Protein Stability Research Articles

SLMAP3 is crucial for organogenesis through mechanisms involving primary cilia formation.

SLMAP3 is a constituent of the centrosome and is known to assemble with the striatin-interacting phosphatase and kinase (STRIPAK) complex, where it has been reported to repress Hippo signalling. The global knockout of SLMAP3 in mice results in embryonic/perinatal lethality and stunted growth without changes in the phosphorylation status of YAP. Diverse phenotypes present in the SLMAP3-/- embryos include reduced body axis, small and abnormal organs resembling defects in planar cell polarity (PCP) signalling, while also displaying the notable polycystic kidneys, a known manifestation of ciliopathies. Analysis of cell polarity in primary mouse embryonic fibroblasts (MEFs) including cell migration, orientation and mitotic spindle angle did not reveal any changes due to SLMAP3 loss in these cells, although the expression of DVL3 was significantly reduced. Furthermore, MEFs lacking FGFR1OP2 or STRN3, two other STRIPAK members, did not reveal any significant changes in any of these parameters either. Significant changes in the number of ciliated cells and primary cilium length in SLMAP3 and FGFR1OP2 deficient MEFs were evident, while a reduced primary cilium length was notable in chondrocytes of SLMAP3 deficient embryos. Our findings suggest that SLMAP3 is essential for mouse embryogenesis through novel mechanisms involving the primary cilium/PCP and protein stability independent of Hippo signalling.

Enhancing Gpx1 palmitoylation to inhibit angiogenesis by targeting PPT1

The significance of protein S-palmitoylation in angiogenesis has been largely overlooked, leaving various aspects unexplored. Recent identification of Gpx1 as a palmitoylated protein has generated interest in exploring its potential involvement in novel pathological mechanisms related to angiogenesis. In this study, we demonstrate that Gpx1 undergoes palmitoylation at cysteine-76 and -113, with PPT1 playing a crucial role in modulating the depalmitoylation of Gpx1. Furthermore, we find that PPT1-regulated depalmitoylation negatively impacts Gpx1 protein stability. Interestingly, inhibiting Gpx1 palmitoylation, either through expression of a non-palmitoylated Gpx1 mutant or by expressing PPT1, significantly enhances neovascular angiogenesis. Conversely, in PPT1-deficient mice, angiogenesis is notably attenuated compared to wild-type mice in an Oxygen-Induced Retinopathy (OIR) model, which mimics pathological angiogenesis. Physiologically, under hypoxic conditions, Gpx1 palmitoylation levels are drastically reduced, suggesting that increasing Gpx1 palmitoylation may have beneficial effects. Indeed, enhancing Gpx1 palmitoylation by inhibiting PPT1 with DC661 effectively suppresses retinal angiogenesis in the OIR disease model. Overall, our findings highlight the pivotal role of protein palmitoylation in angiogenesis and propose a novel mechanism whereby the PPT1-Gpx1 axis modulates angiogenesis, thereby providing a potential therapeutic strategy for targeting PPT1 to combat angiogenesis.

Hexosaminidase B-driven cancer cell-macrophage co-dependency promotes glycolysis addiction and tumorigenesis in glioblastoma

Glycolytic metabolic reprogramming in cancer is regulated by both cancer intrinsic variations like isocitrate dehydrogenase 1 (IDH1) status and non-cancerous microenvironment components like tumor associated macrophages (TAMs). However, the detailed mechanism remains elusive. Here, we identify hexosaminidase B (HEXB) as a key regulator for glycolysis in glioblastoma (GBM). HEXB intercellularly manipulates TAMs to promote glycolysis in GBM cells, while intrinsically enhancing cancer cell glycolysis. Mechanistically, HEXB elevation augments tumor HIF1α protein stability through activating ITGB1/ILK/YAP1; Subsequently, HIF1α promotes HEXB and multiple glycolytic gene transcription in GBM cells. Genetic ablation and pharmacological inhibition of HEXB elicits substantial therapeutic effects in preclinical GBM models, while targeting HEXB doesn’t induce significant reduction in IDH1 mutant glioma and inhibiting IDH1 mutation-derived 2-hydroxyglutaric acid (2-HG) significantly restores HEXB expression in glioma cells. Our work highlights a HEXB driven TAMs-associated glycolysis-promoting network in GBM and provides clues for developing more effective therapies against it.

Identification and characterization of ADAR1 mutations and changes in gene expression in human cancers

ADAR1 (Adenosine deaminase action on RNA1) is involved in post-transcriptional RNA editing. ADAR1 mutations have been identified in many cancers but its role tumor formation is still not well understood. Here we used available cancer genomes deposited on CSOMIC and cBioPortal to identify and characterize mutations and changes in ADAR1 expression in cancer cells. We identify several high frequency substitutions including one at R767 which is located in one of the dsRNA interacting domains. In silico protein structure analysis suggest the R767 mutations affect the protein stability and are likely to destabilize interaction with dsRNA. Gene expression analysis shows that in samples with under-expressed ADAR1, there is a statistically significant increase in expression of BLCAP (Bladder Cancer Associated Protein). Although BLCAP was initially identified in bladder cancers, more recent evidence shows that it is a tumor suppressor and BLCAP mutations have been identified in many cancer cells. Epistatic analysis using the cBioPortal mutual exclusivity calculator for the TCGA pan-cancer data shows that co-mutations between ADAR1 and other genes regulated by it are likely in cancer cells except for PTEN, AKT1 and BLCAP. This suggests that when ADAR1 function is impaired, PTEN, AKT1 and BLCAP become essential for survival of cancer cells. We also identified several samples with high mutation burden between ADAR1 and other genes regulated primarily in endometrial cancers. Finally, we show that the deaminase domain is highly conserved in metazoans and mutations within conserved residues do occur in human cancers suggesting that destabilization of the enzyme function is contributing to cancer development.

Generation of four human-derived iPSC TorsinA-3xFLAG reporter lines from a DYT-TOR1A patient

A 3-bp deletion (ΔGAG) in TOR1A is a common cause of early-onset isolated dystonia DYT-TOR1A. The exact disease mechanism remains unknown. Here we describe the generation and characterization of four TorsinA-3xFLAG reporter induced pluripotent cell (iPSC) lines derived from a DYT-TOR1A patient. The cell lines carry either a ΔGAG variant or a corrected allele and a mono- or biallelic 3xFLAG-Tag introduced using CRISPR/Cas9 technology. These cells provide an opportunity to study differential protein stability, subcellular localization, and interactors of endogenous WT or ΔE variants of TorsinA in iPSCs, neural progenitor cells (smNPC), and neurons.

Taming hemoglobin chemistry—a new hemoglobin-based oxygen carrier engineered with both decreased rates of nitric oxide scavenging and lipid oxidation

The clinical utility of hemoglobin-based oxygen carriers (HBOC) is limited by adverse heme oxidative chemistry. A variety of tyrosine residues were inserted on the surface of the γ subunit of recombinant fetal hemoglobin to create novel electron transport pathways. This enhanced the ability of the physiological antioxidant ascorbate to reduce ferryl heme and decrease lipid peroxidation. The γL96Y mutation presented the best profile of oxidative protection unaccompanied by loss of protein stability and function. N-terminal deletions were constructed to facilitate the production of recombinant hemoglobin by fermentation and phenylalanine insertions in the heme pocket to decrease the rate of NO dioxygenation. The resultant mutant (αV1del. αL29F, γG1del. γV67F, γL96Y) significantly decreased NO scavenging and lipid peroxidation in vitro. Unlike native hemoglobin or a recombinant control (αV1del, γG1del), this mutation showed no increase in blood pressure immediately following infusion in a rat model of reperfusion injury, suggesting that it was also able to prevent NO scavenging in vivo. Infusion of the mutant also resulted in no meaningful adverse physiological effects apart from diuresis, and no increase in oxidative stress, as measured by urinary isoprostane levels. Following PEGylation via the Euro-PEG-Hb method to increase vascular retention, this novel protein construct was compared with saline in a severe rat reperfusion injury model (45% blood volume removal for 90 minutes followed by reinfusion to twice the volume of shed blood). Blood pressure and survival were followed for 4 h post-reperfusion. While there was no difference in blood pressure, the PEGylated Hb mutant significantly increased survival.

Proteomic analysis of wild boar meat: Effect of storage method and time on muscle protein stability

Oxidation processes affect proteins from various molecular pathways and are crucial for wild boar meat quality, shelf life and human health. This study investigated the effects of different storage methods on the formation and composition of oxygen-induced protein aggregates in the muscles of European wild boar (Sus scrofa scrofa). Vacuum packaging (VAC), modified atmosphere packaging (MAP) and dry-ageing (DA) were compared over a 21-day storage period. The results showed significant differences in protein aggregation depending on the method and storage time. The most intense protein aggregation occurred in the MAP (80 % O2), while air DA (20.9 % O2) resulted in intermediate levels of protein aggregation. Crucial myofibrillar proteins involved in aggregate formation were titin, myosin isoforms (MYH1, MYH2 and MYH7) and nebulin, which were cross-linked with small sarcoplasmic enzymes, such as muscle creatine kinase, isocitrate dehydrogenase and ATPase 1. High‑oxygen storage conditions also promoted the oxidation of ATP synthase, beta-enolase 3, ADP/ATP translocase and myoglobin.

Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase (DUB), characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum (ER). Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from ER-associated protein degradation (ERAD) in a catalytic-independent manner, promote autophagy and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a DUB substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis", by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to secretion of lysosomal proteins, including LGMN.

HIF-1α-HPRT1 axis promotes tumorigenesis and gefitinib resistance by enhancing purine metabolism in EGFR-mutant lung adenocarcinoma

BackgroundThe mutations of oncogenic epidermal growth factor receptor (EGFR) is an important cause of lung adenocarcinoma (LUAD) malignance. It has been knowm that metabolic reprogramming is an important hallmark of malignant tumors, and purine metabolism is a key metabolic pathway for tumor progression and drug resistance, but its relationship with the EGFR-mutant LUAD is unclear.MethodsMetabolic reprogramming was studied through capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS)-based metabolic profiling analysis. Cell proliferation in vitro was evaluated by EdU staining and cell cycle assay. Tumorigenicity in vivo was tested by subcutaneous tumor formation experiment in nude mice. The binding of hypoxia-inducible factor-1 alpha (HIF-1α) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was detected by DNA pull‑down assay and Chromatin immunoprecipitation (ChIP) assays. HIF-1α, HPRT1, DNA damage and cell apoptosis related genes were examined by western blot. In addition, RNA sequencing, mass spectrometry and bioinformatics analysis were performed.ResultsWe found that mutated EGFR (muEGFR) upregulates HPRT1 to promote purine metabolism and tumorigenesis of EGFR-mutant LUAD. Mechanistically, muEGFR increases HIF-1α expression through protein stability. Meanwhile, up-regulated HIF-1α bound to the promoter of HPRT1 and transcriptionally activates HPRT1 expression, enhancing purine metabolism to maintain rapid tumor cell proliferation in EGFR-mutant LUAD. Further, gefitinib inhibited the synthesis of purine nucleotides, and HPRT1 inhibition increased the sensitivity of gefitinib to EGFR-mutant LUAD.ConclusionsOur study reveals that muEGFR-HIF-1α-HPRT1 axis plays a key role in EGFR-mutant LUAD and provides a new strategy-inhibiting purine metabolism for treating EGFR-mutant LUAD.

Predictive Model Building for Aggregation Kinetics Based on Molecular Dynamics Simulations of an Antibody Fragment.

Computational methods including machine learning and molecular dynamics simulations have strong potential to characterize, understand, and ultimately predict the properties of proteins relevant to their stability and function as therapeutics. Such methods would streamline the development pathway by minimizing the current experimental testing required for many protein variants and formulations. The molecular understanding of thermostability and aggregation propensity has advanced significantly along with predictive algorithms based on the sequence-level or structural-level information on a protein. However, these approaches focus largely on a comparison of protein sequence variations to correlate the properties of proteins to their stability, solubility, and aggregation propensity. For therapeutic protein development, it is of equal importance to take into account the impact of the formulation conditions to elucidate and predict the stability of the antibody drugs. At the macroscopic level, changing temperature, pH, ionic strength, and the addition of excipients can significantly alter the kinetics of protein aggregation. The mechanisms controlling aggregation kinetics have been traced back to a combination of molecular features, including conformational stability, partial unfolding to aggregation-prone states, and the colloidal stability governed by surface charges and hydrophobicity. However, very little has been done to evaluate these features in the context of protein dynamics in different formulations. In this work, we have combined a range of molecular features calculated from the Fab A33 protein sequence and molecular dynamics simulations. Using the power of advanced, yet interpretable, statistical tools, it has been possible to uncover greater insights into the mechanisms behind protein stability, validating previous findings, and also develop models that can predict the aggregation kinetics within a range of 49 different solution conditions.

Optimal Preservation of PFFs in Glycerol Enhances Suitability for Modeling Parkinson's Disease.

Injecting α-synuclein pre-formed fibrils (αSyn PFFs) into various tissues and organs involves converting monomeric αSyn into a fibrillar form, inducing extensive αSyn pathology that effectively models Parkinson's disease (PD). However, the distinct physicochemical properties of αSyn amyloid fibrils can potentially reduce their seeding activity, especially during storage. In this study, it is demonstrated that αSyn PFFs exhibit significant sensitivity to low temperatures, with notable denaturation occurring between -20 and 4°C, and gradual disassembly persisted even under storage conditions at -80°C. To mitigate this issue, a commonly used protein stabilizer, glycerol is introduced, which significantly reverses the cold-induced disassembly of PFFs. Remarkably, storing PFFs with 20% glycerol at -80°C for a month preserved their morphology and seeding activity as freshly prepared PFFs. Glycerol-stabilized αSyn PFFs resulted in compromised neuronal survival, with the extent of these impairments correlating with the formation of αSyn pathology both in vivo and in vitro, indistinguishable from freshly prepared PFFs. Storing sonicated PFFs with 20% glycerol at -80°C provides an optimal storage method, as sonication is necessary for activating their seeding potential. This approach reduces the frequency of sonication, simplifies handling, and ultimately lowers the overall workload, enhancing the practicality of using PFFs.

TRIM40 interacts with ROCK1 directly and inhibits colorectal cancer cell proliferation through the c-Myc/p21 axis

BackgroundColorectal cancer (CRC) is the most common malignancy of the digestive tract, and to date, morbidity and mortality rates remain high. While existing therapeutic methods have achieved certain effective outcomes, there are still many problems in treating this disease. Therefore, it is still urgent to constantly find new therapeutic targets in CRC that could lead to new therapeutics. MethodsImmunohistochemistry, Real-time PCR and Western Blot were employed to measure mRNA and protein levels of the target protein, respectively. The proliferation ability of CRC cells was evaluated using ATP assay, Soft agar assay, and nude mouse subcutaneous tumorigenesis assay. Protein Degradation Assay was conducted to determine protein degradation rate, while Ubiquitination assay was used to assess the ubiquitination modification level of target proteins. Immunoprecipitation assay was used to study protein interactions, and pull-down assay was employed to investigate direct interactions between proteins. ResultsTRIM40 was significantly down-regulated in CRC tissues, with its expression levels positively correlating with disease prognosis. Using both in vitro and in vivo approaches, it was demonstrated that TRIM40 could significantly inhibit the proliferation of CRC cells. Molecular mechanism studies showed that TRIM40 directly binds to and ubiquitinates ROCK1 protein, accelerating its degradation and subsequently reducing the stability of c-Myc protein. This cascade of events results in the release of transcriptional inhibition of p21 by c-Myc, leading to increased p21 expression and G0/G1 phase arrest in CRC cells. ConclusionThis research suggests that TRIM40 could be a valuable therapeutic target for the treatment of CRC.

The Role of Protein Post-Translational Modifications in Fruit Ripening

Fruit ripening represents a multifaceted biological process intricately controlled by an array of plant hormones, transcription factors, and epigenetic modifications. These regulatory mechanisms are crucial in determining fruit quality and post-harvest shelf life. Recent advancements in proteomics have shifted the focus toward understanding protein post-translational modifications (PTMs), which play a crucial role in modulating protein function. PTMs enhance protein activity and stability by altering their properties after biosynthesis, thereby adding an additional layer of regulation to the ripening process. This paper provides a comprehensive review of the roles of PTMs, including ubiquitination, phosphorylation, redox modifications, and glycosylation in regulating fruit ripening. Emphasis is placed on the intricate interplay between these PTMs and key regulator factors such as plant hormones, transcriptional mechanisms, and epigenetic modifications. By exploring these interactions, this review seeks to enhance our understanding of the complex regulatory network underlying fruit ripening and to offer novel perspectives on strategies for fruit preservation.

Identification of Homeobox Transcription Factors in a Dimorphic Fungus Talaromyces marneffei and Protein-Protein Interaction Prediction of RfeB.

Talaromyces marneffei is a thermally dimorphic fungus that can cause life-threatening systemic mycoses, particularly in immunocompromised individuals. Fungal homeobox transcription factors control various developmental processes, including the regulation of sexual reproduction, morphology, metabolism, and virulence. However, the function of homeobox proteins in T. marneffei has not been fully explored. Here, we searched the T. marneffei genome for the total homeobox transcription factors and predicted their biological relevance by performing gene expression analysis in different cell types, including conidia, mycelia, yeasts, and during phase transition. RfeB is selected for further computational analysis since (i) its transcripts were differentially expressed in different phases of T. marneffei, and (ii) this protein contains the highly conserved protein-protein interaction region (IR), which could be important for pathobiology and have therapeutic application. To assess the structure-function of the IR region, in silico alanine substitutions were performed at three-conserved IR residues (Asp276, Glu279, and Gln282) of RfeB, generating a triple RfeB mutated protein. Using 3D modeling and molecular dynamics simulations, we compared the protein complex formation of wild-type and mutated RfeB proteins with the putative partner candidate TmSwi5. Our results demonstrated that the mutated RfeB protein exhibited increased free binding energy, elevated protein compactness, and a reduced number of atomic contacts, suggesting disrupted protein stability and interaction. Notably, our model revealed that the IR residues primarily stabilized the RfeB binding sites located in the central region (CR). This computational approach for protein mutagenesis could provide a foundation for future experimental studies on the functional characterization of RfeB and other homeodomain-containing proteins in T. marneffei.

The role of lipoprotein‑associated phospholipase A2 in inflammatory response and macrophage infiltration in sepsis and the regulatory mechanisms.

Lipoproteinassociated phospholipase A2 (Lp-PLA2), encoded by the phospholipase A2 group VII (Pla2g7) gene, has been pertinent to inflammatory responses. This study investigates the correlation between Lp-PLA2 and inflammatory injury in septic mice and explores its regulatory mechanism. Lp-PLA2 was found to be upregulated in the serum of septic mice induced by cecal ligation and puncture and in the culture supernatant of RAW264.7 cells following lipopolysaccharide and adenosine triphosphate treatments. The contents of Lp-PLA2 were positively correlated with increased concentrations of proinflammatory cytokines in patients with sepsis. Both animal and cellular models showed increased concentrations of proinflammatory cytokines. Spi-1 proto-oncogene (Spi1), highly expressed in these models, was found to activate Pla2g7 transcription. Knockdown of Pla2g7 or Spi1 reduced the proinflammatory cytokine production, mitigated organ damage in mice, and suppressed macrophage migration in vitro. Retinoblastoma binding protein 6 (Rbbp6), poorly expressed in both models, was found to reduce Spi1 protein stability through ubiquitination modification. Rbbp6 overexpression similarly suppressed inflammatory activation of RAW264.7 cells, which was counteracted by Pla2g7 or Spi1 upregulation. In summary, this study demonstrates that the Pla2g7 loss and Spi1 upregulation participate in inflammatory responses in sepsis by elevating the Lp-PLA2 levels.

Deubiquitinases in skeletal muscle - The Underappreciated Side of the Ubiquitination Coin.

Ubiquitination is a post-translational modification that plays important roles in regulating protein stability, function, localization, and protein-protein interactions. Proteins are ubiquitinated via a process involving specific E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. Simultaneously, protein ubiquitination is opposed by deubiquitinating enzymes (DUBs). DUB-mediated deubiquitination can change protein function or fate and recycle ubiquitin to maintain the free ubiquitin pool. Approximately 100 DUBs have been identified in the mammalian genome, and characterized into seven classes (USP, OTU, UCH, MJD, JAMM, MINDY and ZUP classes). Of these 100 DUBs, there has only been relatively limited investigation of 19 specifically in skeletal muscle cells, in vitro or in vivo, using overexpression, knockdown, and knockout models. To date, evidence indicates roles for individual DUBs in regulating aspects of myogenesis, protein turnover, muscle mass, and muscle metabolism. However, the exact mechanism by which these DUBs act (i.e. the specific targets of these DUBs and the type of ubiquitin chains they target) is still largely unknown, underscoring how little we know about DUBs in skeletal muscle. This review endeavors to comprehensively summarize the current state of knowledge of the function of DUBs in skeletal muscle and highlight the opportunities for gaining a greater understanding through further research into this important area of skeletal muscle and ubiquitin biology.

Revolutionizing Molecular Design for Innovative Therapeutic Applications through Artificial Intelligence

The field of computational protein engineering has been transformed by recent advancements in machine learning, artificial intelligence, and molecular modeling, enabling the design of proteins with unprecedented precision and functionality. Computational methods now play a crucial role in enhancing the stability, activity, and specificity of proteins for diverse applications in biotechnology and medicine. Techniques such as deep learning, reinforcement learning, and transfer learning have dramatically improved protein structure prediction, optimization of binding affinities, and enzyme design. These innovations have streamlined the process of protein engineering by allowing the rapid generation of targeted libraries, reducing experimental sampling, and enabling the rational design of proteins with tailored properties. Furthermore, the integration of computational approaches with high-throughput experimental techniques has facilitated the development of multifunctional proteins and novel therapeutics. However, challenges remain in bridging the gap between computational predictions and experimental validation and in addressing ethical concerns related to AI-driven protein design. This review provides a comprehensive overview of the current state and future directions of computational methods in protein engineering, emphasizing their transformative potential in creating next-generation biologics and advancing synthetic biology.

β-Citronellol: a potential anti-inflammatory and gastro-protective agent-mechanistic insights into its modulatory effects on COX-II, 5-LOX, eNOS, and ICAM-1 pathways through in vitro, in vivo, in silico, and network pharmacology studies

BackgroundThe current study aimed to evaluate the anti-inflammatory, anti-oxidant, and pronounced gastro-protective activities of β- Citronellol using in vitro, in vivo assays and in silico approaches.MethodsIn vitro assays, denaturation of bovine serum albumin, egg protein, and human Red Blood Cells (RBCs) membrane stabilization were performed, using Piroxicam as standard. For in vivo assessment, Histamine (0.1 ml from 1% w/v) and Formaldehyde (0.1 ml from 2% v/v) were used to mediate inflammation. In silico molecular docking and network pharmacology were employed to probe the possible target genes mediating gastroprotective effect of β-Citronellol at 25, 50, and 100 mg/kg, using indomethacin-induced (25 mg/kg i.p) gastric ulcer in rats. Moreover, Gastric tissues were evaluated for morphological, histopathological, and bio-chemical analysis of PGE2, COX-I, COX-II, 5-LOX, eNOS, ICAM-1, oxygen-free radical scavengers (SOD, CAT), and oxidative stress marker (MDA).Resultsβ-Citronellol prevented denaturation of proteins and RBCs membrane stabilization with maximum effect observed at 6,400 µg/mL. Citronellol decreased rat’s paw edema. Network pharmacology and docking studies revealed gastro-protective potential of Citronellol possibly mediated through arachidonic acid pathways by targeting COX-I, COX-II, PGE2, and 5-LOX. Citronellol reduced the ulcer indices, and histopathological changes. Further, β-Citronellol (50 and 100 mg/kg) increased gastric PGE2, COX-1, and eNOS; while suppressing COX-2, 5-LOX and ICAM-1. Citronellol markedly enhanced the oxidative balance in isolated rat stomach tissues.ConclusionsThe anti-inflammatory, anti-oxidant, and gastro-protective effects of β-Citronellol against indomethacin-induced gastric ulcer model in rats through mediating COX-I, COX-II, PGE2, 5-LOX, eNOS, and ICAM-1 inflammatory markers.Graphical abstract

Crotonylation of NAE1 Modulates Cardiac Hypertrophy via Gelsolin Neddylation.

Cardiac hypertrophy and its associated remodeling are among the leading causes of heart failure. Lysine crotonylation is a recently discovered posttranslational modification whose role in cardiac hypertrophy remains largely unknown. NAE1 (NEDD8 [neural precursor cell expressed developmentally downregulated protein 8]-activating enzyme E1 regulatory subunit) is mainly involved in the neddylation modification of protein targets. However, the function of crotonylated NAE1 has not been defined. This study aims to elucidate the effects and mechanisms of NAE1 crotonylation on cardiac hypertrophy. Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. Tandem mass tag (TMT)-labeled quantitative lysine crotonylome analysis was performed to identify the crotonylated proteins in a mouse cardiac hypertrophic model induced by transverse aortic constriction. We generated NAE1 knock-in mice carrying a crotonylation-defective K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking K238Q (lysine to glutamine mutation at site 238) mutation (NAE1 K238Q) to assess the functional role of crotonylation of NAE1 at K238 in pathological cardiac hypertrophy. Furthermore, we combined coimmunoprecipitation, mass spectrometry, and dot blot analysis that was followed by multiple molecular biological methodologies to identify the target GSN (gelsolin) and corresponding molecular events contributing to the function of NAE1 K238 (lysine residue at site 238) crotonylation. The crotonylation level of NAE1 was increased in mice and patients with cardiac hypertrophy. Quantitative crotonylomics analysis revealed that K238 was the main crotonylation site of NAE1. Loss of K238 crotonylation in NAE1 K238R knock-in mice attenuated cardiac hypertrophy and restored the heart function, while hypercrotonylation mimic in NAE1 K238Q knock-in mice significantly enhanced transverse aortic constriction-induced pathological hypertrophic response, leading to impaired cardiac structure and function. The recombinant adenoviral vector carrying NAE1 K238R mutant attenuated, while the K238Q mutant aggravated Ang II (angiotensin II)-induced hypertrophy. Mechanistically, we identified GSN as a direct target of NAE1. K238 crotonylation of NAE1 promoted GSN neddylation and, thus, enhanced its protein stability and expression. NAE1 crotonylation-dependent increase of GSN promoted actin-severing activity, which resulted in adverse cytoskeletal remodeling and progression of pathological hypertrophy. Our findings provide new insights into the previously unrecognized role of crotonylation on nonhistone proteins during cardiac hypertrophy. We found that K238 crotonylation of NAE1 plays an essential role in mediating cardiac hypertrophy through GSN neddylation, which provides potential novel therapeutic targets for pathological hypertrophy and cardiac remodeling.

CircSEC24B activates autophagy and induces chemoresistance of colorectal cancer via OTUB1-mediated deubiquitination of SRPX2

Circular RNAs (circRNAs) are a type of regulatory RNA that feature covalently closed single-stranded loops. Evidence suggested that circRNAs play important roles in the progression and development of various cancers. However, the impact of circRNA on autophagy-mediated progression of colorectal cancer (CRC) remains unclear. The objective of this project was to investigate the influence of circSEC24B on autophagy and its underlying mechanisms in CRC. To validate the presence and circular structure of circSEC24B in CRC cells and tissues, PCR and Sanger sequencing techniques were employed. Drug resistance and invasive phenotype of CRC cells were evaluated using CCK8, transwell, and Edu assays. Gain- and loss-of-function experiments were conducted to assess the effects of circSEC24B and its protein partner on the growth, invasion, and metastasis of CRC cells in vitro and in vivo. Interactions between circSEC24B, OTUB1, and SRPX2 were analyzed through immunofluorescence, RNA-pulldown, and RIP assays. Mass spectrometry analysis was used to identify potential binding proteins of circRNA in CRC cells. Vectors were constructed to investigate the specific structural domain of the deubiquitinating enzyme OTUB1 that binds to circSEC24B. Results showed that circSEC24B expression was increased in CRC tissues and cell lines, and it enhanced CRC cell proliferation and autophagy levels. Mechanistically, circSEC24B promoted CRC cell proliferation by regulating the protein stability of SRPX2. Specifically, circSEC24B acted as a scaffold, facilitating the binding of OTUB1 to SRPX2 and thereby enhancing its protein stability. Additionally, evidence suggested that OTUB1 regulated SRPX2 expression through an acetylation-dependent mechanism. In conclusion, this study demonstrated that circSEC24B activated autophagy and induced chemoresistance in CRC by promoting the deubiquitination of SRPX2, mediated by the deubiquitinating enzyme OTUB1.