The SWI/SNF complex plays an important role in mouse embryonic stem cells (mESCs), but it remains to be determined whether this complex is required for the pluripotency of human ESCs (hESCs). Using RNAi, we demonstrated that depletion of BRG1, the catalytic subunit of the SWI/SNF complex, led to impaired self-renewing ability and dysregulated lineage specification of hESCs. A unique composition of the BRG1-SWI/SNF complex in hESCs was further defined by the presence of BRG1, BAF250A, BAF170, BAF155, BAF53A, and BAF47. Genome-wide expression analyses revealed that BRG1 participated in a broad range of biological processes in hESCs through pathways different from those in mESCs. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) demonstrated that BRG1 played a repressive role in transcriptional regulation by modulating the acetylation levels of H3K27 at the enhancers of lineage-specific genes. Our data thus provide valuable insights into molecular mechanisms by which transcriptional repression affects the self-renewal and differentiation of hESCs.
Endoplasmic reticulum-associated degradation (ERAD) maintains protein homeostasis by retrieving misfolded proteins from the endoplasmic reticulum (ER) lumen into the cytosol for degradation. The retrotranslocation of misfolded proteins across the ER membrane is an energy-consuming process, with the detailed transportation mechanism still needing clarification. We determined the cryo-EM structures of the hetero-decameric complex formed by the Derlin-1 tetramer and the p97 hexamer. It showed an intriguing asymmetric complex and a putative coordinated squeezing movement in Derlin-1 and p97 parts. With the conformational changes of p97 induced by its ATP hydrolysis activities, the Derlin-1 channel could be torn into a "U" shape with a large opening to the lipidic environment, thereby forming an entry for the substrates in the ER membrane. The EM analysis showed that p97 formed a functional protein complex with Derlin-1, revealing the coupling mechanism between the ERAD retrotranslocation and the ATP hydrolysis activities.
Endoplasmic-reticulum-associated protein degradation
Background/Objectives: In chronic hepatitis B infection (CHB), the hepatitis B surface antigen (HBsAg) continuously exhausts the hepatitis B surface antibody (HBsAb), which leads to the formation of immune tolerance. Accordingly, the hepatitis B virus (HBV) infection can be blocked by inhibiting the binding of the hepatitis B surface pre-S1/pre-S2 antigen to the hepatocyte receptor NTCP, but the clinical cure rate of pre-S-based vaccines for CHB is limited. Methods: In this study, we designed and prepared multivalent hepatitis B therapeutic mRNA vaccines encoding three hepatitis B surface antigen proteins (L, M, and S) at the cell membrane, verified via in vitro transfection and expression experiments. An in vivo immunization experiment in HBV transgenic (Tg) mice was first completed. Subsequently, an adeno-associated virus plasmid vector carrying the HBV1.2-fold genome (pAAV HBV1.2) model and the adeno-associated virus vector carrying HBV1.3-fold genome (rAAV HBV1.3) model were constructed and immunized with mRNA vaccines. The HBV antigen, antibodies, and HBV DNA in serum were detected. Indirect (enzyme-linked immunosorbent assay) ELISA were made to analyze the activated antigen-specific IgG in HBV Tg mice. Antigen-dependent T-cell activation experiments were carried out, as well as the acute toxicity tests in mice. Results: The L protein/pre-S antigens could be stably presented at the cell membrane with the support of the S protein (and M protein). After vaccinations, the vaccines effectively reactivated the production of high levels of HBsAb, disrupted immune tolerance, and activated the production of high-affinity antibodies against structural pre-S antigen in HBV Tg mice. The HBsAg seroconversion and serum HBV DNA clearance were achieved in two HBV mice models. Furthermore, pre-S antigen-dependent T-cell response against HBV infection was confirmed. The therapeutic vaccine also showed safety in mice. Conclusions: A novel therapeutic mRNA vaccine was developed to break through HBsAg-mediated immune tolerance and treat CHB by stably presenting the pre-S antigen at the membrane, and the vaccine has great potential for the functional cure of CHB.
Sodium excretion, a critical process in sodium homeostasis, occurs in many tissues, including the kidney and intestine. Unlike in the kidney, the hormonal regulation of intestinal sodium excretion remains unclear. Atrial natriuretic peptide (ANP) is a crucial hormone in renal natriuresis. Corin is a protease critical for ANP activation. Corin and ANP are expressed mainly in the heart. In this study, we investigated corin, ANP, and natriuretic peptide receptor A (Npra) expression in mouse intestines. Corin and ANP expression was co-localized in enteroendocrine cells, whereas Npra expression was on the luminal epithelial cells. In Corin knockout (KO) mice, fecal Na+ and Cl- excretion decreased compared with that in wild-type (WT) mice. Such a decrease was not found in conditional Corin KO mice lacking cardiac corin selectively. In kidney conditional Corin KO mice lacking renal corin, fecal Na+ and Cl- excretion increased, compared to that in WT mice. When WT, Corin KO, and the kidney conditional KO mice were treated with aldosterone, the differences in fecal Na+ and Cl- levels disappeared. These results suggest that intestinal corin may promote fecal sodium excretion in a paracrine mechanism independent of the cardiac corin function. The increased fecal sodium excretion in the kidney conditional Corin KO mice likely reflected an intestinal compensatory response to renal corin deficiency. Our results also suggest that intestinal corin activity may antagonize aldosterone action in the promotion of fecal sodium excretion. These findings help us understand the hormonal mechanism controlling sodium excretion the intestinal tract.
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