Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.
Abstract Background Historically, malaria due to Plasmodium vivax has been epidemic in Henan Province, China, with Anopheles sinensis as the main vector. The most effective measures to prevent malaria transmission are based on vector control through the use of insecticides. However, insecticides exert a strong selective pressure on mosquito populations for insecticide resistance. The aim of this study was to investigate the susceptibility profile and population genetic characteristics of An. sinensis to provide basic data and scientific guidance for the study of resistance mechanisms and the control of An. sinensis in Henan Province. Methods Adult Anopheles mosquitoes were collected at sites near local farmers' sheepfolds, pigsties and/or cowsheds located in Pingqiao, Xiangfu, Xiangcheng and Tanghe counties/districts of Henan Province during July–September 2021 for insecticide susceptibility testing. Molecular identification of collected mosquitoes as belonging to genus Anopheles was by PCR, and the frequencies of mutations in the knockdown resistance gene ( kdr ) and acetylcholinesterase-1 gene ( ace-1 ) were detected using gene amplification. The mitochondrial DNA cytochrome oxidase subunit I (COI) gene was amplified in deltamethrin-resistant and deltamethrin-sensitive mosquitoes to analyze the genetic evolutionary relationship. Results A total of 1409 Anopheles mosquitoes were identified by molecular identification, of which 1334 (94.68%) were An. sinensis , 28 (1.99%) were An. yatsushiroensis , 43 (3.05%) were An. anthropophagus and four (0.28%) were An. belenrae . The 24-h mortality rates of An. sinensis in Pingqiao, Tanghe, Xiangcheng and Xiangfu counties/districts exposed to deltamethrin were 85.85%, 25.38%, 29.73% and 7.66%, respectively; to beta-cyfluthrin, 36.24%, 70.91%, 34.33% and 3.28%, respectively; to propoxur, 68.39%, 80.60%, 37.62% and 9.29%, respectively; and to malathion, 97.43%, 97.67%, 99.21% and 64.23%, respectively. One mutation, G119S, was detected in the ace-1 gene. The frequencies of the main genotypes were 84.21% of specimens collected in Xiangfu (G/S), 90.63% of speciments collected in Xiangcheng (G/G) and 2.44% of speciments collected in Tanghe (S/S). Significantly higher G119S allele frequencies were observed in both propoxur- and malathion-resistant mosquitoes than in their sensitive counterparts in the Tanghe population ( P < 0.05). Three mutations, L1014F (41.38%), L1014C (9.15%) and L1014W (0.12%), were detected in the kdr gene. The genotypes with the highest frequency in the populations of An. sinensis in Xiangfu and Tanghe were the mutant TTT (F/F) and wild-type TTG (L/L), at 67.86% (57/84) and 74.29% (52/70), respectively. In Pingqiao and Xiangfu, higher frequencies of the L1014F allele and lower frequencies of the L1014C allele were observed in mosquitoes resistant for beta-cyfluthrin than in those which were sensitive for this insecticide ( P < 0.05). The results of Tajima's D and of Fu and Li's D and F were not significantly negative ( P > 0.10), and each haplotype was interlaced and did not form two distinct branches. Conclusions High resistance to pyrethroids and propoxur was observed at four sites, but the resistance to malathion varied according to the location. Anopheles belenrae and the L1014W (TGG) mutation in An. sinensis were first discovered in Henan Province. The deltamethrin-resistant and deltamethrin-sensitive mosquito populations showed no genetic differentiation. The generation of resistance might be the result of the combination of multiple factors. Graphical Abstract
Dysregulated lineage commitment of mesenchymal stem cells (MSCs) contributes to impaired bone formation and an imbalance between adipogenesis and osteogenesis during skeletal aging and osteoporosis. The intrinsic cellular mechanism that regulates MSC commitment remains unclear. Here, we identified Cullin 4B (CUL4B) as a critical regulator of MSC commitment. CUL4B is expressed in bone marrow MSCs (BMSCs) and downregulated with aging in mice and humans. Conditional knockout of Cul4b in MSCs resulted in impaired postnatal skeletal development with low bone mass and reduced bone formation. Moreover, depletion of CUL4B in MSCs aggravated bone loss and marrow adipose accumulation during natural aging or after ovariectomy. In addition, CUL4B deficiency in MSCs reduced bone strength. Mechanistically, CUL4B promoted osteogenesis and inhibited adipogenesis of MSCs by repressing KLF4 and C/EBPδ expression, respectively. The CUL4B complex directly bound to Klf4 and Cebpd and epigenetically repressed their transcription. Collectively, this study reveals CUL4B-mediated epigenetic regulation of the osteogenic or adipogenic commitment of MSCs, which has therapeutic implications in osteoporosis.
Abnormal methylation of secreted frizzled‑related proteins (SFRPs) has been observed in various human cancer types. The loss of SFRP gene expression induces the activation of the Wnt pathway and is a vital mechanism for tumorigenesis and development. The aim of the present systematic review was to assess the association between SFRP methylation and cancer risk. A meta‑analysis was systematically conducted to assess the clinicopathological significance of SFRP methylation in cancer risk. The Cochrane Library, PubMed and Web of Science databases were comprehensively searched, and 83 publications with a total of 21,612 samples were selected for the meta‑analysis. The pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to evaluate the degree of associations between SFRP promoter methylation and cancer risk. Subgroup analysis, meta regression and sensitivity analysis were used to identify the potential sources of heterogeneity. SFRP1, SFRP2, SFRP4 and SFRP5 hypermethylation was significantly associated with cancer risk, with ORs of 8.48 (95% CI, 6.26‑11.49), 8.21 (95% CI, 6.20‑10.88), 11.41 (95% CI, 6.42‑20.30) and 6.34 (95% CI, 3.86‑10.42), respectively. SFRP2 methylation was significantly associated with differentiation in colorectal cancer (OR, 2.16; 95% CI, 1.02‑4.56). The results of the present study demonstrated that SFRP methylation may contribute to carcinogenesis, especially in certain cancer types, including hepatocellular carcinoma and colorectal cancer.
The secreted frizzled related proteins (SFRPs) are extracellular inhibitors of WNT pathway signaling. Methyl‑CpG binding domain protein 2 (MBD2) and enhancer of zeste homolog 2 (EZH2) are core members of the methylated DNA binding domain (MBD) and polycomb group (PcG) protein families for epigenetic regulation, respectively. This study aimed to ascertain the potential role of MBD2 and EZH2 proteins in colorectal cancer (CRC) and its effects on the expression of SFRP. Bioinformatics, real‑time quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of MBD2, EZH2, and SFRP in CRC cell lines and tissues. The functions of MBD2 and EZH2 in regards to cell proliferation, cell cycle distribution, apoptosis and invasion were examined in CRC cell lines. Methylation‑specific PCR (MSP) was used to detect the methylation status of the SFRP promoter. The results revealed that the mRNA expression levels of SFRP were significantly decreased in CRC tissues and cell lines compared to these levels in the adjacent tissues and NCM460, respectively. However, the mRNA levels of EZH2 and MBD2 genes were highly expressed in CRC cell lines. We found that reducing MBD2 and EZH2 expression together remarkably inhibited and decreased the proliferation, migration and invasion abilities of the CRC cell lines compared to reducing one of each. Flow cytometric analysis showed that knockdown of MBD2 and EZH2 together in CRC affected cell apoptosis and the cell cycle progression more effectively than knockdown of one of each. The mRNA expression of SFRP1 was reactivated by silencing of MBD2 but not EZH2 in SW480 and HCT116 cells. SFRP4 and SFRP5 mRNA expression was reactivated by silencing of EZH2 but not MBD2 only in SW480 cells. However, depletion of both MBD2 and EZH2 restored SFRP1, SFRP2, SFRP4, and SFRP5 mRNA expression more effectively in CRC cells. Interestingly, there was no significant change in the methylation status of SFRP1, SFRP2, SFRP4, and SFRP5 gene promoter between before and after interference with MBD2, EZH2, and both. In conclusion, our results suggest that silencing of MBD2 and EZH2 simultaneously was able to rescue the expression of SFRP and inhibit the proliferation of CRC cells more effectively. However, the underlying regulatory mechanism system of MBD2 and EZH2 for SFRP in CRC requires further research.
Transcriptional elongation is a universal and critical step during gene expression. The super elongation complex (SEC) regulates the rapid transcriptional induction by mobilizing paused RNA polymerase II (Pol II). Dysregulation of SEC is closely associated with human diseases. However, the physiological role of SEC during development and homeostasis remains largely unexplored. Here we studied the function of SEC in adipogenesis by manipulating an essential scaffold protein AF4/FMR2 family member 4 (AFF4), which assembles and stabilizes SEC. Knockdown of AFF4 in human mesenchymal stem cells (hMSCs) and mouse 3T3-L1 preadipocytes inhibits cellular adipogenic differentiation. Overexpression of AFF4 enhances adipogenesis and ectopic adipose tissue formation. We further generate Fabp4-cre driven adipose-specific Aff4 knockout mice and find that AFF4 deficiency impedes adipocyte development and white fat depot formation. Mechanistically, we discover AFF4 regulates autophagy during adipogenesis. AFF4 directly binds to autophagy-related protein ATG5 and ATG16L1, and promotes their transcription. Depleting ATG5 or ATG16L1 abrogates adipogenesis in AFF4-overepressing cells, while overexpression of ATG5 and ATG16L1 rescues the impaired adipogenesis in Aff4-knockout cells. Collectively, our results unveil the functional importance of AFF4 in regulating autophagy and adipogenic differentiation, which broaden our understanding of the transcriptional regulation of adipogenesis.
The Keap1-Nrf2-ARE pathway regulates the constitutive and inducible transcription of various genes that encode detoxification enzymes, antioxidant proteins and anti-inflammatory proteins and has pivotal roles in the defence against cellular oxidative stress. In this study, we investigated the therapeutic potential of CPUY192018, a potent small-molecule inhibitor of the Keap1-Nrf2 protein-protein interaction (PPI), in renal inflammation. In human proximal tubular epithelial HK-2 cells, CPUY192018 treatment significantly increased Nrf2 protein level and Nrf2 nuclear translocation, which enhanced Nrf2-ARE transcription capacity and the downstream protein content in a Nrf2 dependent manner. In lipopolysaccharide (LPS)-challenged human HK-2 cells, CPUY192018 exhibited cytoprotective effects by enhancing the Nrf2-ARE regulated antioxidant system and diminished the LPS-induced inflammatory response by hindering the ROS-mediated activation of the NF-κB pathway. In the LPS-induced mouse model of chronic renal inflammation, by activating Nrf2, CPUY192018 treatment balanced renal oxidative stress and suppressed inflammatory responses. Hence, administration of CPUY192018 reduced kidney damage and ameliorated pathological alterations of the glomerulus. Taken together, our study suggested that small-molecule Keap1-Nrf2 PPI inhibitors can activate the Nrf2-based cytoprotective system and protect the kidney from inflammatory injury, raising a potential application of Keap1-Nrf2 PPI inhibitors in the treatment of inflammatory kidney disorders.
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