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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]

The C–H⋯S–S interactions are fundamentally important to understand the stability of biomacromolecules and their binding with small molecules, but they are still underappreciated. Herein, we characterized the C–H⋯S–S interactions in model molecular complexes. The rotational spectra of the complexes of diethyl disulfide with CH2CH2 and CH2CHF were measured and analyzed. All the detected structures are mainly stabilized by a C–H⋯S–S hydrogen bond, providing stabilization energies of 2.3–7.2 kJ mol−1. Incidental C–H⋯π or C–H⋯F interactions enhance the stabilization of the complexes. London dispersion, which accounts for 54%–68% of the total attractions, is the main driving force of stabilization. The provided bonding features of C–H⋯S–S are crucial for understanding the stabilizing role of this type of interaction in diverse processes such as supramolecular recognition, protein stability, and enzyme activity. [ABSTRACT FROM AUTHOR]

The full-length prefusion-stabilized SARS-CoV-2 spike (S) is the principal antigen of COVID-19 vaccines. Vaccine efficacy has been impacted by emerging variants of concern that accumulate most of the sequence modifications in the immunodominant S1 subunit. S2, in contrast, is the most evolutionarily conserved region of the spike and can elicit broadly neutralizing and protective antibodies. Yet, S2s usage as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design S2-only immunogens stabilized in a closed prefusion conformation. Molecular simulations provide a mechanistic characterization of the S2 trimers opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Structural characterization via cryo-EM shows the molecular basis of S2 stabilization in the closed prefusion conformation. Informed by molecular simulations and corroborated by experiments, we report an engineered S2 immunogen that exhibits increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses.

The identification and quantification of misfolded proteins from complex mixtures is important for biological characterization and disease diagnosis, but remains a major bioanalytical challenge. We have developed Hsp40 Affinity Profiling as a bioanalytical approach to profile protein stability in response to cellular stress. In this assay, we ectopically introduce the Hsp40 FlagDNAJB8H31Q into cells and use quantitative proteomics to determine how protein affinity for DNAJB8 changes in the presence of cellular stress, without regard for native clients. Herein, we evaluate potential approaches to improve the performance of this bioanalytical assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNAJB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hsp40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress.

Protein stability plays a crucial role in a variety of applications, such as food processing, therapeutics, and the identification of pathogenic mutations. Engineering campaigns commonly seek to improve protein stability, and there is a strong interest in streamlining these processes to enable rapid optimization of highly stabilized proteins with fewer iterations. In this work, we explore utilizing a mega-scale dataset to develop a protein language model optimized for stability prediction. ESMtherm is trained on the folding stability of 528k natural and de novo sequences derived from 461 protein domains and can accommodate deletions, insertions, and multiple-point mutations. We show that a protein language model can be fine-tuned to predict folding stability. ESMtherm performs reasonably on small protein domains and generalizes to sequences distal from the training set. Lastly, we discuss our models limitations compared to other state-of-the-art methods in generalizing to larger protein scaffolds. Our results highlight the need for large-scale stability measurements on a diverse dataset that mirrors the distribution of sequence lengths commonly observed in nature.

Differential scanning fluorimetry (DSF) is a method to determine the apparent melting temperature (Tma) of a purified protein. In DSF, the raw unfolding curves from which Tma is calculated vary widely in shape and complexity. However, the tools available for calculating Tma are only compatible with the simplest of DSF curves, hindering many otherwise straightforward applications of the technology. To overcome this limitation, we designed new mathematical models for Tma calculation that accommodate common forms of variation in DSF curves, including the number of transitions, the presence of high initial signal, and temperature-dependent signal decay. When tested these models against DSFbase, an open-source database of 6235 raw, real-life DSF curves, these models outperformed the existing standard approaches of sigmoid fitting and maximum of the first derivative. To make these models accessible, we created an open-source software and website, DSFworld (https://gestwickilab.shinyapps.io/dsfworld/). In addition to these improved fitting capabilities, DSFworld also includes features that overcome the practical limitations of many analysis workflows, including automatic reformatting of raw data exported from common qPCR instruments, labeling of data based on experimental variables, and flexible interactive plotting. We hope that DSFworld will enable more streamlined and accurate calculation of Tma values for DSF experiments.

Deep eutectic solvents (DESs) emerged as potential alternative solvent media in multiple areas, including biomolecular (cryo)preservation. Herein, we studied the stability of a small protein (ubiquitin) in water and a betaine-glycerol-water (B:G:W) (1:2:ζ; ζ = 0, 1, 2, 5, 10) DES, through molecular dynamics. An AMBER-based model that accurately describes the density and shear viscosity of the DES is proposed. We find that water molecules are largely trapped in the solvent, precluding the formation of a full hydration layer, seemingly opposite to osmolytes' preferential exclusion/preferential hydration mechanism. Although the protein is stable in the DES, structural fluctuations are largely suppressed and only recovered upon sufficient hydration. This is explained by a solvent-slaving mechanism where β-fluctuations are key, with the non-monotonic hydration of some amino acids with the water content providing an explanation to the non-monotonic folding of some proteins in aqueous DESs. A major thermal stability enhancement in the DES is also observed, caused by a similar slowdown of the backbone torsional dynamics. Our results support a kinetic stabilization of the protein in the DES, whereas a possible thermodynamic stabilization does not follow a preferential hydration or water entrapment mechanism. [ABSTRACT FROM AUTHOR]

Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear membraneless organelles named photobodies (PBs). However, the function of PBs in PHYB signaling remains frustratingly elusive. Here, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) to PBs. Surprisingly, PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal that PB formation spatially segregates two antagonistic PHYB signaling actions - PIF5 stabilization in PBs and PIF5 degradation in the surrounding nucleoplasm - which could enable an environmentally sensitive, counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

The transition of IgA antibodies into clinical development is crucial because they have the potential to create a new class of therapeutics with superior pathogen neutralization, cancer cell killing, and immunomodulation capacity compared to IgG. However, the biological role of IgA glycans in these processes needs to be better understood. This study provides a detailed biochemical, biophysical, and structural characterization of recombinant monomeric human IgA2, which varies in the amount/locations of attached glycans. Monomeric IgA2 antibodies were produced by removing the N-linked glycans in the CH1 and CH2 domains. The impact of glycans on oligomer formation, thermal stability, and receptor binding was evaluated. In addition, we performed a structural analysis of recombinant IgA2 in solution using Small Angle X-Ray Scattering (SAXS) to examine the effect of glycans on protein structure and flexibility. Our results indicate that the absence of glycans in the Fc tail region leads to higher-order aggregates. SAXS, combined with atomistic modeling, showed that the lack of glycans in the CH2 domain results in increased flexibility between the Fab and Fc domains and a different distribution of open and closed conformations in solution. When binding with the Fcα-receptor, the dissociation constant remains unaltered in the absence of glycans in the CH1 or CH2 domain, compared to the fully glycosylated protein. These results provide insights into N-glycans' function on IgA2, which could have important implications for developing more effective IgA-based therapeutics in the future.

Nanoceria have demonstrated a wide array of catalytic activity similar to natural enzymes, holding considerable significance in the colorimetric detection of alkaline phosphatase (ALP), which is a biomarker of various biological disorders. However, the issues of physiological stability and formation of protein corona, which are strongly related to their surface chemistry, limit their practical application. In this work, CeO2 nanoparticles characterized by enhanced dimensional uniformity and specific surface area were synthesized, followed by encapsulation with various polymers to further increase catalytic activity and physiological stability. Notably, the CeO2 nanoparticles encapsulated within each polymer exhibited improved catalytic characteristics, with PAA-capped CeO2 exhibiting the highest performance. We further demonstrated that the PAA-CeO2 obtained with enhanced catalytic activity was attributed to an increase in surface negative charge. PAA-CeO2 enabled the quantitative assessment of AA activity within a wide concentration range of 10 to 60 μM, with a detection limit of 0.111 μM. Similarly, it allowed for the evaluation of alkaline phosphatase activity throughout a broad range of 10 to 80 U/L, with a detection limit of 0.12 U/L. These detection limits provided adequate sensitivity for the practical detection of ALP in human serum. [ABSTRACT FROM AUTHOR]

Background: Protein instability has been reported to be an important cause of haze in white wines; to investigate the feasibility of protease as a substitute for bentonite in the protein stability of white grape liqueurs, the effect of protease combined with heat treatment on the composition and protein stability of liqueur wines was investigated by chemical analysis, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), and amino acid analysis. Results: The results showed that the contents of total acid, volatile acid, pH, and glycerol were not significantly different from the blank control; the effect on total protein content and thermal stability were similar to the bentonite treatment; the L∗, b∗, and Cab∗ values of liqueur wines increased by 5.59%, 4.33%, and 4.33% after protease combined with heat treatment (p < 0.05); SDS‐PAGE results confirmed that protease combined with heat treatment had good hydrolysis for thaumatin‐like proteins (TLPs) and chitinases (CHIs) in liqueur wines. Amino acid analysis revealed that the levels of threonine, serine, tyrosine, arginine, and proline had increased, respectively, and isoleucine (1.26 mg/L) and leucine (1.16 mg/L) were newly detected after protease combined with heat treatment. Particle size analysis showed that the protease combined with heat treatment had less effect on the particle size distribution of the wines. Conclusion: Protease combined with heat treatment had a certain protein stabilizing effect and had no significant negative impact on other nutrients of liqueur wines. Thus, it can be used as an alternative to bentonite for the protein stabilization of liqueur wines. [ABSTRACT FROM AUTHOR]

ABSTRACT Wheat is a crucial food crop, yet its production is continually threatened by abiotic stresses, particularly salt stress. Understanding the molecular mechanisms by which wheat responds to salt stress is essential for developing salt‐tolerant varieties. In this study, we investigated the molecular pathway involving the wheat TaDHN7 in response to salt stress. The overexpression of TaDHN7 enhances salt tolerance and reactive oxygen species (ROS) scavenging in wheat, while the knockout of TaDHN7 significantly impairs salt tolerance. Furthermore, we identified that TaWRKY44 promotes the expression of TaDHN7 by binding to the W‐box within the TaDHN7 promoter. Additionally, TaWRKY17 interacts with TaWRKY44, and this interaction enhances the protein stability of TaWRKY44 under salt stress, thereby enhancing its transcriptional regulatory capacity on TaDHN7. This study elucidates the TaWRKY17‐TaWRKY44‐TaDHN7 pathway in response to salt stress in wheat, providing valuable insights for the development of salt‐tolerant wheat cultivars. [ABSTRACT FROM AUTHOR]

SUMMARY ROS/redox signaling plays an important role in the regulation of signal transduction and acclimation pathways activated by multiple abiotic stresses and leaf senescence. However, the regulatory events that produce ROS under different stimuli are far from clear. Here, we report the elucidation of the molecular mechanism of an h type thioredoxin, AhTRX h2, positively regulates Al sensitivity and leaf senescence by promoting ROS. AhTRX h2 transcript levels increased greatly during both natural senescence and Al stress condition in peanut. Ectopic expression of AhTRX h2 in Arabidopsis conferred Al sensitivity as well as premature leaf senescence, manifested by multiple indices, including inhibiting root elongation, severe cell death, and accelerated expression of MC1 and CEX17. AhTRX h2 exhibited similar functions to AtTRX h2, as AhTRX h2 was able to restore the phenotypes of the AtTRX h2 defective mutant (trxh2‐4) which showed Al tolerant and late senescence phenotypes. The knock down of AhTRX h2 markedly suppressed Al‐ and senescence‐induced cell death in peanut. AhTRX h2 could recruit catalytic subunit of protein phosphatase 2A (PP2AC2) to form a stable complex. The interaction between AhTRX h2 and AtPP2AC2, as well as AhPP2AC2 and AtTRX h2 was also proved. Overexpression of AhPP2AC2 significantly enhanced Al sensitivity and leaf senescence in Arabidopsis. Protein stability assay revealed that AhTRX h2 was more stable during aging or aluminum stress. Moreover, PP2AC2 could greatly enhance the stability of AhTRX h2 in vivo. Consistent with these observations, overexpression of AhPP2AC2 effectively enhanced AhTRX h2‐induced Al sensitivity and precocious leaf senescence. AhTRX h2 and AhPP2AC2 required ABA and ROS in response to cell death under Al stress and senescence, and it was evidence to suggest that ABA acted upstream of ROS in this process. Together, AhTRX h2 and AhPP2AC2 constitute a stable complex that promotes the accumulation of ABA and ROS, effectively regulate cell death. These findings suggest that TRX h2‐PP2AC2‐mediated pathway may be a widespread mechanism in regulating Al stress and leaf senescence. [ABSTRACT FROM AUTHOR]

The expression of TRIM47, a member of the TRIM protein and E3 ubiquitin ligase families, is elevated in various cancers, such as non-small cell lung cancer and colorectal cancer, and is linked to poor prognosis. This study aimed to investigate the role of TRIM47 in gastric cancer development. Using The Cancer Genome Atlas-Stomach Adenocarcinoma (TCGA-STAD) dataset and analysis of 20 patient samples from our center, TRIM47 was found to be significantly up-regulated in gastric cancer tissues and associated with advanced N-stage and poor prognosis. We constructed stable TRIM47 knockdown and overexpressing gastric cancer cell lines. CCK8, EDU, colony formation, wound healing, and Transwell tests were used to evaluate the effects on cell proliferation, invasion, and migration. The results showed that TRIM47 knockdown inhibited the proliferation, migration and invasion of gastric cancer cells, while TRIM47 overexpression promoted these behaviors. These results were further confirmed in vivo. In the mechanism part, we found that TRIM47 interacts with CYLD protein. Moreover, TRIM47 promotes K48-linked ubiquitination, leading to the degradation of CYLD by the proteasome, thereby activating the NF-κB pathway and regulating the biological behavior of gastric cancer cells. Taken together, our study demonstrated that TRIM47 is involved in the proliferation and metastasis of gastric cancer through the CYLD/NF-κB pathway. [ABSTRACT FROM AUTHOR]

The progression and outcome of bladder cancer (BLCA) are critically affected by the propensity of tumor metastasis. Our previous study revealed that activation of the mevalonate (MVA) pathway promoted migration of BLCA cells; however, the exact mechanism is unclear. Here we show that elevated expression of MVA pathway enzymes in BLCA cells, correlating with poorer patient prognosis by analyzing single-cell and bulk-transcriptomic datasets. Inhibition of the MVA pathway, either through knockdown of farnesyl diphosphate synthase (FDPS) or using inhibitors such as zoledronic acid or simvastatin, led to a marked reduction in BLCA cell migration. Notably, this effect was reversed by administering geranylgeranyl pyrophosphate (GGPP), not farnesyl pyrophosphate (FPP) or cholesterol, indicating the specificity of geranylgeranylation for cell motility. Moreover, we found that RhoB, a Rho GTPase family member, was identified as a key effector of the impact of the MVA pathway on BLCA metastasis. The post-translational modification of RhoB by GGPP-mediated geranylgeranylation influenced its protein stability through the ubiquitin-proteasome pathway. Additionally, overexpression of RhoB was found to block the membrane translocation of integrin β1 in BLCA cells. In summary, our findings underscore the role of the MVA pathway in BLCA metastasis, providing insights into potential therapeutic targets of this malignancy. RhoB plays an important role in the effect of MVA pathway inhibition on the migratory capacity of bladder cancer cells and its protein stability is closely related to GGPP-mediated geranylgeranyl modification. [ABSTRACT FROM AUTHOR]

DNA methylation is an important epigenetic mechanism essential for transposon silencing and genome integrity. Across evolution, the substrates of DNA methylation have diversified between kingdoms. In plants, chromomethylase3 (CMT3) and CMT2 mediate CHG and CHH methylation, respectively. However, how these two methyltransferases diverge on substrate specificities during evolution remains unknown. Here, we reveal that CMT2 originates from a duplication of an evolutionarily ancient CMT3 in flowering plants. Lacking a key arginine residue recognizing CHG in CMT2 impairs its CHG methylation activity in most flowering plants. An engineered V1200R mutation empowers CMT2 to restore CHG and CHH methylations in Arabidopsis cmt2cmt3 mutant, testifying a loss-of-function effect for CMT2 during evolution. CMT2 has evolved a long and unstructured amino terminus critical for protein stability, especially under heat stress, and is plastic to tolerate various natural mutations. Together, this study reveals the mechanism of chromomethylase divergence for context-specific DNA methylation in plants and sheds important lights on DNA methylation evolution and function. [ABSTRACT FROM AUTHOR]

Changes in protein homeostasis are broadly implicated in many disease states, including amyloidoses, neurodegenerative diseases, cancer, and normal aging. Although this relationship has been fruitful for identifying and developing therapeutic strategies, it is challenging to identify which proteins are misfolding. New technologies have recently emerged that enable proteome‐wide interrogation of protein conformation and stability. In this review, we describe these technologies, and how they have been used to identify proteins whose folding changes between disease states. We discuss some of the challenges in this emerging field, and the potential for misfolded protein profiling to provide insight into human disease. [ABSTRACT FROM AUTHOR]

FKBP4, a member of the FK506-binding protein (FKBP) family, is a promising target for a variety of disorders, including cancer. However, its underlying molecular mechanism and potential function in hepatocellular carcinoma (HCC) are largely elusive. Therefore, we aimed to investigate the expression status, functional implications and underlying mechanisms of FKBP4 in HCC. Our bioinformatics analysis of TCGA LIHC datasets, ICGC LIRI-JP datasets and GEO datasets results showed FKBP4 was upregulated in HCC tissues. We also confirmed the elevated FKBP4 in clinical HCC samples. Additionally, quantitative RT-PCR results revealed FKBP4 was highly expressed in all five tested HCC cell lines. We also observed a correlation between elevated FKBP4 expression and poor prognosis in HCC patients. Loss of FKBP4 can inhibit the proliferation and migration in HCC cells. Furthermore, we found that silencing FKBP4 suppressed glucose uptake, lactic acid production and 18F-FDG uptake compared with the control group. Mechanistically, our funding indicated that FKBP4 participates in glycolysis through p53 mediated HK2 signaling pathway, specially, FKBP4 knockdown promotes the expression and stability of p53 protein rather than affecting the transcription level. Finally, rescue experiments revealed that simultaneous knockdown of both FKBP4 and p53 partially reversed the inhibitory effects on HK2 protein levels and 18F-FDG uptake. Our study elucidates a novel role of FKBP4 in promoting HCC development and glycolysis by modulating the p53/HK2 signaling pathway. Given the critical role of aerobic glycolysis in the progression of HCC, targeting FKBP4 may offer a new therapeutic strategy for treating this malignancy. [ABSTRACT FROM AUTHOR]

Clinical samples are routinely inactivated before molecular assays to prevent pathogen transmission. Antibody-based assays are sensitive to changes in analyte conformation, but the impact of inactivation on the analyte detectability has been overlooked. This study assessed the effects of commonly used inactivation-methods, Triton X-100 (0.5%) and heat (60 °C, 1 h), on cytokine/chemokine detection in plasma, lung aspirates, and nasopharyngeal samples. Heat significantly reduced analyte detectability in plasma (IL-12p40, IL-15, IL-16, VEGF, IL-7, TNF-β) by 33–99% (p ≤ 0.02), while Triton X-100 minimally affected analytes in plasma and nasopharyngeal samples (11–37%, p ≤ 0.04) and had no significant impact on lung aspirates. Structural analysis revealed that cytokines affected by heat had more hydrophobic residues and higher instability-indices. As the protein-detectability was affected differently in different sample types, the sample environment could also influence protein stability. This underscores the importance of selecting the most suitable inactivation methods for clinical samples to ensure accurate cytokine/chemokine analysis in both clinical and research settings. [ABSTRACT FROM AUTHOR]

A small‐angle X‐ray scattering study on the pressure‐dependent behavior of human serum albumin (HSA) is presented. HSA is able to absorb cosolvents such as drugs or other hydrophobic substances and transport them, for example, in the human body. It is shown that the uptake of various substances is associated with increased pressure stability, which can be used as an indicator of protein–substance interaction. Especially the interaction between the substances and the hydrophobic pockets of the protein is an important aspect, which mainly depends on the hydrophobicity as well as the size of the used cosolvents. Specifically, drugs such as ibuprofen and theophylline increase the pressure stability of the protein enormously, while caffeine or TMAO, for example, has no strong influence. [ABSTRACT FROM AUTHOR]

Introduction: Matrix Metalloproteinase 3 (MMP3) is a vital member of the MMP family, known for its wide range of substrate specificity and proteolytic activity against Extracellular Matrix (ECM) components. The role of functional polymorphisms in the MMP genes has been previously investigated in relation to cancer susceptibility, particularly breast cancer. Several Single- Nucleotide Polymorphisms (SNPs) in the MMP3 gene have been linked to a number of clinical illnesses, such as Coronary Artery Disease (CAD); however, the results were not entirely conclusive. Aim: To identify pathogenic missense SNPs in the human MMP3 gene and analyse their effects on structure and function. Materials and Methods: This was a record-based cross-sectional study performed using data retrieved from online resources. The analysis was conducted using a series of different bioinformatic tools, for which ethical clearance was obtained from the institution. The online tools used included Sorting Intolerant from Tolerant (SIFT), PolyPhen-2, PhD-SNP, PANTHER, PROVEAN, and SNPs and GO to predict harmful non synonymous SNPs (nsSNPs). Further analysis was performed using I-Mutant 2.0, MutPred2, Consurf, and HOPE software. These tools were able to filter out damaging SNPs and predict the impact of deleterious SNPs on the structure and function of the MMP3 protein. Results: This study predicted two potentially pathogenic SNPs (D175Y and Y116C) out of 443 missense SNPs from dbSNP, which is a database of SNPs available on the National Centre for Biotechnology Information website. Further analysis revealed that these SNPs were located in highly conserved regions and were predicted to decrease protein stability. Conclusion: In this study, two potentially pathogenic SNPs (D175Y and Y116C) were identified. Characterisation of these SNPs can help us gain a better understanding of the molecular basis of clinical conditions. The results of this study can be further validated by designing population-based studies and wet lab experiments. This will help in augmenting research and personalised medicine. [ABSTRACT FROM AUTHOR]

Triosephosphate isomerase (TPI) dysfunction is a critical factor in diverse pathological conditions. Deficiencies in TPI lead to the accumulation of toxic methylglyoxal (MGO), which induces non-enzymatic post-translational modifications, thus compromising protein stability and leading to misfolding. This study investigates how specific TPI mutations (E104D, N16D, and C217K) affect the enzyme's structural stability when exposed to its substrate glyceraldehyde 3-phosphate (G3P) and MGO. We employed circular dichroism, intrinsic fluorescence, native gel electrophoresis, and Western blotting to assess the structural alterations and aggregation propensity of these TPI mutants. Our findings indicate that these mutations markedly increase TPI's susceptibility to MGO-induced damage, leading to accelerated loss of enzymatic activity and enhanced protein aggregation. Additionally, we observed the formation of MGO-induced adducts, such as argpyrimidine (ARGp), that contribute to enzyme inactivation and aggregation. Importantly, the application of MGO-scavenging molecules partially mitigated these deleterious effects, highlighting potential therapeutic strategies to counteract MGO-induced damage in TPI-related disorders. [ABSTRACT FROM AUTHOR]

Understanding the effect of single-missense mutations on protein stability is crucial for clinical decision-making and therapeutic development. The impact of these mutations on protein stability and 3D structure remains underexplored. Here, we developed a program to investigate the relationship between pathogenic mutations with protein unfolding and compared seven machine learning (ML) models to predict the clinical significance of single-missense mutations with unknown impacts, based on protein stability parameters. We analyzed seven proteins associated with ocular disease-causing genes. The program revealed an R-squared value of 0.846 using Decision Tree Regression between pathogenic mutations and decreased protein stability, with 96.20% of pathogenic mutations in RPE65 leading to protein instability. Among the ML models, Random Forest achieved the highest AUC (0.922) and PR AUC (0.879) in predicting the clinical significance of mutations with unknown effects. Our findings indicate that most pathogenic mutations affecting protein stability occur in alpha-helices, beta-pleated sheets, and active sites. This study suggests that protein stability can serve as a valuable parameter for interpreting the clinical significance of single-missense mutations in ocular proteins. [ABSTRACT FROM AUTHOR]

Human phosphoglycerate kinase 1(hPGK1) is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus respectively. These changes in subcellular locations drive tumorigenesis and are likely associated with site‐specific changes in protein stability. In this work, we investigate the effects of site‐specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen–deuterium exchange (HDX) and molecular dynamics (MD) simulations. We also investigate the binding of 3‐phosphoglycerate and Mg‐ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50‐fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg‐ADP in the S256D variant, whereas Mg‐ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX and MD simulations confirmed the destabilization caused by the mutation S256D (with some long‐range effects on stability) and its reduced affinity for Mg‐ADP due to the strong destabilization of its binding site (particularly in the apo‐state). Our research provides evidence suggesting that modifications in protein stability could potentially enhance the translocation of hPGK1 to the nucleus in cancer. While the structural and energetic basis of its mitochondrial import remain unknown. [ABSTRACT FROM AUTHOR]

The paper presents in silico mutational research on the energetics of the most resolved protein structure of HIV-1 NNRTIs, the 4G1Q HIV-1 reverse transcriptase protein. Twenty nearby residues that are less than 6 Å from the center of the embedded ligand are subjected to in silico alterations. For the current study, 380 unique proteins are generated in silico using a set of 380 surrounding residues that have been altered. Analyses and comparison with the parent protein have been done to determine the impact of mutation on the change in folding-unfolding free energy (G), protein stability, and solvation energy. To evaluate the impact of mutation (i) by and (ii) on a particular amino acid residue, a two-fold investigation is conducted. The findings imply that in 12 designed proteins (G − 3.0), folding-unfolding is significantly favored, resulting in the creation of a highly stable conformation. The 11 designed proteins are unstable because the positive values of G > 0.5 in these proteins point to unfavorable mutations. However, the G value in 171 designed proteins is − 1.0, which suggests that mutations cause designed proteins to adopt a stable conformation. According to the findings, out of the 380 created proteins, 11 had extremely unfavorable, 69 less-favorable, and 270 favorable folding-unfolding changes. [ABSTRACT FROM AUTHOR]

Chemotherapy resistance is a major obstacle to effective cancer treatment, and promotion of ferroptosis can suppress cisplatin resistance in tumor cells. TCF12 plays a suppressive role in oral squamous cell carcinoma (OSCC), but whether it participates in the regulation of cisplatin resistance by modulating ferroptosis remains unclear. Here, we found that TCF12 expression was decreased in OSCC cells compared with normal oral cells, and it was reduced in cisplatin (DDP)‐resistant OSCC cells compared with parental cells. Moreover, overexpression of TCF12 sensitized DDP‐resistant cells to DDP by promoting ferroptosis. Intriguingly, silencing TCF12 reversed the promotion effect of the ferroptosis activator RSL3 on ferroptosis and DDP sensitivity, and overexpressing TCF12 antagonized the effect of the ferroptosis inhibitor liproxstatin‐1 on ferroptosis and DDP resistance. Mechanically, TCF12 promoted ubiquitination of SLC7A11 and decreased SLC7A11 protein stability through transcriptional repression of OTUB1, thereby facilitating ferroptosis. Consistently, SLC7A11 overexpression neutralized the promotion effect of TCF12 on ferroptosis and DDP sensitivity. Additionally, upregulation of TCF12 hindered the growth of mouse OSCC xenografts and enhanced the DDP sensitivity of xenografts by inducing ferroptosis. In conclusion, TCF12 enhanced DDP sensitivity in OSCC cells by promoting ferroptosis, which was achieved through modulating SLC7A11 expression via transcriptional regulation of OTUB1. [ABSTRACT FROM AUTHOR]

In our previous research, microporous packaging combined with chitosan active coating (MP-CH) treatment extended the shelf life of passion fruit at 25 °C to 12 days by regulating water loss and physiological metabolism. However, proteomic investigation and metabolic changes in passion fruit by MP-CH treatment are unclear. In this study, tandem mass tag (TMT) labeling combined with two-dimensional liquid chromatography tandem mass spectrometry (2D LC‒MS/MS) was used to monitor the metabolic changes in passion fruit treated with the MP-CH group and the control group. A total of 730 differentially expressed proteins (DEPs) were identified. Compared with the control group stored for 12 days, there were 359 DEPs in the MP-CH group, including 179 upregulated proteins and 180 downregulated proteins. The upregulated DEPs were mainly involved in the processes of L-ascorbic acid biosynthesis, anthocyanin-containing compound biosynthesis, aspartate family amino acid biosynthesis, and regulation of protein stability. The downregulated DEPs were mainly involved in oxaloacetate metabolism, carbohydrate metabolism, organic substance metabolism, cellular lipid metabolism, pectin catabolism, and galacturonan metabolism. Results showed that the expression of antioxidant enzymes in the MP-CH group was upregulated, which could enhance the antioxidant capacity and stress resistance of passion fruit. In addition, the expression of cell wall-degrading enzymes was downregulated, which could delay the degradation of cell wall components and maintain the relative stability of its cell wall structure, making for a further extended shelf life of passion fruit. [ABSTRACT FROM AUTHOR]

Summary: Bighead carp has the largest production amount among freshwater fish in China, but its physicochemical properties change rapidly with the denaturation of their myofibril protein (MP). The protein–polysaccharide complexes have shown interesting physicochemical properties as stabilisers and emulsifiers. In this study, arabinoxylans (AXs) were prepared and mixed with bighead carp MP to form a composite gel. The effect of AXs on the thermal stability, emulsification and emulsion stability of the composite gel was investigated. The results showed that fraction precipitated by 60% ethanol (F60) and by 90% (F90) improved the denaturation temperature of bighead carp MP from 65.99 °C to 67.39 °C and 68.22 °C, respectively. The emulsifying capacity of F60 (0.54 m2 g−1) was similar to that of sucrose (0.52 m2 g−1). The effect of F90 on the emulsion stability of bighead carp MP was better than that of F60. The elasticity of bighead carp MP was improved by F60 and F90. The hydroxyl group interactions, hydrophobic interactions and electrostatic forces exist between the macromolecules. AXs of fresh maize bran could prevent protein denaturation and improve the emulsion capacity and gel properties of bighead carp MP. [ABSTRACT FROM AUTHOR]

Summary: Vitamin A (VA) is a fat‐soluble compound that is essential for physiological functions including vision, fetal growth, immune response, cell differentiation and proliferation. However, its susceptibility to environmental conditions leading to poor stability limits its application in food. This study investigated the interaction between egg white protein (EWP) and VA by spectroscopy and molecular docking. The results indicated one EWP could bind 16.00 ± 0.89 VA molecules and the binding constant is (2.59 ± 0.71) × 105 m−1. What's more, there was a fluorescence resonance energy transfer between EWP and VA, and the Förster radius is 5.28 nm. In addition, VA enhanced the hydrophobic interactions with the EWP, resulting in protein aggregation, larger average particle size and more uniform particle size distribution. Finally, the combination of VA with EWP could significantly improve the photothermal stability and storage stability of VA. These studies indicate that EWP nanocomplexes can be used as ideal delivery carriers for bioactive substances. [ABSTRACT FROM AUTHOR]

The human innate immune system recognizes dsRNA as a pathogen‐associated molecular pattern that induces a potent inflammatory response. The primary source of pathogenic dsRNA is cells infected with replicating viruses, but can also be released from uninfected necrotic cells. Here, we show that the dsRNA poly(I:C) challenge in human macrophages activates the p38 MAPK‐MK2 signalling pathway and subsequently the phosphorylation of tristetraprolin (TTP/ZFP36). The latter is an mRNA decay‐promoting protein that controls the stability of AU‐rich mRNAs (AREs) that code for many inflammatory mediators. Hydroxychloroquine (HCQ), a common anti‐malaria drug, is used to treat inflammatory and autoimmune disorders and, controversially, during acute COVID‐19 disease. We found that HCQ reduced the dsRNA‐dependent phosphorylation of p38 MAPK and its downstream kinase MK2. Subsequently, HCQ reduced the abundance and protein stability of the inactive (phosphorylated) form of TTP. HCQ reduced the levels and the mRNA stability of poly (I:C)‐induced cytokines and inflammatory mRNAs like TNF, IL‐6, COX‐2, and IL‐8 in THP‐1 and primary blood monocytes. Our results demonstrate a new mechanism of the anti‐inflammatory role of HCQ at post‐transcriptional level (TTP phosphorylation) in a model of dsRNA activation, which usually occurs in viral infections or RNA release from necrotic tissue. [ABSTRACT FROM AUTHOR]

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1 (HSFA1) family. Members of this family interact with heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role of cochaperones, such as the HSP70‐HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown. Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a‐responsive‐responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability. Summary statement: This work describes that HOP cochaperones play a major role in thermomorphogenesis by modulating the stability of the HSFA1a in Arabidopsis. [ABSTRACT FROM AUTHOR]

Antizyme (AZ) is a regulatory protein that plays a crucial role in modulating the activity of ornithine decarboxylase (ODC), which is the initial and rate‐limiting enzyme in the complex pathway of polyamine biosynthesis. AZ facilitates the swift degradation of ODC, thereby modulating the levels of cellular polyamines. This study unveils a new ubiquitin‐independent mechanism for AZ degradation, emphasizing the essential role of N‐terminal degrons. Contrary to traditional ubiquitin‐dependent degradation, our findings reveal that AZ degradation is significantly influenced by its N‐terminal region. By conducting a series of experiments, including in vitro degradation assays, cycloheximide chase experiments, differential scanning calorimetry, and measurement of cellular concentrations of polyamines, we demonstrate that N‐terminal truncation significantly enhances AZ's stability and facilitates the reduction of polyamine levels by accelerating ODC degradation. The removal of the N‐terminal portion of AZ results in a reduced degradation rate and enhanced thermal stability of the protein, leading to a more efficient inhibition of polyamine synthesis. These findings are corroborated by the analysis of AZ isoforms, AZ1, AZ2, and AZ3, which display differential degradation patterns based on the specific N‐terminal segments. This substantiates a degradation mechanism driven by an intrinsically disordered N‐terminal region acting as a degron, independent of lysine ubiquitination. These results underscore the significant regulatory function of the N‐terminal domain in the activity of AZ and the maintenance of polyamine homeostasis. [ABSTRACT FROM AUTHOR]

Background Hypothesis/Objectives Materials and Methods Results Conclusions and Clinical Relevance Few studies have investigated optimal storage conditions or expiration dates for sublingual immunotherapy (SLIT) formulations prepared from glycerinated allergen extracts.The objective of this study was to compare concentrations of short ragweed major allergen (Amb a 1) and total protein in SLIT formulations stored at two different temperatures. It was hypothesised that protein concentrations would show greater decline over time in a formulation stored at room temperature (RT) than in one stored under refrigeration.Two SLIT samples containing equal volumes of 20,000 PNU Amb a 1 extract were prepared and stored at refrigerated cold (CT) (2–8°C) or RT (20–24°C) for 140 days. Changes in total protein and major allergen concentration and composition were measured by Bradford assay, two‐site enzyme‐linked immunosorbent assay and SDS–PAGE. Presence of Amb a 1 was confirmed with Western immunoblot. Data were analysed using an analysis of covariance, with p < 0.05 considered significant.SDS–PAGE showed compositional changes in a ~26–30 kDa protein band under RT and not CT storage. The Amb a 1 concentration of the RT SLIT sample declined significantly over time, compared to that of the CT SLIT sample (F(1,8) = 47.69, p < 0.0001). There was no significant difference in total protein concentration over time between groups (F(1,8) = 1.79, p = 0.22).These results demonstrate that storage of glycerinated SLIT formulations in refrigerated CT preserved the highest concentration of the specific allergen Amb a 1, suggesting that SLIT formulations containing short ragweed should be stored under refrigeration. [ABSTRACT FROM AUTHOR]

The objective was to determine whether USP13 stabilizes WISP1 protein and contributes to tumorigenicity and metastasis in ESCC through the Wnt/CTNNB1 signaling pathway. ESCC cell lines (KYSE150 and TE10) were treated with the proteasome inhibitor MG‐132, followed by siRNA screening of deubiquitinases (DUBs) to identify regulators of WISP1. Mass spectrometry, immunoprecipitation, and in vitro functional assays were conducted to explore the interaction between USP13 and WISP1 and to assess the effects of USP13 downregulation on cell proliferation, migration, invasion, epithelial–mesenchymal transition (EMT), and apoptosis. Additionally, in vivo experiments using mouse models were performed to evaluate the impact of USP13 knockdown on tumor growth and metastasis. USP13 was identified as a key regulator of WISP1, stabilizing its protein levels through deubiquitination. Downregulation of USP13 resulted in reduced WISP1 protein stability, decreased cell proliferation, migration, and EMT, and increased apoptosis in vitro. In vivo, USP13 knockdown significantly inhibited tumor growth and lung metastasis. WISP1 overexpression in USP13‐knockdown cells partially rescued these phenotypes, confirming the functional role of the USP13/WISP1 axis. Furthermore, knockdown of USP13 or WISP1 impaired the activation of the Wnt/CTNNB1 signaling pathway and reduced immune checkpoint marker expression, indicating a mechanism by which USP13 promotes immune evasion in ESCC. USP13 stabilizes WISP1 through deubiquitination, enhancing ESCC progression by activating the Wnt/CTNNB1 pathway and promoting immune evasion, making USP13 a potential therapeutic target in ESCC. [ABSTRACT FROM AUTHOR]

Adenylosuccinate synthetase (AdSS), which catalyses the GTP‐dependent conversion of inosine monophosphate (IMP) and aspartic acid to succinyl‐AMP, plays a major role in purine biosynthesis. In some bacterial AdSS, it is implicated that IMP binding is important to organize the active site, but in certain plant AdSS, GTP performs this role. Here, we report that in Leishmania donovani AdSS, IMP binding favoured dimerization, induced greater conformational change and improved the protein stability more than GTP binding. IMP binding, which resulted in a network of hydrogen bonds, stabilized the conformation of active site loops and brought the switch loop to a closed conformation, which then facilitated GTP binding. Our results provide a basis for designing better inhibitors of leishmanial AdSS. [ABSTRACT FROM AUTHOR]

Circadian clocks rely on transcriptional/translational feedback loops involving clock genes and their corresponding proteins. While the primary oscillations originate from gene expression, the precise control of clock protein stability plays a pivotal role in establishing the 24-hour circadian rhythms. Most clock proteins are degraded through the ubiquitin/26S proteasome pathway, yet the enzymes responsible for ubiquitination and deubiquitination remain poorly characterised. We identified a missense allele (ubp12-3, S327F) of the UBP12 gene/protein in Arabidopsis. Despite ubp12-3 exhibited a short period phenotype similar to that of a loss-of-function allele, molecular analysis indicated elevated protease activity in ubp12-3. We demonstrated that early flowering of ubp12 mutants is a result of the shortened circadian period rather than a direct alteration of UBP12 function. Analysis of protease activity of non-phosphorylatable (S327A, S327F) and phosphomimetic (S327D) derivatives in bacteria suggested that phosphorylation of serine 327 inhibits UBP12 enzymatic activity, which could explain the over-functioning of S327F in vivo. We showed that phosphomimetic mutations of the conserved serine in the Neurospora and human orthologues reduced ubiquitin cleavage activity suggesting that not only the primary structures of UBP12-like enzymes are phylogenetically conserved across a wide range of species, but also the molecular mechanisms governing their enzymatic activity. [ABSTRACT FROM AUTHOR]

SUMMARY Flavonoids represent a diverse group of plant specialised metabolites which are also discussed in the context of dietary health and inflammatory response. Numerous studies have revealed that flavonoids play a central role in plant acclimation to abiotic factors like low temperature or high light, but their structural and functional diversity frequently prevents a detailed mechanistic understanding. Further complexity in analysing flavonoid metabolism arises from the different subcellular compartments which are involved in biosynthesis and storage. In the present study, non‐aqueous fractionation of Arabidopsis leaf tissue was combined with metabolomics and proteomics analysis to reveal the effects of flavonoid deficiencies on subcellular metabolism during cold acclimation. During the first 3 days of a 2‐week cold acclimation period, flavonoid deficiency was observed to affect pyruvate, citrate and glutamate metabolism which indicated a role in stabilising C/N metabolism and photosynthesis. Also, tetrahydrofolate metabolism was found to be affected, which had significant effects on the proteome of the photorespiratory pathway. In the late stage of cold acclimation, flavonoid deficiency was found to affect protein stability, folding and proteasomal degradation, which resulted in a significant decrease in total protein amounts in both mutants. In summary, these findings suggest that flavonoid metabolism plays different roles in the early and late stages of plant cold acclimation and significantly contributes to establishing a new protein homeostasis in a changing environment. [ABSTRACT FROM AUTHOR]

BACKGROUND RESULTS CONCLUSION Arabinoxylan is commonly used as a hydrocolloid in frozen dough to improve the texture and the sensory qualities of the products. The effects of vinegar residue arabinoxylan (VRAX) on the secondary structures and microstructures of gluten proteins during freeze–thaw storage were studied, and the underlying mechanism governing these effects was clarified.The results revealed that VRAX improved the textural properties of gluten proteins, but had a negative impact on their viscoelasticity. Additionally, the addition of VRAX increased the number of disulfide bonds and also improved the freezing tolerance of the gluten proteins. It was found that the enthalpy of the gluten proteins decreased by 19.78% following VRAX addition. As a result of the use of VRAX, the freezing procedure resulted in reduced formation of ice crystals, protecting the gluten network structure and preserving the dough's elasticity. The network structure of gluten proteins after VRAX treatment was more ordered and integrated relative to that of frozen blank control gluten proteins.Overall, the freeze–thaw stability of the gluten proteins was enhanced by VRAX. These results suggest that VRAX has potential as an effective cryoprotectant in frozen dough. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Dopamine transfers information to striatal neurons, and disrupted neurotransmission leads to motor deficits observed in movement disorders. Striatal dopamine converges downstream to Adenylyl Cyclase Type 5 (AC5)-mediated synthesis of cAMP, indicating the essential role of signal transduction in motor physiology. However, the relationship between dopamine decoding and AC5 regulation is unknown. Here, we utilized an unbiased global protein stability screen to identify Potassium Channel Tetramerization Domain 1 (KCTD1) as a key regulator of AC5 level that is mechanistically tied to N-linked glycosylation. We then implemented a CRISPR/SaCas9 approach to eliminate KCTD1 in striatal neurons expressing a Förster resonance energy transfer (FRET)-based cAMP biosensor. 2-photon imaging of striatal neurons in intact circuits uncovered that dopaminergic signaling was substantially compromised in the absence of KCTD1. Finally, knockdown of KCTD1 in genetically defined dorsal striatal neurons significantly altered motor behavior in mice. These results reveal that KCTD1 acts as an essential modifier of dopaminergic signaling by stabilizing striatal AC5. [ABSTRACT FROM AUTHOR]

Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs. [Display omitted] • Vinculin is mechanosensitively recruited to tricellular junctions • Vinculin's actin-binding function is needed for tricellular actomyosin organization • Vinculin reinforces tricellular junction integrity in response to increased tension • Vinculin is required to maintain barrier function at tricellular junctions Epithelial tricellular junctions are vulnerable to disruption. Here, van den Goor et al. reveal that Vinculin is mechanosensitively recruited to vertebrate tricellular junctions, where it regulates actomyosin organization and barrier function. Vinculin reinforces tricellular junction integrity when epithelia experience increased mechanical force. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) have achieved impressive antitumor clinical outcomes. However, the limited response rates suggest the incomplete understanding of PD-L1 regulation. Here, we demonstrate that vacuole protein sorting 11 and 18 (VPS11/18), two key players in vesicular trafficking, positively regulate PD-L1 and confer resistance to immune checkpoint blockade therapy. VPS11/18 interact with PD-L1 in endosome recycling accompanied by promoting PD-L1 glycosylation and protein stability. VPS18 deficiency enhances antitumor immune response. Pharmacological inhibition by VPS18 inhibitor RDN impaired PD-L1 member trafficking and protein stability. Combination treatment of RDN and anti-cytotoxic T lymphocyte-associated antigen 4 synergistically enhances antitumor efficacy in aggressive and drug-resistant tumors. RDN exerted lung-preferred distribution and good bioavailability, suggesting a favorable drug efficacy. Together, our study links VPS18/11-mediated trans-Golgi network recycling of PD-L1 and points to a promising treatment strategy for the enhancement of antitumor immunity. [ABSTRACT FROM AUTHOR]

ABSTRACT Sucrose functions as a signaling molecule in several metabolic pathways as well as in various developmental processes. However, the molecular mechanisms by which sucrose regulates these processes remain largely unknown. In the present study, we demonstrate that sucrose promotes flowering by mediating the stability of a regulatory protein that represses flowering in rice. Exogenous application of sucrose promoted flowering by inducing florigen gene expression. Reduction of sucrose levels in the phloem through genetic modifications, such as the overexpression of the vacuolar invertase OsVIN2 or the mutation of OsSUT2, a sucrose transporter, delayed flowering. Analysis of relative transcript levels of floral regulatory genes showed that sucrose activated Ehd1 upstream of the florigen, with no significant effect on the expression of other upstream genes. Examination of protein stability after sucrose treatment of major floral repressors revealed that the Ghd7 protein was specifically degraded. The Ghd7 protein interacted with the E3 ligase IPA INTERACTING PROTEIN1 (IPI1), and sucrose‐induced K48‐linked polyubiquitination of Ghd7 via IPI1, leading to protein degradation. Mutants defective in IPI1 delayed flowering, confirming its role in modulating proteins involved in flowering. We conclude that sucrose acts as a signaling molecule to induce flowering by promoting Ghd7 degradation via IPI1. [ABSTRACT FROM AUTHOR]

In the aging process, skin morphology might be affected by wrinkle formation due to the loss of elasticity and resilience of connective tissues linked to the cleavage of elastin by the enzymatic activity of elastase. Little information is available about the structural requirements to efficiently inhibit elastase 1 (EC 3.4.21.36) expressed in skin keratinocytes. In this study, a structure-based approach led to the identification to the pharmacophoric hypotheses that described the main structural requirements for binding to porcine pancreatic elastase as a valuable tool for the development of skin therapeutic agents due to its similarity with human elastase 1. The obtained models were subsequently refined through the application of computational alanine-scanning mutagenesis to evaluate the effect of single residues on the binding affinity and protein stability; in turn, molecular dynamic simulations were carried out; these procedures led to a simplified model bearing few essential features, enabling a reliable collection of chemical features for their interactions with elastase. Then, a virtual screening campaign on the in-house library of synthetic compounds led to the identification of a nonpeptide-based inhibitor (IC50 = 60.4 µM) belonging to the class of N-substituted-1H-benzimidazol-2-yl]thio]acetamides, which might be further exploited to obtain more efficient ligands of elastase for therapeutic applications. [ABSTRACT FROM AUTHOR]

Autoimmune polyglandular syndrome (APS) comprises a complex association of autoimmune pathological conditions. APS Type 1 originates from loss-of-function mutations in the autoimmune regulator (AIRE) gene. APS2, APS3 and APS4 are linked to specific HLA alleles within the major histocompatibility complex, with single-nucleotide polymorphisms (SNPs) in non-HLA genes also contributing to disease. In general, variability in the AIRE locus and the presence of heterozygous loss-of-function mutations can impact self-antigen presentation in the thymus. In this study, whole-exome sequencing (WES) was performed on a sixteen-year-old female APS3A/B patient to investigate the genetic basis of her complex phenotype. The analysis identified two variants (p.Arg111Trp and p.Thr101Ile) of the hepatitis A virus cell receptor 2 gene (HAVCR2) encoding for the TIM-3 (T cell immunoglobulin and mucin domain 3) protein. These variants were predicted, through in silico analysis, to impact protein structure and stability, potentially influencing the patient's autoimmune phenotype. While confocal microscopy analysis revealed no alteration in TIM-3 fluorescence intensity between the PBMCs isolated from the patient and those of a healthy donor, RT-qPCR showed reduced TIM-3 expression in the patient's unfractionated PBMCs. A screening conducted on a cohort of thirty APS patients indicated that the p.Thr101Ile and p.Arg111Trp mutations were unique to the proband. This study opens the pathway for the search of TIM-3 variants possibly linked to complex autoimmune phenotypes, highlighting the potential of novel variant discovery in contributing to APS classification and diagnosis. [ABSTRACT FROM AUTHOR]

Peri-implantitis and osseointegration failure present considerable challenges to the prolonged stability of oral implants. To address these issues, there is an escalating demand for a resilient implant surface coating that seamlessly integrates antimicrobial features to combat bacteria-induced peri‑implantitis, and osteogenic properties to promote bone formation. In the present study, a bio-inspired poly(amidoamine) dendrimer (DA-PAMAM-NH 2) is synthesized by utilizing a mussel protein (DA) known for its strong adherence to various materials. Conjugating DA with PAMAM-NH 2 , inherently endowed with antibacterial and osteogenic properties, results in a robust and multifunctional coating. Robust adhesion between DA-PAMAM-NH 2 and the titanium alloy surface is identified using confocal laser scanning microscopy (CLSM) and attenuated total reflectance-infrared (ATR-IR) spectroscopy. Following a four-week immersion of the coated titanium alloy surface in simulated body fluid (SBF), the antimicrobial activity and superior osteogenesis of the DA-PAMAM-NH 2 -coated surface remain stable. In contrast, the bifunctional effects of the PAMAM-NH 2 -coated surface diminish after the same immersion period. In vivo animal experiments validate the enduring antimicrobial and osteogenic properties of DA-PAMAM-NH 2 -coated titanium alloy implants, significantly enhancing the long-term stability of the implants. This innovative coating holds promise for addressing the multifaceted challenges associated with peri‑implantitis and osseointegration failure in titanium-based implants. Prolonged stability of oral implants remains a clinically-significant challenge. Peri-implantitis and osseointegration failure are two important contributors to the poor stability of oral implants. The present study developed a mussel-bioinspired poly(amidoamine) dendrimer (DA-PAMAM-NH 2) for a resilient implant surface coating that seamlessly integrates antimicrobial features to combat bacteria-induced peri‑implantitis, and osteogenic properties to promote bone formation to extend the longevity of oral implants. [Display omitted] [ABSTRACT FROM AUTHOR]

The photolysis of disulfide bonds is implicated in denaturation of proteins exposed to ultraviolet light. Despite this biological relevance in stabilizing the structure of many proteins, the mechanisms of disulfide photolysis are still contested after decades of research. Herein, we report new insight into the photochemistry of L-cystine in aqueous solution by femtosecond X-ray absorption spectroscopy at the sulfur K-edge. We observe homolytic bond cleavage upon ultraviolet irradiation and the formation of thiyl radicals as the single primary photoproduct. Ultrafast thiyl decay due to geminate recombination proceeds at a quantum yield of >80 % within 20 ps. These dynamics coincide with the emergence of a secondary product, attributed to the generation of perthiyl radicals. From these findings, we suggest a mechanism of perthiyl radical generation from a vibrationally excited parent molecule that asymmetrically fragments along a carbon-sulfur bond. Our results point toward a dynamic photostability of the disulfide bridge in condensed-phase. Disulfide bonds play a key role in the stability of proteins. Here, the authors show such bonds are efficiently reformed after UV photolysis in L-cysteine in solution using femtosecond X-ray absorption spectroscopy and theoretical calculations. [ABSTRACT FROM AUTHOR]

Breast cancer remains a significant health concern, with triple-negative breast cancer (TNBC) being an aggressive subtype with poor prognosis. Epithelial-mesenchymal transition (EMT) is important in early-stage tumor to invasive malignancy progression. Snail, a central EMT component, is tightly regulated and may be subjected to proteasomal degradation. We report a novel proteasomal independent pathway involving chaperone-mediated autophagy (CMA) in Snail degradation, mediated via its cytosolic interaction with HSC70 and lysosomal targeting, which prevented its accumulation in luminal-type breast cancer cells. Conversely, Snail predominantly localized to the nucleus, thus evading CMA-mediated degradation in TNBC cells. Starvation-induced CMA activation downregulated Snail in TNBC cells by promoting cytoplasmic translocation. Evasion of CMA-mediated Snail degradation induced EMT, and enhanced metastatic potential of luminal-type breast cancer cells. Our findings elucidate a previously unrecognized role of CMA in Snail regulation, highlight its significance in breast cancer, and provide a potential therapeutic target for clinical interventions. [ABSTRACT FROM AUTHOR]

MITF, a basic Helix-Loop-Helix Zipper (bHLHZip) transcription factor, plays vital roles in melanocyte development and functions as an oncogene. We perform a genetic screen for suppressors of the Mitf-associated pigmentation phenotype in mice and identify an intragenic Mitf mutation that terminates MITF at the K316 SUMOylation site, leading to loss of the C-end intrinsically disordered region (IDR). The resulting protein is more nuclear but less stable than wild-type MITF and retains DNA-binding ability. As a dimer, it can translocate wild-type and mutant MITF partners into the nucleus, improving its own stability thus ensuring nuclear MITF supply. smFRET analysis shows interactions between K316 SUMOylation and S409 phosphorylation sites across monomers; these interactions largely explain the observed effects. The recurrent melanoma-associated E318K mutation in MITF, which affects K316 SUMOylation, also alters protein regulation in concert with S409. This suggests that residues K316 and S409 of MITF are impacted by SUMOylation and phosphorylation, respectively, mediating effects on nuclear localization and stability through conformational changes. Our work provides a novel mechanism of genetic suppression, and an example of how apparently deleterious mutations lead to normal phenotypes. Synopsis: An intragenic Mitf suppressor mutation was identified that terminates MITF at the K316 SUMOylation site, leading to loss of the C-end intrinsically disordered region (IDR). This mutation provides novel information on how the dynamic IDR mediates MITF localization and stability. The MITF suppressor mutation uncovers a novel mechanism of genetic suppression that opens unexpected insights into MITF subcellular localization and stability. The MITF suppressor mutation provides one explanation for how humans with knockout mutations in essential genes are viable and normal. An intragenic Mitf suppressor mutation was identified that terminates MITF at the K316 SUMOylation site, leading to loss of the C-end intrinsically disordered region (IDR). This mutation provides novel information on how the dynamic IDR mediates MITF localization and stability. [ABSTRACT FROM AUTHOR]

The transcription factor E2F1 serves as a regulator of the cell cycle and promotes cell proliferation. It is highly expressed in cancer tissues and contributes to their malignant transformation. Degradation by the ubiquitin-proteasome system may help to prevent such overexpression of E2F1 and thereby to suppress carcinogenesis. A detailed understanding of the mechanisms underlying E2F1 degradation may therefore inform the development of new cancer treatments. We here identified SCFFBXW7 as a ubiquitin ligase for E2F1 by comprehensive analysis. We found that phosphorylation of E2F1 at serine-403 promotes its binding to FBXW7 (F-box/WD repeat-containing protein 7) followed by its ubiquitination and degradation. Furthermore, calcineurin, a Ca2+/calmodulin-dependent serine-threonine phosphatase, was shown to stabilize E2F1 by mediating its dephosphorylation at serine-403 and thereby preventing FBXW7 binding. Treatment of cells with Ca2+ channel blockers resulted in downregulation of both E2F1 protein and the expression of E2F1 target genes, whereas treatment with the Ca2+ ionophore ionomycin induced upregulation of E2F1. Finally, the calcineurin inhibitor FK506 attenuated xenograft tumor growth in mice in association with downregulation of E2F1 in the tumor tissue. Impairment of the balance between the opposing actions of FBXW7 and calcineurin in the regulation of E2F1 abundance may therefore play an important role in carcinogenesis. [ABSTRACT FROM AUTHOR]