A divergent synthetic strategy for generating helical p53 peptides bearing functionalised staple linkages, allowing for efficient optimisation of cellular activity.
An extensive series of histological sections reflecting the various states of normal breast tissue, and a range of benign and malignant lesions, were examined for the expression of the p53 protein using a panel of anti-p53 antibodies. In 2 separate series the results of using frozen or methacarn-fixed, paraffin-embedded sections were compared. Strong positive staining for p53 was detected in over 50% of the malignant lesions when frozen sections were used. This number fell to just over 20% when methacarn-fixed sections were examined. In neither series was any p53 staining seen in normal breast or in the benign lesions. Studies by Western blotting on breast cell lines confirmed that this histological signal is due to a pronounced over-expression of the p53 protein. Earlier studies show that this over-expression is associated with mutation of the p53 gene. Mutation of the p53 gene with over-expression of the mutant protein is therefore one of the most frequent specific genetic changes in malignant breast cancer.
These results demonstrate that a p16-derived peptide can mediate three of the known functions of p16: firstly, it interacts with cdk4 and cdk6; secondly, it inhibits pRb phosphorylation in vitro and in vivo; and thirdly, it blocks entry into S phase. The fact that one small synthetic peptide can enter the cells directly from the tissue culture medium to inhibit pRb phosphorylation and block cell-cycle progression makes this an attractive approach for future peptidometic drug design. Our results suggest a novel and exciting means by which the function of the p16 suppressor gene can be restored in human tumours.
Using cell-autonomous markers we have traced the origins of prespore cells and two types of prestalk cells (pstA and pstB cells) during slug formation. We show that cell sorting and positional information both contribute to Dictyostelium morphogenesis. The initial pattern established at the mound stage is topologically quite different from that of the slug. Confirming previous studies, we find that prespore cells occupy most of the aggregate but are absent from a thin layer at the base and from the emerging tip. PstB cells are almost entirely localized to the basal region during the early stages of tip formation. Thus prespore and pstB cell differentiation appear to occur in response to localized morphogenetic signals. In the case of pstB cells, these signals presumably emanate from the base and not, as might be expected, from the tip. When first detectable, pstA cells are scattered throughout the aggregate. They then appear to migrate to the apex, where the tip forms.
Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)—engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.
The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus Elb protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.
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