl-SH3 protein, which is responsible for the development of chronic myelogenous leukemia. Interactions with proteins seem to be significant for the cytotoxic and photocytotoxic activity of these complexes. [ABSTRACT FROM AUTHOR]

The spectacular yield increases in rice and wheat during the green revolution were partly realized by reduced gibberellin (GA) synthesis or sensitivity, both causing the accumulation of DELLA proteins. Although insights into the regulation of plant growth and development by DELLA proteins advanced rapidly in arabidopsis ( Arabidopsis thaliana ), DELLA-mediated regulation of downstream responses in cereals has received little attention to date. Furthermore, translating this research from arabidopsis to cereals is challenging given their different growth patterns and our phylogenetic analysis which reveals that DELLA-related DGLLA proteins exist in cereals but not in arabidopsis. Therefore, understanding the molecular basis of DELLA function in cereals holds great potential to improve yield. In this review, we propose to extend the focus of DELLA functional research to cereals, and highlight the appropriate tools that are now available to achieve this. [ABSTRACT FROM AUTHOR]

The work deals with optimizing a methodology for fabrication of monodisperse metallic nanoparticles (active against microbes) using micellar core of amine based metallosurfactant. Novel double chained amine metallosurfactants of the type [M(C 12 H 25 NH 2 ) 2 ] (where M is copper, nickel and cobalt) have been synthesized and characterized with elemental analysis, Fourier Transform Infrared spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR) and thermogravimetric analysis (TGA). Further, study of theaggregated structures ( i.e . bilayer) of these lipophilic metallosurfactants in various alcohols has been carried out. Thermodynamics parameters of reverse micellization have also been estimated. The process of micellization is spontaneous and entropy driven. Prepared metallosurfactants have been utilized as precursors for the fabrication of metallic nanoparticles (NPs) of Co, Ni and Cu. The method is validated for all the three studied transition metals for the preparation of metallic nanoparticles. This approach offers better results in terms of size, shape, morphology, distribution and stability of NPs. Characterization of NPs is done by UV–vis, X-Ray Diffraction (XRD), Transmission Emission Microscopy (TEM) and Dynamic light scattering (DLS). Prepared metallosurfactants and NPs have been trialled for antimicrobial activity against three pathogenic microbes viz. Bacillus Cereus (gram +ve bacterium), Klebsiella Pneumoniae (gram −ve bacterium) and Curvularia lunata (fungus) and best results are shown by Cu NPs. Furthermore, interaction of metallic NPs with bovine serum albumin (BSA) has been estimated. [ABSTRACT FROM AUTHOR]

Summary Dynactin and NudE/Nudel are prominent regulators of cytoplasmic dynein motility and cargo-binding activities. Both interact with the intrinsically disordered N-terminal domain of dynein intermediate chain (IC), which also contains phosphorylation sites that apparently regulate these interactions. Nuclear magnetic resonance and isothermal calorimetry studies demonstrate that the Ser84 phosphorylation site identified in cells is in a disordered linker distant from the N-terminal helix that contains both the dynactin- and the Nudel-binding interfaces. Structural studies of a phosphomimetic Ser84Asp imply that phosphorylation stabilizes an electrostatic cluster that docks the disordered linker containing Ser84 against the N-terminal helix, resulting in a conformation that blocks access of IC to dynactin, but not to NudE/Nudel. Formation of this cluster is dependent on the length and sequence of the disordered linkers. This model explains the selective binding of mammalian IC to dynactin versus NudE/Nudel and why this selection is specific for IC-2C and not the IC-1A isoform. [ABSTRACT FROM AUTHOR]

The aqueous two-phase extraction (ATPE) of therapeutic proteins is a promising separation alternative to cost-intensive chromatography, still being the workhorse of nowadays downstream processing. As shown in many publications, using NaCl as displacement agent in salt-polymer ATPE allows for a selective purification of the target protein immunoglobulin G ( IgG ) from human serum albumin ( HSA , represents the impurity). However a high yield of the target protein is only achievable as long as the protein is stabilized in solution and not precipitated. In this work the combined influence of NaCl and polyethylene glycol (Mw = 2000 g/mol) on the IgG - IgG interactions was determined using composition gradient multi-angle light scattering (CG-MALS) demonstrating that NaCl induces a solubilization of IgG in polyethylene glycol 2000 solution. Moreover it is shown that the displacement agent NaCl has a significant and beneficial influence on the IgG solubility in polyethylene glycol 2000-citrate aqueous two-phase system (ATPS) which can also be accessed by these advanced B 22 measurements. By simultaneous consideration of IgG solubility data with results of the ATPS phase behavior (especially volume fraction of the respective phases) allows for the selection of process tailored ATPS including identification of the maximum protein feed concentration. Through this approach an ATPS optimization is accessible providing high yields and selectivity of the target protein ( IgG ). [ABSTRACT FROM AUTHOR]

In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregation. [ABSTRACT FROM AUTHOR]