A human cancer-predisposing polymorphism in Cdc25A is embryonic lethal in the mouse and promotes ASK-1 mediated apoptosis

<p>Abstract</p> <p>Background</p> <p>Failure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-β-TrCP (SCF^β-TrCP) complex. This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur.</p> <p>Results</p> <p>We now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both <it>in vitro </it>and in cultured cells. It fails to interact with the apoptosis signal-regulating kinase 1 (ASK1), however, and therefore does not suppress ASK1-mediated apoptosis.</p> <p>Conclusions</p> <p>These data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis.</p

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.</p

Host Immune Responses to Surface S-Layer Proteins (SLPs) of Clostridioides difficile

Clostridioides difficile, a nosocomial pathogen, is an emerging gut pathobiont causing antibiotic-associated diarrhea. C. difficile infection involves gut colonization and disruption of the gut epithelial barrier, leading to the induction of inflammatory/immune responses. The expression of two major exotoxins, TcdA and TcdB is the major cause of C. difficile pathogenicity. Attachment of bacterial abundant cell wall proteins or surface S-layer proteins (SLPs) such as SlpA with host epithelial cells is critical for virulence. In addition to being toxins, these surface components have been shown to be highly immunogenic. Recent studies indicate that C. difficile SLPs play important roles in the adhesion of the bacteria to the intestinal epithelial cells, disruption of tight junctions, and modulation of the immune response of the host cells. These proteins might serve as new targets for vaccines and new therapeutic agents. This review summarizes our current understanding of the immunological role of SLPs in inducing host immunity and their use in the development of vaccines and novel therapeutics to combat C. difficile infection

Host Immune Responses to Surface S-Layer Proteins (SLPs) of <i>Clostridioides difficile</i>

Clostridioides difficile, a nosocomial pathogen, is an emerging gut pathobiont causing antibiotic-associated diarrhea. C. difficile infection involves gut colonization and disruption of the gut epithelial barrier, leading to the induction of inflammatory/immune responses. The expression of two major exotoxins, TcdA and TcdB is the major cause of C. difficile pathogenicity. Attachment of bacterial abundant cell wall proteins or surface S-layer proteins (SLPs) such as SlpA with host epithelial cells is critical for virulence. In addition to being toxins, these surface components have been shown to be highly immunogenic. Recent studies indicate that C. difficile SLPs play important roles in the adhesion of the bacteria to the intestinal epithelial cells, disruption of tight junctions, and modulation of the immune response of the host cells. These proteins might serve as new targets for vaccines and new therapeutic agents. This review summarizes our current understanding of the immunological role of SLPs in inducing host immunity and their use in the development of vaccines and novel therapeutics to combat C. difficile infection

Su(H)^WARE binding capacity to Hairless mutants.

<p>Yeast two-hybrid assay to test for binding activity of wild type Su(H), CTD and mutant CTD^WARE constructs with Hairless NTCT, NTCT^ΔNT Dand NTCT^LD; empty vector served as a negative control. Note the lack of binding of CTD^WARE with the ΔNT deletion or the NTCT^LD mutation.</p

In vivo influence of the Su(H) and Hairless protein variants on the Notch target <i>wingless</i>.

<p>Wingless (wg) protein is expressed along the dorso-ventral boundary of the wing imaginal disc as a result of Notch signalling (see arrow in lac-Z control) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027986#pone.0027986-DasBenjumea1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027986#pone.0027986-Rulifson1" target="_blank">[18]</a>. Wingless protein is shown in green in all panels. Su(H) and Hairless variants were overexpressed using the Gal4/UAS system within the central part of the wing disc (red in lac-Z control). Apart from the beta-galactosidase control, red depicts Su(H) protein. Hairless protein is shown in blue; overlap with Su(H) appears magenta and in addition with wg, it appears white. The control disc shows the expression domain (red, beta-galactosidase) of the <i>omb</i>-Gal4 driver. Wingless (Wg) protein outlines the wing pouch and dissects it along the dorso-ventral boundary (arrow). The latter expression is induced by a Notch signal. Overexpression of wild type Hairless (blue) represses Wg expression (blunt bar). In contrast, the mutant H^LD is unable to repress Wg. Overexpression of wild type Su(H) (red) causes proliferation of the wing blade and a subtle expansion of Wg expression. A combined overexpression of Su(H) and Hairless gives a super-additive effect: the expression domain becomes very small and Wg expression is inhibited. The mutant H^LD has no such effect; the combination with Su(H) resemble the sole Su(H) overexpression reflecting lack of binding of the two proteins. Overexpression of mutant Su(H)^WARE results also in a slight overproliferation of the wing disc and subtle Wg expansion. Also in combination with Hairless, Su(H)^WARE strongly impedes proliferation and Wg expression. However, no such effect of H^LD on Su(H)^WARE can be observed.</p