Neurogenin3 phosphorylation controls reprogramming efficiency of pancreatic ductal organoids into endocrine cells

β-cell replacement has been proposed as an effective treatment for some forms of diabetes, and in vitro methods for β-cell generation are being extensively explored. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of three regulators of pancreatic endocrine formation and β-cell identity, Ngn3, Pdx1 and MafA. Pancreatic ductal organoid cultures have recently been developed that can be expanded indefinitely, while maintaining the potential to differentiate into the endocrine lineage. Here, using mouse pancreatic ductal organoids, we see that co-expression of Ngn3, Pdx1 and MafA are required and sufficient to generate cells that express insulin and resemble β-cells transcriptome-wide. Efficiency of β-like cell generation can be significantly enhanced by preventing phosphorylation of Ngn3 protein and further augmented by conditions promoting differentiation. Taken together, our new findings underline the potential of ductal organoid cultures as a source material for generation of β-like cells and demonstrate that post-translational regulation of reprogramming factors can be exploited to enhance β-cell generation

Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes

© 2015 Elsevier Inc. Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming

The ASPP proteins complex and cooperate with p300 to modulate the transcriptional activity of p53.

Understanding how p53 is able to specifically respond to particular stress signals and regulate many different signalling pathways remains a challenge. Several studies have demonstrated that p53's interactions with different protein partners are essential for it to be able to coordinate specific responses. In particular, the apoptotic pathway is regulated by p53 in cooperation with the Apoptosis Stimulating Proteins of p53 (ASPP) proteins. In this study, we showed that the ASPP proteins are able to bind and cooperate with p300, a well defined co-factor of p53, to selectively regulate p53's transcriptional activity on promoters such as p53-inducible gene 3 but not on p21waf1. This is the first demonstration that the ASPPs can function together with p300 in regulating the transcriptional activity of p53

The ASPP proteins complex and cooperate with p300 to modulate the transcriptional activity of p53

Understanding how p53 is able to specifically respond to particular stress signals and regulate many different signalling pathways remains a challenge. Several studies have demonstrated that p53's interactions with different protein partners are essential for it to be able to coordinate specific responses. In particular, the apoptotic pathway is regulated by p53 in cooperation with the Apoptosis Stimulating Proteins of p53 (ASPP) proteins. In this study, we showed that the ASPP proteins are able to bind and cooperate with p300, a well defined co-factor of p53, to selectively regulate p53's transcriptional activity on promoters such as p53-inducible gene 3 but not on p21waf1. This is the first demonstration that the ASPPs can function together with p300 in regulating the transcriptional activity of p53. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

Mutation at Ser392 specifically sensitizes mutant p53H175 to mdm2-mediated degradation.

Mdm2 is one of the main E3 ubiquitin ligases, which targets both wild type and mutant p53 for degradation. The ability of post-translational modifications, such as phosphorylation, to modulate the function and stability of wild type p53 has been extensively studied. However, their ability to modulate the functions and stability of mutant forms of p53 remains poorly documented. Here we show, for the first time, that the stability of mutant p53 can be regulated by phosphorylation. Mutation of serine 392 to alanine shortens the half life of p53H175, and renders p53H175A392 more sensitive to mdm2-mediated degradation than p53H175. This effect of Ser392 phosphorylation specifically affects p53H175, a misfolded mutant, and does not affect p53W248 which maintains a native conformation. Detailed analysis subsequently showed that the reduced stability of p53H175A392 is not due to an increase in mdm2/p300 binding or polyubiquitin chain formation, uncoupling the extent of polyubiquitin chain formation and the stability of mutant p53. This is supported by the observation that Ser392 mutation enhances polyubiquitin chain formation on p53W248, without reducing its stability. These results suggest that the inhibition of phosphorylation at Ser392 of p53, together with the use of an mdm2-enhancing agent such as nutlin, could present a new therapeutic strategy with which to treat tumors expressing mutant p53H175

The N-terminus of a novel isoform of human iASPP is required for its cytoplasmic localization.

ASPP1 and ASPP2 are both proteins that interact with p53 and enhance its ability to induce apoptosis by selectively elevating the expression of proapoptotic p53-responsive genes. iASPP(RAI) is a third member of the family that is the most conserved inhibitor of p53-mediated apoptosis. Here, we have described iASPP, a longer form of iASPP(RAI), which at 828 amino acids is more than twice the size of iASPP(RAI). Using two antibodies that recognize both iASPP and iASPP(RAI), we report that this longer form of iASPP is the predominant form of the molecule expressed in cells. Like iASPP(RAI), iASPP also binds to p53 and inhibits apoptosis induced by p53 overexpression. However, whereas iASPP(RAI) is predominantly nuclear, the N-terminus of iASPP is entirely cytoplasmic, and the longer iASPP is located in both the cytoplasm and the nucleus. The effect upon subcellular localization of the longer N-terminus of iASPP means that this new, longer form of the molecule may be subject to greater regulation and provides another layer in the control of p53-induced apoptosis

Pharmacokinetic Study of Human Immunodeficiency Virus Protease Inhibitors Used in Combination with Amprenavir

In an open-label, randomized, multicenter, multiple-dose pharmacokinetic study, we determined the steady-state pharmacokinetics of amprenavir with and without coadministration of indinavir, nelfinavir, or saquinavir soft gel formulation in 31 human immunodeficiency virus type 1-infected subjects. The results indicated that amprenavir plasma concentrations were decreased by saquinavir soft gel capsule (by 32% for area under the concentration-time curve at steady state [AUC(ss)] and 37% for peak plasma concentration at steady state [C(max,ss)]) and increased by indinavir (33% for AUC(ss)). Nelfinavir significantly increased amprenavir minimum drug concentration at steady state (by 189%) but did not affect amprenavir AUC(ss) or C(max,ss). Nelfinavir and saquinavir steady-state pharmacokinetics were unchanged by coadministration with amprenavir compared with the historical monotherapy data. Concentrations of indinavir, coadministered with amprenavir, in plasma decreased in both single-dose and steady-state evaluations. The changes in amprenavir steady-state pharmacokinetic parameters, relative to those for amprenavir alone, were not consistent among protease inhibitors, nor were the changes consistent with potential interactions in CYP3A4 metabolism or P-glycoprotein transport. No dose adjustment of either protease inhibitor in any of the combinations studied is needed

Neurogenin3 phosphorylation controls reprogramming efficiency of pancreatic ductal organoids into endocrine cells

β-cell replacement has been proposed as an effective treatment for some forms of diabetes, and in vitro methods for β-cell generation are being extensively explored. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of three regulators of pancreatic endocrine formation and β-cell identity, Ngn3, Pdx1 and MafA. Pancreatic ductal organoid cultures have recently been developed that can be expanded indefinitely, while maintaining the potential to differentiate into the endocrine lineage. Here, using mouse pancreatic ductal organoids, we see that co-expression of Ngn3, Pdx1 and MafA are required and sufficient to generate cells that express insulin and resemble β-cells transcriptome-wide. Efficiency of β-like cell generation can be significantly enhanced by preventing phosphorylation of Ngn3 protein and further augmented by conditions promoting differentiation. Taken together, our new findings underline the potential of ductal organoid cultures as a source material for generation of β-like cells and demonstrate that post-translational regulation of reprogramming factors can be exploited to enhance β-cell generation