Protein Stability Research Articles

Cryoprotective effect of magnetic field-assisted freezing in combination with curdlan on myofibrillar protein of Litopenaeus vannamei: Oxidative aggregation, protein structure and thermal stability

To counteract the negative effects of conventional freezing methods on frozen surimi products, the present study investigated the effects of magnetic field (MF) freezing in combination with different amounts of curdlan (CUR) on oxidative denaturation, structural alterations, and thermal stability of myofibrillar protein (MP) in shrimp surimi. The results indicated that the intervention of magnetic fields alone improved the quality of frozen muscle proteins. Under the dual intervention of magnetic field-assisted freezing and CUR, small ice crystals formed with MF6 treatment resulted in significantly lower solubility, turbidity, and mean particle size than the control group (p < 0.05). Moreover, the oxidation of MP was also significantly slowed down by inhibiting the formation of hydrophobic groups and carbonyl groups, maintaining a high content of sulfhydryl groups. MF6 also exhibited a high Ca2+-ATPase activity. The α-helix content, the increase in fluorescence intensity under this condition also tend to make the secondary and tertiary structures of myofibrillar protein more stable. Meanwhile, electrophoretic profiles and CLSM showed that this condition maintains the integrity of the MP. In addition, the MF6 samples also had high protein thermal stability. However, excess CUR reduces the excellence of MP cryoprotection. As a result, MF6 has better protection and improvement effects on protein function, structure and thermal stability, and CUR was also found to have the potential for cryoprotectant development.

High-throughput screening for small-molecule stabilizers of misfolded glucocerebrosidase in Gaucher disease and Parkinson’s disease

Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease, PD); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small proluminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and noninhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: The fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 directly visualized GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of small molecules targeting GCase, ultimately leading to a viable therapeutic for GD and PD.

Identification and analysis of differentially expressed lncRNAs and their ceRNA networks in DMD/mdx primary myoblasts

This study explored the significance of long non-coding RNAs (lncRNAs), particularly their role in maintaining dystrophin protein stability and regulating myocyte proliferation and differentiation. The investigation focused on DMD/mdx mouse skeletal muscle primary myoblasts, aiming to identify lncRNAs potential as biomarkers and therapeutic targets for Duchenne muscular dystrophy (DMD). Utilizing CLC Genomics Workbench software, 554 differentially expressed lncRNAs were identified in DMD/mdx mice compared to wild-type (WT) control. Among them, 373 were upregulated, and 181 were downregulated. The study highlighted specific lncRNAs (e.g., 5930430L01Rik, Gm10143, LncRNA1490, LncRNA580) and their potential regulatory roles in DMD key genes like IGF1, FN1, TNNI1, and MYOD1. By predicting miRNA and their connections with lncRNA and mRNA (ceRNA network) using tools such as miRNet, miRSYSTEM and miRCARTA, the study revealed potential indirect regulation of Dystrophin, IGF1R and UTRN genes by identified lncRNAs (e.g. 2310001H17Rik-203, C130073E24Rik-202, LncRNA2767, 5930430L01Rik and LncRNA580). These findings suggest that the identified lncRNAs may play crucial roles in the development and progression of DMD through their regulatory influence on key gene expression, providing valuable insights for potential therapeutic interventions.

Shikonin induces ferroptosis in osteosarcomas through the mitochondrial ROS-regulated HIF-1α/HO-1 axis

BackgroundThe most common malignant bone tumour is osteosarcoma, which has an unsatisfactory prognosis and unsatisfactory treatment. Ferroptosis shows promise as an effective OS therapy. A substance extracted from the Lithospermum erythrorhizon, Shikonin (SHK), inhibits a number of tumours, including ovarian, gastric, and lung cancers. However, whether SHK induces OS ferroptosis and its mechanisms are not clear. PurposeOur study is aimed at investigating whether SHK causes ferroptosis and elucidating its molecular mechanism. MethodsCell counting Kit-8, cell cycle and cell apoptosis assay were utilised to assess therapeutic effect of SHK against OS. Normal cells, including H9C2, AML-12 and HK-2, haematoxylin and eosin staining and mice serum biomarker tests were used to assess SHK biosafety. Malondialdehyde (MDA) levels, the glutathione (GSH)/oxidized glutathione (GSSG) ratio, reactive oxygen species (ROS), lipid peroxide (LPO), and intracellular Fe2+ detection, quantitative reverse transcription PCR (qRT-PCR), Western blotting (WB) and rescue experiments were employed to confirm whether SHK induced ferroptosis in OS cells. Molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay were used to evaluate the direct binding between SHK and hypoxia-inducible factor-1α (HIF-1α). Protein stability and degradation analysis, small RNA interference, flow cytometry, qRT-PCR, and WB were used to investigate the molecular mechanism of ferroptosis. In vivo xenografts of nude mouse was used to study the anti-OS effect of SHK. ResultsSHK significantly reduced OS cell viability and induced apoptosis and G2/M arrest. SHK increased intracellular levels of MDA, ROS, LPO, and Fe2+ while simultaneously reducing the GSH/GSSG ratio and GPX4 and SLC7A11 expression. CETSA and DARTS results demonstrated that SHK did not bind targetly to HIF-1α. Instead, mitochondrial ROS (MitoROS) promoted HIF-1α expression, resulting in HO-1 overexpression, excess Fe2+ production, ROS accumulation and GPX depletion, and ferroptosis. Furthermore, inhibition of MitoROS using Mito-TEMPO downregulated HIF-1α/HO-1 axis and mitigated the SHK-induced ferroptosis. In vivo, SHK effectively suppressed OS growth with favourable biosafety, confirming the molecular mechanism underlying SHK-induced ferroptosis in OS. ConclusionWe observe that HIF-1α/HO-1 axis is the crucial factor in SHK-induced OS ferroptosis. Importantly, we demonstrate that HIF-1α is indirectly regulated by MitoROS rather than SHK bound directly to HIF-1α. Our research suggest that SHK is a potential drug candidate for OS treatment and may help in identifying novel therapeutic targets for OS.

A molecular dynamics investigation into the inhibitory function of hydroxypropyl-beta-cyclodextrin (HPBCD) in its interaction with amyloid-beta (Aβ) plaques near the cell membrane in the context of Alzheimer's disease.

Although Alzheimer`s disease has been known for a long time, it is interesting that scientists are still doing widespread research on it. Meanwhile, in parallel with experimental research, computational research is also yielding interesting results. In this study, we investigated the inhibitory behavior of hydroxypropyl-beta-cyclodextrin (HPBCD) drug candidates, which are more soluble than beta-cyclodextrin (BCD), using molecular dynamics simulations and compared them with AC0107 new drug. Parameters such as cell membrane stability, protein stability, drug inhibition rate, protein permeability, hydrogen bonding agents, the study of the energy content of interactions between different groups, and interactions between different species were of interest. The outcomes indicate that the drug candidate HPBCD has a role in inhibiting protein membrane penetration and has better performance than new AC0107 drug. In other words, HPBCD not only act as a drug carrier of Alzheimer's disease, but also as an inhibitor of it and can play a double role in its improvement.

Deciphering the acidophilia and acid resistance in Acetilactobacillus jinshanensis dominating baijiu fermentation through multi-omics analysis

Lactic acid bacteria (LAB) are pivotal in constructing the intricate bio-catalytic networks underlying traditional fermented foods such as Baijiu. However, LAB and their metabolic mechanisms are partially understood in Moutai flavor Baijiu fermentation. Here, we found that Acetilactobacillus jinshanensis became the· dominant species with relative abundance reaching 92%, where the acid accumulated rapidly and peaked at almost 30 g/kg in Moutai flavor Baijiu. After separation, purification, and cultivation, A. jinshanensis exhibited pronounced acidophilia and higher acid resistance compared to other LAB. Further integrated multi-omics analysis revealed that fatty acid synthesis, cell membrane integrity, pHi and redox homeostasis maintenance, protein and amide syntheses were possibly crucial acid-resistant mechanisms in A. jinshanensis. Structural proteomics indicated that the surfaces of A. jinshanensis proteases contained more positively charged amino acid residues to maintain protein stability in acidic environments. The genes HSP20 and acpP were identified as acid-resistant genes for A. jinshanensis by heterologous expression analysis. These findings not only enhance our understanding of LAB in Baijiu, providing a scientific basis for acid regulation for production process, but also offer valuable insights for studying core species in other fermentation systems.

Quantum computational and experimental spectroscopic investigation (FT-IR, Raman, UV–Vis), PES, LHE and topological investigations of 7-[(2R)-2-hydroxypropyl]-1,3-dimethylpurine-2,6-dione

The current research explored the structural optimization, electrical and vibrational characteristics, and quantum chemical calculations via the procedure of DFT for a purine derivative, 7-[(2R)-2-hydroxypropyl]-1,3-dimethylpurine-2,6-dione (2HDPD). Functional groups were found by correlating FT-IR with simulated spectra. TD-DFT calculations were done for UV–vis absorption and to ascertain the electronic properties of both the solvent and the gas phase, enabling additional study of the compounds LHE. The experimental outcomes and theoretic parameters are in good agreement. LUMO and HOMO band gaps spectacle that the molecule is chemically reactive and that there is adequate charge transfer. Polar solvent exhibits the highest band gap value, 5.057 eV. Numerous topological considerations were accomplished using the Multiwfn tool. Charge transfer investigations have demonstrated the most imperative states. Weak intermolecular interactions were investigated using RDG analysis, LOL, NBO, and ELF. The 2HDPD compounds reactive areas were discovered by applying the MEP and Fukui investigations. Hyperpolarizability characteristics were used to calculate NLO behavior. The drug-like characteristics of the molecule follow Lipinski five rules. After docking with the proteins 1NG2, 2DVV, 3CAF, and 7OEX, the compound 2HDPD showed moderate binding affinities of −5.28, −5.04, −4.96, and −5.66 kcal/mol, in that order. The Ramachandran plot verified the proteins stability and advantageous locations. The Docking results show compound 2HDPD exhibited a COPD property.

COSMIC Database and Structural Modeling Analysis of CYP2D6 Mutations in Human Cancers.

Single nucleotide polymorphisms (SNPs) in cytochrome P450 (CYP450) enzymes alter the metabolism of a variety of drugs. Numerous medications, including chemotherapies, are metabolized by CYP450 enzymes, making the expression of this suite of enzymes in tumor cells relevant to prescription regimens for cancer patients. We analyzed the characteristics of mutations of the CYP2D6 enzymes in cancer patients obtained from the Catalogue of Somatic Mutations in Cancer (COSMIC), including mutation type, age of the patient, tissue type, and histology. Mutations were analyzed through the Cancer-Related Analysis of Variants Toolkit (CRAVAT) software along with CHASM and VEST4 algorithms to determine the likelihood of being a driver and/or pathogenic mutation. For mutations with significant CHASM and VEST4 scores, structural analysis of each corresponding mutant protein was performed. The effect of each mutation was evaluated for its impact on the overall protein stability and ligand binding using Foldit Standalone and SwissDock, respectively. Structural analysis revealed that several missense mutations in CYP2D6 resulted in altered stability after energy minimization. Three missense mutations of CYP2D6 significantly altered docking stability and those located on alpha-helices near the docking site had a more significant impact than those not found in secondary protein structures. In conclusion, we have identified a series of mutations to CYP2D6 enzymes with possible relevance to cancer pathologies. Significance Statement CYP2D6 is responsible for the metabolism of many anti-cancer drugs. This study identified and characterized a series of mutations in the CYP2D6 enzyme that occurred in tumors. We found it likely that many of these mutations would alter enzyme function, leading to changes in drug metabolism in the tumor. We provide a basis for predicting the likelihood of a patient carrying these mutations to identify patients who may benefit from a precision medicine approach to drug selection and dosing.

Identification of Pathogenic Missense Mutations of NF1 Using Computational Approaches

Neurofibromatosis type 1 (NF1) is a prevalent autosomal dominant disorder caused by mutations in the NF1 gene, leading to multisystem disorders. Given the critical role of cysteine residues in protein stability and function, we aimed to identify key NF1 mutations affecting cysteine residues that significantly contribute to neurofibromatosis pathology. To identify the most critical mutations in the NF1 gene that contribute to the pathology of neurofibromatosis, we employed a sophisticated computational pipeline specifically designed to detect significant mutations affecting the NF1 gene. Our approach involved an exhaustive search of databases such as the Human Gene Mutation Database (HGMD), UniProt, and ClinVar for information on missense mutations associated with NF1. Our search yielded a total of 204 unique cysteine missense mutations. We then employed in silico prediction tools, including PredictSNP, iStable, and Align GVGD, to assess the impact of these mutations. Among the mutations, C379R, R1000C, and C1016Y stood out due to their deleterious effects on the biophysical properties of the neurofibromin protein, significantly destabilizing its structure. These mutations were subjected to further phenotyping analysis using SNPeffect 4.0, which predicted disturbances in the protein’s chaperone binding sites and overall structural stability. Furthermore, to directly visualize the impact of these mutations on protein structure, we utilized AlphaFold3 to simulate both the wild-type and mutant NF1 structures, revealing the significant effects of the R1000C mutation on the protein’s conformation. In conclusion, the identification of these mutations can play a pivotal role in advancing the field of precision medicine and aid in the development of effective drugs for associated diseases.

Increasing Vitamin K2 Synthesis in Bacillus subtilis by Controlling the Expression of MenD and Stabilizing MenA.

As an indispensable member of the family of lipid vitamins, vitamin K2 (MK-7) plays an important role in blood coagulation, cardiovascular health, and kidney health. Microbial fermentation is favored due to its high utilization rate of raw materials, simple operation, and moderate conditions. However, the biosynthesis pathway of vitamin K2 in microorganisms is highly complex, which hinders its industrial production in microbial cell factories. One of the major challenges is the stable expression and deregulation of key enzymes in the vitamin K2 biosynthesis pathway, which remains unclear and has undergone little investigation. In this study, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid synthase (MenD) and 1,4-dihydroxy-2-naphthoate polyprenyltransferase (MenA) were identified as pivotal enzymes in the biosynthesis of vitamin K2. To investigate the catalytic efficiency of MenD in the biosynthesis pathway of vitamin K2, structure-based mutation design and site-directed mutagenesis were performed. Three mutation sites were identified in MenD: A115Y, R96 M, and R323M, which improve the expression level and protein stability. Meanwhile, the MenA mutant T290M, which exhibits improved protein stability, was obtained by modifying its hydrophobic stacking structure. Finally, an engineered strain noted ZQ13 that combinatorially overexpressed MenD (A115Y) and MenA (T290M) mutants was constructed and achieved 338.37 mg/L vitamin K2 production in a 3-L fermenter.

The Feline calicivirus capsid protein VP1 is a client of the molecular chaperone Hsp90.

Feline calicivirus (FCV) icosahedral viral capsids are composed of dozens of structural subunits that rely on cellular chaperones to self-assemble in an orderly fashion. Here, we report that the heat shock protein 90 (Hsp90) inhibition significantly reduced FCV particle production, suggesting a role in the replicative cycle. We found that Hsp90 inhibition was not related to the synthesis or stability of the early proteins that translate from the gRNA nor to the minor capsid protein VP2 but with a reduction in the major capsid protein VP1 levels, both translated late in infection from the subgenomic RNAs. Reduction in VP1 levels was observed despite an augment of the leader of the capsid (LC)-VP1 precursor levels, from which the LC and VP1 proteins are produced after proteolytic processing by NS6/7. The direct interaction of VP1 with Hsp90 was observed in infected cells. These results suggest that upon release from the polyprotein precursor, VP1 becomes a client of Hsp90 and that this interaction is required for an efficient FCV replicative cycle.

NEDD4L affects stability of the CHEK2/TP53 axis through ubiquitination modification to enhance osteogenic differentiation of periodontal ligament stem cells

ABSTRACT Background Checkpoint kinase 2 (CHEK2) and its regulated tumor protein p53 (TP53) have been correlated with osteogenic differentiation of osteoblast-like cells. Based on bioinformatics predictions, this study aims to investigate the effect of the CHEK2/TP53 axis on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to explore the regulatory mechanism. Methods PDLSCs were isolated from human impacted wisdom teeth, and they were cultured in normal medium (NM) or osteogenic medium (OM). Protein levels of CHEK2 and TP53 were examined using western blot analysis. Osteogenic differentiation ability of PDLSCs was analyzed by measuring marker proteins (RUNX2, OCN, and OSX), ALP activity, and ALP staining. Molecular interaction between NEDD4 like E3 ubiquitin protein ligase (NEDD4L) and CHEK2 was examined by ubiquitination and co-immunoprecipitation assays. Gain- and loss-of function assays of NEDD4L, CHEK2, and TP53 were performed to analyze their function in osteogenic differentiation of PDLSCs. A rat model of mandibular bone defect was generated for in vivo validation. Results NEDD4L was upregulated, while CHEK2 and TP53 were downregulated in PDLSCs cultured in OM. CHEK2 protected TP53 from degradation, while NEDD4L reduced CHEK2 protein level by ubiquitination modification. NEDD4L silencing reduced osteogenic differentiation ability of PDLSCs both in vitro and in vivo, which was restored by CHEK2 silencing. By contrast, CHEK2 overexpression blocked the osteogenic differentiation of PDLSCs in vitro. Conclusion This study demonstrates that NEDD4L affects protein stability of the CHEK2/TP53 axis through ubiquitination modification, thus increasing osteogenic differentiation of PDLSCs.

AI Prediction of Structural Stability of Nanoproteins Based on Structures and Residue Properties by Mean Pooled Dual Graph Convolutional Network.

The structural stability of proteins is an important topic in various fields such as biotechnology, pharmaceuticals, and enzymology. Specifically, understanding the structural stability of protein is crucial for protein design. Artificial design, while pursuing high thermodynamic stability and rigidity of proteins, inevitably sacrifices biological functions closely related to protein flexibility. The thermodynamic stability of proteins is not always optimal when they are highest to perfectly perform their biological functions. Extensive theoretical and experimental screening is often required to obtain stable protein structures. Thus, it becomes critically important to develop a stability prediction model based on the balance between protein stability and bioactivity. To design protein drugs with better functionality in a broader structural space, a novel protein structural stability predictor called PSSP has been developed in this study. PSSP is a mean pooled dual graph convolutional network (GCN) model based on sequence characteristics and secondary structure, distance matrix, graph, and residue properties of a nanoprotein to provide rapid prediction and judgment. This model exhibits excellent robustness in predicting the structural stability of nanoproteins. Comparing with previous artificial intelligence algorithms, the results indicate this model can provide a rapid and accurate assessment of the structural stability of artificially designed proteins, which shows the great promises for promoting the robust development of protein design.

Using AI-predicted protein structures as a reference to predict loss-of-function activity in tumor suppressor breast cancer genes

BackgroundThe loss-of-function (LOF) classification of most missense variants in tumor suppressor breast cancer genes BRCA1, BRCA2, PALB2, and RAD51C remains unclassified and confounds clinical actionability. Classifying these variants is challenging due to their rarity, leading clinicians to rely on in silico predictive methods. Protein stability changes are associated with function, making stability predictors valuable. Stability predictions upon missense variant perturbations require high-resolution protein structures. However, the availability of these high-resolution structures is lacking. This study explores using generative AI to predict high-resolution protein structures, which can then be analyzed with in silico protein stability prediction methods to assess LOF activity in ordered regions of the protein. This study also determines the appropriate in silico protein stability and dedicated in silico missense prediction methods in dbNSFP v4.7 database to predict LOF activity in ordered regions of these four genes. Functional classifications from homology recombination DNA repair (HDR) assays and variant classifications from the ClinVar database provide a reliable dataset for evaluating the performance of these in silico prediction methods. ResultsComplex AlphaFold2 structures of the BRCA1-C terminal (BRCT) domain and the DNA-binding (DB) domain of BRCA2, analyzed using protein stability tool FoldX predicts LOF activity from missense variants significantly better than experimentally-derived structures in ordered regions. The BRCT domain achieved an Area Under the Curve (AUC)= 0.861 (95 % CI:0.858–0.863) and AUC= 0.842 (95 % CI:0.840–0.845), while the DB domain achieved an AUC= 0.836 (95 % CI:0.8322–0.841), compared to AUC= 0.847 (95 % CI:0.844–0.850) and AUC= 0.835 (95 % CI:0.832–0.837) from the BRCT domain, and AUC= 0.830 (95 % CI:0.821–0.8320) from the DB domain from experimentally-derived structures. Protein stability does not predict LOF activity from missense variants better than dedicated in silico missense predictors. Overall, we find that AlphaMissense ranks highly, with an average AUC= 0.890 (95 % CI 0.886–0.895) from ordered regions across these four cancer genes, compared to all other in silico missense predictors present in the dbNSFP database. ConclusionsThe study reveals that generative AI protein predicted structures can outperform experimentally-derived structures in evaluating LOF activity from predicted protein stability in ordered regions of genes BRCA1, BRCA2, PALB2 and RAD51C. The study also highlights the predictive performance of AlphaMissense as the premier in silico missense prediction method to predict LOF activity from missense variants in these four tumor suppressor breast cancer genes. The code for this study can be downloaded for free on GitHub (https://github.com/rohandavidg/CarePred)

SENP1 mediates zinc-induced ZnT6 deSUMOylation at Lys-409 involved in the regulation of zinc metabolism in Golgi apparatus

Zinc (Zn) transporters contribute to the maintenance of intracellular Zn homeostasis in vertebrate, whose activity and function are modulated by post-translational modification. However, the function of small ubiquitin-like modifier (SUMOylation) in Zn metabolism remains elusive. Here, compared with low Zn group, a high-Zn diet significantly increases hepatic Zn content and upregulates the expression of metal-response element-binding transcription factor-1 (MTF-1), Zn transporter 6 (ZnT6) and deSUMOylation enzymes (SENP1, SENP2, and SENP6), but inhibits the expression of SUMO proteins and the E1, E2, and E3 enzymes. Mechanistically, Zn triggers the activation of the MTF-1/SENP1 pathway, resulting in the reduction of ZnT6 SUMOylation at Lys 409 by small ubiquitin-like modifier 1 (SUMO1), and promoting the deSUMOylation process mediated by SENP1. SUMOylation modification of ZnT6 has no influence on its localization but reduces its protein stability. Importantly, deSUMOylation of ZnT6 is crucial for controlling Zn export from the cytosols into the Golgi apparatus. In conclusion, for the first time, we elucidate a novel mechanism by which SUMO1-catalyzed SUMOylation and SENP1-mediated deSUMOylation of ZnT6 orchestrate the regulation of Zn metabolism within the Golgi apparatus.

12554 Human Sex Determination: SRY Length Regulates Its Cellular Stability And Hence The Robustness Of Testis Differentiation

Abstract Disclosure: M.A. Weiss: None. The abundance of transcription factors (TFs) mediated by the rates of degradation are subjected to be a robust to an appropriate level. This regulation via the proteasome is largely controlled by the stability of individual proteins and then could determine the direction of a gene-regulatory network. Insight is obtained through studies of bistable genetic circuits mediated by initiating transcription factors. A model is provided by SRY, a Y-encoded TF that initiates testicular differentiation. Known functions in human SRY (204 residues) majorly cluster in its high mobility group (HMG) box whereas the functions of the N- and C-terminal non-box segments are not well characterized. Here, we have used cell-based and mouse transgenic studies to measure the transcriptional threshold of SRY regulating the balance between development and dysgenesis. Our findings demonstrate a threshold length in the C-terminal domain of human SRY that determines the protein’s proteosome-enforced half-life. In a pre-Sertoli cell model, truncation of SRY resulted in the reduction of intracellular concentration and twofold attenuation of the male-specific GRN. Expression of the 1-164 fragment of human SRY in CRISPR-Cas9-edited XX mice failed to drive male differentiation whereas the 1-200 of SRY initiated male GRN development. This study provides insight into the robustness of human SRY and illustrates a powerful strategy to link biochemical properties in cultured cells and in vivo developmental outcomes. Our study reveals a checkpoint in a key TF initiating a sex-specific GRN, functioning as an experimental “control knob” in development. Our approach probes molecular determinants of cell fate and so promises to extend structure-function studies of SRY to the flanking and relatively obscure non-box domains. This result implies the balance between robustness and evolvability in metazoan is a game of numbers of initial transcription factor in the networks. Presentation: 6/1/2024

Berberine modulates ovarian cancer autophagy and glycolysis through the LINC01123/P65/MAPK10 signaling axis

BackgroundBerberine, a readily accessible natural compound known for its ease of synthesis and low toxicity, exhibits anti-tumor properties by modulating inflammatory responses. Recent studies have revealed that berberine can also treat malignant tumors by influencing tumor metabolic reprogramming, making it a potential candidate for metabolic therapy in ovarian cancer. MethodsThe anti-proliferative and anti-metastatic effects of berberine on ovarian cancer cells were investigated using CCK-8 assays, scratch assays, EDU proliferation assays, and assays related to glycolysis and autophagy. Differentially expressed lncRNAs in ovarian cancer were identified using data from the TCGA database. A specific lncRNA's role was delineated through RNA pulldown assays, silver staining, mass spectrometry analysis, CHIP assays, and immunoprecipitation experiments, focusing on its involvement in glycolysis and autophagy regulation in ovarian cancer. Additionally, the inhibitory mechanism of berberine on ovarian cancer cells was validated through cell thermal shift assays and cycloheximide protein degradation experiments to confirm its interaction with key targets. ResultsIn vitro experiments revealed that berberine reduces glycolysis and autophagy levels, leading to the inhibition of ovarian cancer cell proliferation and metastasis. Bioinformatics analysis of TCGA data identified LINC00123 as associated with poor prognosis in ovarian cancer. Experimental validation, including RNA pulldown assays, confirmed that the LINC00123/P65/MAPK10 signaling axis regulates glycolysis and autophagy in ovarian cancer. Furthermore, at the molecular level, berberine inhibits the interaction between LINC00123 and P65, thereby reducing P65 protein stability and impeding its transcriptional regulation of downstream MAPK10. These findings were further validated in animal models. ConclusionOur study highlights berberine's dual benefits of anti-inflammatory effects and inhibition of ovarian cancer proliferation and metastasis by modulating autophagy and glycolysis levels. Mechanistically, berberine targets the LINC00123/P65/MAPK10 signaling pathway to regulate glycolysis and autophagy in ovarian cancer. These insights not only expand the potential of berberine in ovarian cancer therapy but also provide new targets and therapeutic strategies for metabolic therapy in this cancer type.

Small Molecule Compound DHPA Screened by Computer-Aided Drug Design and Molecular Dynamics Simulation Inhibits Neuroblastoma Cell Proliferation by Targeting TrkB.

Neuroblastoma (NB) is a rare and malignant pediatric solid tumor. Due to its heterogeneity, it poses significant challenges for treatment, resulting in a high mortality rate. This study aimed to identify new therapeutic drugs by modeling the TrkB receptor from PDB 4AT5 and conducting virtual screening of compounds from the YaTCM database (containing 47,696 compounds derived from 6220 Traditional Chinese Medicines). The screening utilized the E-pharmacophore approach to select compounds with potential binding affinity to TrkB. The binding abilities of these compounds were tested through molecular dynamics simulations, stretch dynamics simulations, and US simulations. Among the top 11 optimized hit compounds, DHPA and 3″-demethylhexahydrocurcumin are prominent. Further simulations reveal that they form stable receptor-ligand binary complexes with TrkB. In subsequent in vitro cell experiments, 3″-demethylhexahydrocurcumin is eliminated due to its high IC50 for killing NB cells. Low concentrations of DHPA can significantly kill NB cells. Additionally, DHPA can inhibit the expression of TrkB, the activation of TrkB's downstream signaling pathways, and affect the thermal stability of TrkB protein and its response to streptase protease degradation. DHPA may be a potential TrkB inhibitor.

A Deficiency in Glutamine-Fructose-6-Phosphate Transaminase 1 (Gfpt1) in Skeletal Muscle Results in Reduced Glycosylation of the Delta Subunit of the Nicotinic Acetylcholine Receptor (AChRδ).

The neuromuscular junction (NMJ) is the site where the motor neuron innervates skeletal muscle, enabling muscular contraction. Congenital myasthenic syndromes (CMS) arise when mutations in any of the approximately 35 known causative genes cause impaired neuromuscular transmission at the NMJ, resulting in fatigable muscle weakness. A subset of five of these CMS-causative genes are associated with protein glycosylation. Glutamine-fructose-6-phosphate transaminase 1 (Gfpt1) is the rate-limiting enzyme within the hexosamine biosynthetic pathway (HBP), a metabolic pathway that produces the precursors for glycosylation. We hypothesized that deficiency in Gfpt1 expression results in aberrant or reduced glycosylation, impairing the proper assembly and stability of key NMJ-associated proteins. Using both in vitro and in vivo Gfpt1-deficient models, we determined that the acetylcholine receptor delta subunit (AChRδ) has reduced expression and is hypo-glycosylated. Using laser capture microdissection, NMJs were harvested from Gfpt1 knockout mouse muscle. A lower-molecular-weight species of AChRδ was identified at the NMJ that was not detected in controls. Furthermore, Gfpt1-deficient muscle lysates showed impairment in protein O-GlcNAcylation and sialylation, suggesting that multiple glycan chains are impacted. Other key NMJ-associated proteins, in addition to AChRδ, may also be differentially glycosylated in Gfpt1-deficient muscle.

Alternate RNA decoding results in stable and abundant proteins in mammals.

Amino acid substitutions may substantially alter protein stability and function, but the contribution of substitutions arising from alternate translation (deviations from the genetic code) is unknown. To explore it, we analyzed deep proteomic and transcriptomic data from over 1,000 human samples, including 6 cancer types and 26 healthy human tissues. This global analysis identified 60,024 high confidence substitutions corresponding to 8,801 unique sites in proteins derived from 1,990 genes. Some substitutions are shared across samples, while others exhibit strong tissue-type and cancer specificity. Surprisingly, products of alternate translation are more abundant than their canonical counterparts for hundreds of proteins, suggesting sense codon recoding. Recoded proteins include transcription factors, proteases, signaling proteins, and proteins associated with neurodegeneration. Mechanisms contributing to substitution abundance include protein stability, codon frequency, codon-anticodon mismatches, and RNA modifications. We characterize sequence motifs around alternatively translated amino acids and how substitution ratios vary across protein domains, tissue types and cancers. The substitution ratios are positively associated with intrinsically disordered regions and genetic polymorphisms in gnomAD, though the polymorphisms cannot account for the substitutions. Both the sequence and the tissue-specificity of alternatively translated proteins are conserved between human and mouse. These results demonstrate the contribution of alternate translation to diversifying mammalian proteomes, and its association with protein stability, tissue-specific proteomes, and diseases.