Alzheimer’s disease (AD) is characterized by overproduction of β amyloid peptides in the brain with progressive loss of neuronal cells. The 42-aa form of the β amyloid peptide (Aβ42) is implied as a major causative factor, because it is toxic to neurons and elicits inflammatory responses in the brain by activating microglial cells. Despite the overproduction of Aβ42, AD brain tissue also generates protective factor(s) that may antagonize the neurodestructive effect of Aβ42. Humanin is a gene cloned from an apparently normal region of an AD brain and encodes a 24-aa peptide. Both secreted and synthetic Humanin peptides protect neuronal cells from damage by Aβ42, and the effect of Humanin may involve putative cellular receptor(s). To elucidate the molecular identity of such receptor(s), we examined the activity of synthetic Humanin on various cells and found that Humanin induced chemotaxis of mononuclear phagocytes by using a human G protein-coupled formylpeptide receptor-like-1 (FPRL1) and its murine counterpart FPR2. Coincidentally, FPRL1 and FPR2 are also functional receptors used by Aβ42 to chemoattract and activate phagocytic cells. Humanin reduced the aggregation and fibrillary formation by suppressing the effect of Aβ42 on mononuclear phagocytes. In neuroblast cells, Humanin and Aβ42 both activated FPRL1; however, only Aβ42 caused apoptotic death of the cells, and its cytopathic effect was blocked by Humanin. We conclude that Humanin shares human FPRL1 and mouse FPR2 with Aβ42 and suggest that Humanin may exert its neuroprotective effects by competitively inhibiting the access of FPRL1 to Aβ42.
The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.
Sumoylation is one of the most essential mechanisms of reversible protein post-translational modifications and is a crucial biochemical process in the regulation of a variety of important biological functions. Sumoylation is also closely involved in various human diseases. The accurate computational identification of sumoylation sites in protein sequences aids in experimental design and mechanistic research in cellular biology. In this study, we introduced amino acid hydrophobicity as a parameter into a traditional binary encoding scheme and developed a novel sumoylation site prediction tool termed SUMOhydro. With the assistance of a support vector machine, the proposed method was trained and tested using a stringent non-redundant sumoylation dataset. In a leave-one-out cross-validation, the proposed method yielded an excellent performance with a correlation coefficient, specificity, sensitivity and accuracy equal to 0.690, 98.6%, 71.1% and 97.5%, respectively. In addition, SUMOhydro has been benchmarked against previously described predictors based on an independent dataset, thereby suggesting that the introduction of hydrophobicity as an additional parameter could assist in the prediction of sumoylation sites. Currently, SUMOhydro is freely accessible at http://protein.cau.edu.cn/others/SUMOhydro/.
Lysine crotonylation (Kcr) is a newly discovered type of protein post-translational modification and has been reported to be involved in various pathophysiological processes. High-resolution mass spectrometry is the primary approach for identification of Kcr sites. However, experimental approaches for identifying Kcr sites are often time-consuming and expensive when compared with computational approaches. To date, several predictors for Kcr site prediction have been developed, most of which are capable of predicting crotonylation sites on either histones alone or mixed histone and nonhistone proteins together. These methods exhibit high diversity in their algorithms, encoding schemes, feature selection techniques and performance assessment strategies. However, none of them were designed for predicting Kcr sites on nonhistone proteins. Therefore, it is desirable to develop an effective predictor for identifying Kcr sites from the large amount of nonhistone sequence data. For this purpose, we first provide a comprehensive review on six methods for predicting crotonylation sites. Second, we develop a novel deep learning-based computational framework termed as CNNrgb for Kcr site prediction on nonhistone proteins by integrating different types of features. We benchmark its performance against multiple commonly used machine learning classifiers (including random forest, logitboost, naïve Bayes and logistic regression) by performing both 10-fold cross-validation and independent test. The results show that the proposed CNNrgb framework achieves the best performance with high computational efficiency on large datasets. Moreover, to facilitate users’ efforts to investigate Kcr sites on human nonhistone proteins, we implement an online server called nhKcr and compare it with other existing tools to illustrate the utility and robustness of our method. The nhKcr web server and all the datasets utilized in this study are freely accessible at http://nhKcr.erc.monash.edu/.
The prognostic nomogram provided individualized risk estimate of survival in patients after esophagectomy followed by adjuvant chemotherapy.
The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.
The application of poly(lactic acid) for sustained protein delivery is restricted by the harsh pH inside carriers. In this study, we synthesized a pH-responsive comb-shaped block copolymer, polyethylene glycol monomethyl ether-ε-polylysine-g-poly (lactic acid) (PEP)to deliver protein (bovine serum albumin (BSA)). The PEP nanoparticles could automatically adjust the internal pH to a milder level, as shown by the quantitative ratio metric results. The circular dichroism spectra showed that proteins from the PEP nanoparticles were more stable than those from poly(lactic acid) nanoparticles. PEP nanoparticles could achieve sustained BSA release in both in vitro and in vivo experiments. Cytotoxicity results in HL-7702 cells suggested good cell compatibility of PEP carriers. Acute toxicity results showed that the PEP nanoparticles induced no toxic response in Kunming mice. Thus, PEP nanoparticles hold potential as efficient carriers for sustained protein release.
Background: Cancer metastasis-related chemoresistance and tumour progression are the leading causes of death among CRC patients. Therefore, it is urgent to identify reliable novel biomarkers for predicting the metastasis of CRC.Methods: The gene expression and corresponding clinical data of CRC patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Univariate and multivariate analyses were performed to identify prognostic metastasis-related lncRNAs. Nomograms were constructed, and the predictive accuracy of the nomogram model was assessed by ROC curve analysis. Then, the R package “pRRophetic” was used to predict chemotherapeutic response in CRC patients. In addition, the CIBERSORT database was introduced to evaluate tumour infiltrating immune cells between the high—and low-risk groups. The potential roles of SNHG7 and ZEB1-AS1 in CRC cell lines were further confirmed by in vitro experiments.Results: An 8-lncRNA (LINC00261, RP1-170O19.17, CAPN10-AS1, SNHG7, ZEB1-AS1, U47924.27, NIFK-AS1, and LINC00925) signature was constructed for CRC prognosis prediction, which stratified patients into two risk groups. Kaplan-Meier analysis revealed that patients in the higher-risk group had a lower survival probability than those in the lower-risk group [p < 0.001 (TCGA); P = 0.044 (GSE39582); and P = 0.0078 (GSE29621)] The AUCs of 1-, 3-, and 5-year survival were 0.678, 0.669, and 0.72 in TCGA; 0.58, 0.55, and 0.56 in GSE39582; and 0.75, 0.54, and 0.56 in GSE29621, respectively. In addition, the risk score was an independent risk factor for CRC patients. Nomograms were constructed, and the predictive accuracy was assessed by ROC curve analysis. This signature could effectively predict the immune status and chemotherapy response in CRC patients. Moreover, SNHG7 and ZEB1-AS1 depletion significantly suppressed the colony formation, migration, and invasion of CRC cells in vitro.Conclusion: We constructed a signature that could predict the metastasis of CRC and provide certain theoretical guidance for novel therapeutic approaches for CRC.
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