The role of vitamin D through SphK1/S1P in the regulation of MS progression.

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-ß, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-ß, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

An acceptability study of the introduction of total online or partial online PBL in a large classroom setting in biochemistry.

BACKGROUND: Traditional problem-based learning (PBL) relying on tutored learning in small groups is very resource-intensive. Little is known about the benefits of PBL in a large classroom setting. This paper introduced a PBL case into the traditional didactic biochemistry course and investigated the acceptability of total online or partial online PBL in a large classroom setting introduced during the coronavirus pandemic. METHODS: The students were allocated into either total online Group 1, partial online Group 2, or partial online and with poorer academic performance Group 3. A questionnaire comprising of 8 closed-ended questions and 2 open-ended questions and final exam performances were used to evaluate the acceptability of total online or partial online PBL in a large classroom setting. The 8 closed-ended questions were analysed by the Kruskal-Wallis test or chi-square tests. The word cloud analysis of the 2 open-ended questions were conducted by Wenjuanxing. Students' performances in the final examination were analysed by One-way Anova. RESULTS: Both total online and partial online PBL were rated highly by the students. Overall, there were no significant differences in the effectiveness evaluation of PBL between Group 2 and Group 3. There were no significant differences in final exam performances between Group 1 and Group 2. However, Group 1 rated the effectiveness of PBL much higher than Group 2 and 3. Word cloud analysis of the 2 open-ended questions showed students' positive perspectives of PBL. In biochemistry teaching, from the perspective of the students, the expected optimal number of useful PBL cases might be 2. CONCLUSIONS: Both total online and partial online PBL in a large classroom setting were widely accepted as a beneficial supplement to traditional biochemistry classes.

Meta-analyzing the efficacy of 3D printed models in anatomy education.

Three-dimensional printing models (3DPs) have been widely used in medical anatomy training. However, the 3DPs evaluation results differ depending on such factors as the training objects, experimental design, organ parts, and test content. Thus, this systematic evaluation was carried out to better understand the role of 3DPs in different populations and different experimental designs. Controlled (CON) studies of 3DPs were retrieved from PubMed and Web of Science databases, where the participants were medical students or residents. The teaching content is the anatomical knowledge of human organs. One evaluation indicator is the mastery of anatomical knowledge after training, and the other is the satisfaction of participants with 3DPs. On the whole, the performance of the 3DPs group was higher than that of the CON group; however, there was no statistical difference in the resident subgroup, and there was no statistical difference for 3DPs vs. 3D visual imaging (3DI). In terms of satisfaction rate, the summary data showed that the difference between the 3DPs group (83.6%) vs. the CON group (69.6%) (binary variable) was not statistically significant, with p > 0.05. 3DPs has a positive effect on anatomy teaching, although there are no statistical differences in the performance tests of individual subgroups; participants generally had good evaluations and satisfaction with 3DPs. 3DPs still faces challenges in production cost, raw material source, authenticity, durability, etc. The future of 3D-printing-model-assisted anatomy teaching is worthy of expectation.

The benefits of using atypical presentations and rare diseases in problem-based learning in undergraduate medical education.

BACKGROUND: The nature of student learning in problem-based learning (PBL) largely depends on the quality of the case scenarios presented to them. The effect of case scenarios with higher challenge degree, especially common disease with atypical symptoms (CDAS)- and rare disease (RD)-based case scenarios, on undergraduate medical students remains unclear. This study compared the impact of all scenarios pertaining to common disease with typical symptoms (CDTS) case scenarios, CDTS interspersed with CDAS case scenarios, and CDTS interspersed with RD case scenarios on perceptions of undergraduate students studying organ/system integration curriculum via PBL. METHODS: After finishing four CDTS case scenarios, 294 third-year medical students were randomly allocated into three groups: CDTS, CDAS and RD, studying via CDTS, CDAS and RD case scenarios, respectively. A questionnaire with 15 items was conducted to evaluate the students' perceptions. The students' responses were scored using a 4-point rating scale. The data were analysed using the Kruskal-Wallis test. RESULTS: Among the three PBL conditions, the ones with a higher degree of challenge were rated higher by the students, which included the quality of the case scenarios and the overall performances of the students. The CDAS and RD cases were more effective in developing students' self-directed learning skills, stimulating them to acquire more knowledge required for future work. The satisfaction percentage of RD case scenario sessions was higher. CONCLUSIONS: Of all the three kinds of case scenarios, both CDTS interspersed with CDAS and RD case scenarios had more positive effects on the self-evaluated performance of students. Increasing the challenge and variety of case scenarios by the inclusion of CDAS and RD especially RD might be an effective stimulus in improving students' performance in PBL sessions.

Crystal structure and catalytic activity of the PPM1K N94K mutant.

Protein Phosphatase Mg2+ /Mn2+ -Dependent 1K (PPM1K),also named as PP2Cm or branched-chain α-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg2+ -dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg2+ in the active site. Specifically, three Mg2+ were located in the active site of the PPM1K N94K instead of two Mg2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.

Computational Strategy for Bound State Structure Prediction in Structure-Based Virtual Screening: A Case Study of Protein Tyrosine Phosphatase Receptor Type O Inhibitors.

Accurate protein structure in the ligand-bound state is a prerequisite for successful structure-based virtual screening (SBVS). Therefore, applications of SBVS against targets for which only an apo structure is available may be severely limited. To address this constraint, we developed a computational strategy to explore the ligand-bound state of a target protein, by combined use of molecular dynamics simulation, MM/GBSA binding energy calculation, and fragment-centric topographical mapping. Our computational strategy is validated against low-molecular weight protein tyrosine phosphatase (LMW-PTP) and then successfully employed in the SBVS against protein tyrosine phosphatase receptor type O (PTPRO), a potential therapeutic target for various diseases. The most potent hit compound GP03 showed an IC50 value of 2.89 µM for PTPRO and possessed a certain degree of selectivity toward other protein phosphatases. Importantly, we also found that neglecting the ligand energy penalty upon binding partially accounts for the false positive SBVS hits. The preliminary structure-activity relationships of GP03 analogs are also reported.

Allosteric modulation of the catalytic VYD loop in Slingshot by its N-terminal domain underlies both Slingshot auto-inhibition and activation.

Slingshots are phosphatases that modulate cytoskeleton dynamics, and their activities are tightly regulated in different physiological contexts. Recently, abnormally elevated Slingshot activity has been implicated in many human diseases, such as cancer, Alzheimer's disease, and vascular diseases. Therefore, Slingshot-specific inhibitors have therapeutic potential. However, an enzymological understanding of the catalytic mechanism of Slingshots and of their activation by actin is lacking. Here, we report that the N-terminal region of human Slingshot2 auto-inhibits its phosphatase activity in a noncompetitive manner. pH-dependent phosphatase assays and leaving-group dependence studies suggested that the N-terminal domain of Slingshot2 regulates the stability of the leaving group of the product during catalysis by modulating the general acid Asp361 in the catalytic VYD loop. F-actin binding relieved this auto-inhibition and restored the function of the general acid. Limited tryptic digestion and biophysical studies identified large conformational changes in Slingshot2 after the F-actin binding. The dissociation of N-terminal structural elements, including Leu63, and the exposure of the loop between α-helix-2 and ß-sheet-3 of the phosphatase domain served as the structural basis for Slingshot activation via F-actin binding in vitro and via neuregulin stimulation in cells. Moreover, we designed a FlAsH-BRET-based Slingshot2 biosensor whose readout was highly correlated with the in vivo phosphatase activities of Slingshot2. Our results reveal the auto-inhibitory mechanism and allosteric activation mechanisms of a human Slingshot phosphatase. They also contribute to the design of new strategies to study Slingshot regulation in various cellular contexts and to screen for new activators/inhibitors of Slingshot activity.

Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

The protein tyrosine phosphatase nonreceptor type 12 (PTPN12) is a multifunctional protein and has elicited much research attention because its decreased protein level has been associated with poor prognosis of several types of cancers. Recently, we have solved the crystal structure of the phosphatase domain of PTPN12, which disclosed a specific PTPN12-insert-loop harboring a cyclin-dependent kinase 2 (CDK2) phosphorylation site. However, the functional significance of this phosphorylation is undefined. In the present study, we found that S19 site phosphorylation of PTPN12 by CDK2 discharged its antitumor activity by down-regulation of its inhibitory role in cell migration, but not affecting its other regulatory functions. Phosphorylation of PTPN12 at the S19 site changed its substrate interface, and by doing so, selectively decreased its activity toward the human epidermal growth factor receptor 2 (HER2)- pY1196 site, but not other HER2 phosphorylation sites or other known PTPN12 substrates. A further in-depth mechanism study revealed that the phosphorylation of PTPN12 by CDK2 impaired recruitment of the serine/threonine-protein kinase 1 (PAK1) to HER2, resulted in the blockade of the HER2-pY1196-PAK1-T423 signaling pathway, thus increased tumor cell motility. Taken together, our results identified a new phosphorylation-based substrate recognition mechanism of PTPN12 by CDK2, which orchestrated signaling crosstalk between the oncogenic CDK2 and HER2 pathways. The newly identified governing mechanism of the substrate selectivity of a particular phosphatase was previously unappreciated and exemplifies how a phospho-network is precisely controlled in different cellular contexts.-Li, H., Yang, D., Ning, S., Xu, Y., Yang, F., Yin, R., Feng, T., Han, S., Guo, L., Zhang, P., Qu, W., Guo, R., Song, C., Xiao, P., Zhou, C., Xu, Z., Sun, J.-P., Yu, X. Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

Discovery of novel FFA4 (GPR120) receptor agonists with ß-arrestin2-biased characteristics.

BACKGROUND: Free fatty acid 4 (FFA4) (GPR120) receptor functions as a receptor for unsaturated long-chain free fatty acids by regulating the secretion of glucagon-like peptide-1 and suppressing the inflammatory process, in which these two distinct biological functions are modulated by two signaling pathways, Gq and ß-arrestin2, respectively. RESULTS: By using pharmacophore modeling and virtual screening methods, several compounds are found with excellent activities for agonizing FFA4 receptor. It needs to be noted that among them, some molecules demonstrate appealing ß-arrestin2-biased properties for the FFA4 receptor. CONCLUSION: These compounds may serve as the useful toolkits for detecting differential biased mechanism and developing new candidate therapeutic agents of the FFA4 receptor.

Identification of para-Substituted Benzoic Acid Derivatives as Potent Inhibitors of the Protein Phosphatase Slingshot.

Slingshot proteins form a small group of dual-specific phosphatases that modulate cytoskeleton dynamics through dephosphorylation of cofilin and Lim kinases (LIMK). Small chemical compounds with Slingshot-inhibiting activities have therapeutic potential against cancers or infectious diseases. However, only a few Slingshot inhibitors have been investigated and reported, and their cellular activities have not been examined. In this study, we identified two rhodanine-scaffold-based para-substituted benzoic acid derivatives as competitive Slingshot inhibitors. The top compound, (Z)-4-((4-((4-oxo-2-thioxo-3-(o-tolyl)thiazolidin-5-ylidene)methyl)phenoxy)methyl)benzoic acid (D3) had an inhibition constant (Ki) of around 4 µm and displayed selectivity over a panel of other phosphatases. Moreover, compound D3 inhibited cell migration and cofilin dephosphorylation after nerve growth factor (NGF) or angiotensin II stimulation. Therefore, our newly identified Slingshot inhibitors provide a starting point for developing Slingshot-targeted therapies.

The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes.

The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY(1112), the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12.