Apoptin induces pyroptosis of colorectal cancer cells via the GSDME-dependent pathway

Apoptin is a small molecular weight protein encoded by the VP3 gene of chicken anemia virus (CAV). It can induce apoptosis of tumor cells and play anti-tumorigenic functions. In this study, we identified a time-dependent inhibitory role of apoptin on the viability of HCT116 cells. We also demonstrated that apoptin induces pyroptosis through cleaved caspase 3, and with a concomitant cleavage of gasdermin E (GSDME) rather than GSDMD. GSDME knockdown switched the apoptin-induced cell death from pyroptosis to apoptosis in vitro. Furthermore, we demonstrated that the effect of apoptin on GSDME-dependent pyroptosis could be mitigated by caspase-3 and caspase-9 siRNA knockdown. Additionally, apoptin enhanced the intracellular reactive oxygen species (ROS), causing aggregation of the mitochondrial membrane protein Tom20. Moreover, bax and cytochrome c were released to the activating caspase-9, eventually triggering pyroptosis. Therefore, GSDME mediates the apoptin-induced pyroptosis through the mitochondrial apoptotic pathway. Finally, using nude mice xenografted with HCT116 cells, we found that apoptin induces pyroptosis and significantly inhibits tumor growth. Based on this mechanism, apoptin may provide a new strategy for colorectal cancer therapy.     

Epac‐2 ameliorates spontaneous colitis in Il‐10 −/− mice by protecting the intestinal barrier and suppressing NF‐κB/MAPK signalling

Intestinal barrier dysfunction and intestinal inflammation interact in the progression of Crohn''s disease (CD). A recent study indicated that Epac‐2 protected the intestinal barrier and had anti‐inflammatory effects. The present study examined the function of Epac‐2 in CD‐like colitis. Interleukin‐10 gene knockout (Il‐10 ^−/−) mice exhibit significant spontaneous enteritis and were used as the CD model. These mice were treated with Epac‐2 agonists (Me‐cAMP) or Epac‐2 antagonists (HJC‐0350) or were fed normally (control), and colitis and intestinal barrier structure and function were compared. A Caco‐2 and RAW 264.7 cell co‐culture system were used to analyse the effects of Epac‐2 on the cross‐talk between intestinal epithelial cells and inflammatory cells. Epac‐2 activation significantly ameliorated colitis in mice, which was indicated by reductions in the colitis inflammation score, the expression of inflammatory factors and intestinal permeability. Epac‐2 activation also decreased Caco‐2 cell permeability in an LPS‐induced cell co‐culture system. Epac‐2 activation significantly suppressed nuclear factor (NF)‐κB/mitogen‐activated protein kinase (MAPK) signalling in vivo and in vitro. Epac‐2 may be a therapeutic target for CD based on its anti‐inflammatory functions and protective effects on the intestinal barrier.     

LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110–CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110–CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.     

Design,synthesis, and evaluation of chalcone-Vitamin E-donepezil hybrids as multi-target-directed ligands for the treatment of Alzheimer’s disease

A novel series of chalcone-Vitamin E-donepezil hybrids was designed and developed based on multitarget-directed ligands (MTDLs) strategy for treating Alzheimer’s disease (AD). The biological results revealed that compound 17f showed good AChE inhibitory potency (ratAChE IC₅₀ = 0.41 µM; eeAChE IC₅₀ = 1.88 µM). Both the kinetic analysis and docking study revealed that 17f was a mixed type AChE inhibitor. 17f was also a good antioxidant (ORAC = 3.3 eq), selective metal chelator and huMAO-B inhibitor (IC₅₀ = 8.8 µM). Moreover, it showed remarkable inhibition of self- and Cu²⁺-induced Aβ_1–42 aggregation with a 78.0 and 93.5% percentage rate at 25 µM, respectively, and disassembled self-induced and Cu²⁺-induced aggregation of the accumulated Aβ_1–42 fibrils with 72.3 and 84.5% disaggregation rate, respectively. More importantly, 17f exhibited a good neuroprotective effect on H₂O₂-induced PC12 cell injury and presented good blood-brain barrier permeability in vitro. Thus, 17f was a promising multi-target-directed ligand for treating AD.     

CPEB3 deficiency in mice affect ovarian follicle development and causes premature ovarian insufficiency

Premature ovarian insufficiency (POI) is a heterogeneous and multifactorial disorder. In recent years, there has been an increasing interest in research on the pathogenesis and treatment of POI, owing to the implementation of the second-child policy in China. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that can bind to specific RNA sequences. CPEB3 can bind to and affect the expression, cellular location, and stability of target RNAs. Cpeb3 is highly expressed in the ovary; however, its functions remain unknown. In this study, Cpeb3-mutant mice were used to characterize the physiological functions of CPEB3. Cpeb3-mutant female mice manifested signs of gradual loss of ovarian follicles, ovarian follicle development arrest, increased follicle atresia, and subfertility with a phenotype analogous to POI in women. Further analysis showed that granulosa cell proliferation was inhibited and apoptosis was markedly increased in Cpeb3-mutant ovaries. In addition, the expression of Gdf9, a potential target of CPEB3, was decreased in Cpeb3-mutant ovaries and oocytes. Altogether, these results reveal that CPEB3 is essential for ovarian follicle development and female fertility as it regulates the expression of Gdf9 in oocytes, disruption of which leads to impaired ovarian follicle development and POI.Subject terms: RNA-binding proteins, Infertility     

DNA methylation and single-nucleotide polymorphisms in DDX58 are associated with hand,foot and mouth disease caused by enterovirus 71

BackgroundThis research aimed to explore the association between the RIG-I-like receptor (RIG-I and MDA5 encoded by DDX58 and IFIH1, respectively) pathways and the risk or severity of hand, foot, and mouth disease caused by enterovirus 71 (EV71-HFMD). In this context, we explored the influence of gene methylation and polymorphism on EV71-HFMD.Methodology/Principal findings60 healthy controls and 120 EV71-HFMD patients, including 60 mild EV71-HFMD and 60 severe EV71-HFMD patients, were enrolled. First, MiSeq was performed to explore the methylation of CpG islands in the DDX58 and IFIH1 promoter regions. Then, DDX58 and IFIH1 expression were detected in PBMCs using RT-qPCR. Finally, imLDR was used to detect DDX58 and IFIH1 single-nucleotide polymorphism (SNP) genotypes. Severe EV71-HFMD patients exhibited higher DDX58 promoter methylation levels than healthy controls and mild EV71-HFMD patients. DDX58 promoter methylation was significantly associated with severe HFMD, sex, vomiting, high fever, neutrophil abundance, and lymphocyte abundance. DDX58 expression levels were significantly lower in mild patients than in healthy controls and lower in severe patients than in mild patients. Binary logistic regression analysis revealed statistically significant differences in the genotype frequencies of DDX58 rs3739674 between the mild and severe groups. GeneMANIA revealed that 19 proteins displayed correlations with DDX58, including DHX58, HERC5, MAVS, RAI14, WRNIP1 and ISG15, and 19 proteins displayed correlations with IFIH1, including TKFC, IDE, MAVS, DHX58, NLRC5, TSPAN6, USP3 and DDX58.Conclusions/SignificanceDDX58 expression and promoter methylation were associated with EV71 infection progression, especially in severe EV71-HFMD patients. The effect of DDX58 in EV71-HFMD is worth further attention.     

Strigo-D2—a bio-sensor for monitoring spatio-temporal strigolactone signaling patterns in intact plants

Strigolactones (SLs) are a class of plant hormones that mediate biotic interactions and modulate developmental programs in response to endogenous and exogenous stimuli. However, a comprehensive view on the spatio-temporal pattern of SL signaling has not been established, and tools for a systematic in planta analysis do not exist. Here, we present Strigo-D2, a genetically encoded ratiometric SL signaling sensor that enables the examination of SL signaling distribution at cellular resolution and is capable of rapid response to altered SL levels in intact Arabidopsis (Arabidopsis thaliana) plants. By monitoring the abundance of a truncated and fluorescently labeled SUPPRESSOR OF MAX2 1-LIKE 6 (SMXL6) protein, a proteolytic target of the SL signaling machinery, we show that all cell types investigated have the capacity to respond to changes in SL levels but with very different dynamics. In particular, SL signaling is pronounced in vascular cells but low in guard cells and the meristematic region of the root. We also show that other hormones leave Strigo-D2 activity unchanged, indicating that initial SL signaling steps work in isolation from other hormonal signaling pathways. The specificity and spatio-temporal resolution of Strigo-D2 underline the value of the sensor for monitoring SL signaling in a broad range of biological contexts with highly instructive analytical depth.

Strigo-D2 is a genetically encoded sensor visualizing spatio-temporal patterns of strigolactone signaling levels in intact plants based on the activity ratio of two fluorescent marker proteins.     

The Role of Autophagy in Lamellar Body Formation and Surfactant Production in Type 2 Alveolar Epithelial Cells

The lamellar body (LB), a concentric structure loaded with surfactant proteins and phospholipids, is an organelle specific to type 2 alveolar epithelial cells (AT2). However, the origin of LBs has not been fully elucidated. We have previously reported that autophagy regulates Weibel-Palade bodies (WPBs) formation, and here we demonstrated that autophagy is involved in LB maturation, another lysosome-related organelle. We found that during development, LBs were transformed from autophagic vacuoles containing cytoplasmic contents such as glycogen. Fusion between LBs and autophagosomes was observed in wild-type neonate mice. Moreover, the markers of autophagic activity, microtubule-associated protein 1 light chain 3B (LC3B), largely co-localized on the limiting membrane of the LB. Both autophagy-related gene 7 (Atg7) global knockout and conditional Atg7 knockdown in AT2 cells in mice led to defects in LB maturation and surfactant protein B production. Additionally, changes in autophagic activity altered LB formation and surfactant protein B production. Taken together, these results suggest that autophagy plays a critical role in the regulation of LB formation during development and the maintenance of LB homeostasis during adulthood.     

RAB31 marks and controls an ESCRT-independent exosome pathway

Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.Subject terms: Small GTPases, Endosomes, Multivesicular bodies, Lysosomes, ESCRT