Disruption of epithelium integrity by inflammation-associated fibroblasts through prostaglandin signaling.

Inflammation-associated fibroblasts (IAFs) are associated with progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial cells is unknown. Here, we developed an in vitro model whereby human colon fibroblasts are induced by specific cytokines and recapitulate key features of IAFs in vivo. When cocultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid expansion and barrier disruption due to swelling and rupture of individual epithelial cells. Colonoids cocultured with IAFs also show increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated by a paracrine pathway involving prostaglandin E2 and its receptor EP4, leading to protein kinase A -dependent activation of the cystic fibrosis transmembrane conductance regulator. EP4-specific chemical inhibitors effectively prevented IAF-induced colonoid swelling and restored normal proliferation and genome stability. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a therapeutic avenue to mitigate inflammation-associated epithelial injury.

Differential effects of aneuploidy on growth and differentiation in human intestinal stem cells.

Aneuploidy, a near ubiquitous genetic feature of tumors, is a context-dependent driver of cancer evolution; however, the mechanistic basis of this role remains unclear. Here, by inducing heterogeneous aneuploidy in non-transformed human colon organoids (colonoids), we investigate how the effects of aneuploidy on cell growth and differentiation may promote malignant transformation. Single-cell RNA sequencing reveals that the gene expression signature across over 100 unique aneuploid karyotypes is enriched with p53 responsive genes. The primary driver of p53 activation is karyotype complexity. Complex aneuploid cells with multiple unbalanced chromosomes activate p53 and undergo G1 cell-cycle arrest, independent of DNA damage and without evidence of senescence. By contrast, simple aneuploid cells with 1-3 chromosomes gained or lost continue to proliferate, demonstrated by single cell tracking in colonoids. Notably, simple aneuploid cells exhibit impaired differentiation when niche factors are withdrawn. These findings suggest that while complex aneuploid cells are eliminated from the normal epithelium due to p53 activation, simple aneuploid cells can escape this checkpoint and may contribute to niche factor-independent growth of cancer-initiating cells.

Disruption of Epithelium Integrity by Inflammation-Associated Fibroblasts through Prostaglandin Signaling: IAFs disrupt colon epithelium via PGE2-EP4.

Inflammation-associated fibroblasts (IAFs) are associated with the progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial function and architecture is unknown. In this study, we developed an in vitro model whereby human colon fibroblasts are induced to become IAFs by specific cytokines and recapitulate key features of IAFs in vivo. When co-cultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid swelling and barrier disruption due to swelling and rupture of individual epithelial cells. Epithelial cells co-cultured with IAFs also exhibit increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated through a paracrine pathway involving prostaglandin E2 (PGE2) and the PGE2 receptor EP4, leading to PKA-dependent activation of the CFTR chloride channel. Importantly, EP4-specific chemical inhibitors effectively prevented colonoid swelling and restored normal proliferation and genome stability of IAF-exposed epithelial cells. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a potential treatment to mitigate inflammation-associated epithelial injury.

Publisher Correction: Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody.

The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein, a (gp120-gp41)3 trimer, mediates fusion of viral and host cell membranes after gp120 binding to host receptor CD4. Receptor binding triggers conformational changes allowing coreceptor (CCR5) recognition through CCR5's tyrosine-sulfated amino (N) terminus, release of the gp41 fusion peptide and fusion. We present 3.3 Å and 3.5 Å cryo-EM structures of E51, a tyrosine-sulfated coreceptor-mimicking antibody, complexed with a CD4-bound open HIV-1 native-like Env trimer. Two classes of asymmetric Env interact with E51, revealing tyrosine-sulfated interactions with gp120 mimicking CCR5 interactions, and two conformations of gp120-gp41 protomers (A and B protomers in AAB and ABB trimers) that differ in their degree of CD4-induced trimer opening and induction of changes to the fusion peptide. By integrating the new structural information with previous closed and open envelope trimer structures, we modeled the order of conformational changes on the path to coreceptor binding site exposure and subsequent viral-host cell membrane fusion.

Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis.

Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.