Controlled induction of human pancreatic progenitors produces functional beta‐like cells in vitro

Directed differentiation of human pluripotent stem cells into functional insulin‐producing beta‐like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non‐functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1+ and subsequent PDX1+/NKX6.1+ pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1+/NKX6.1+ progenitors produces glucose‐responsive beta‐like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short‐term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta‐like cells.SynopsisFocusing on developmental mechanisms, the results of this study further accelerate successful differentiation of human ESCs into functional pancreatic beta cells.Exclusion of commonly used BMP inhibitors during human embryonic stem cell to pancreatic progenitor differentiation prevents precocious endocrine induction.Sequential exposure of foregut cells to retinoic acid followed by combined EGF/KGF treatment establishes highly pure PDX1+ and PDX1+/NKX6.1+ progenitor populations, respectively.Precise temporal induction of endocrine differentiation in PDX1+/NKX6.1+ progenitors, but not in PDX1+/NKX6.1− progenitors, results in the generation of functional beta‐like cells in vitro.Beta‐like cells exhibit key features of bona fide human beta cells, remain functional after short‐term transplantation, and reduce blood glucose levels in diabetic mice.Focusing on developmental mechanisms, the results of this study further accelerate successful differentiation of human ESCs into functional pancreatic beta cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111932/1/embj201591058.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/111932/2/embj201591058.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/111932/3/embj201591058-sup-0001-FigsS1-S4.pd

Regulation of Eukaryotic Translation Initiation by Signal Transduction

<p>Eukaryotic translation initiation is a rate-limiting step of protein synthesis and is controlled by signal transduction in response to various extracellular cues and stresses. This thesis is focused on the eukaryotic initiation factor 4G (eIF4G), which participates in multiple steps of initiation: (i) eIF4G interaction with polyA-binding protein (PABP) links the 5' pre-initiation complex to the 3' poly(A) tail of mRNAs; (ii) eIF4G recruits 40S ribosomal subunit to mRNAs through interactions with the m7-G cap binding protein eIF4E; (iii) eIF4G binds to the eIF4E kinase, mitogen activated protein kinase (MAPK) interacting kinase 1 (Mnk1), modulating eIF4E phosphorylation. I studied how eIF4G function is affected by viral infection, mitogenic stimulation and during mitosis.</p><p>First, I reported that herpes simplex virus 1 (HSV-1) infection leads to re-localization of PABP to the nucleus, dissociating it from translation initiation machinery. Next, I showed that MAPK-mediated phosphorylation of Mnk1 leads to Mnk1 conformational changes, enhancing its binding to eIF4G and, hence, increasing phosphorylation of its substrate, eIF4E. Finally, I demonstrated that Cdk1/cyclin B1 directly phosphorylates eIF4G in mitosis and speculated on the role of this phosphorylation event in mitotic translation. In summary, my work demonstrated that eIF4G plays key roles in the regulation of the translational response to viral infection, growth signaling and cell cycle progression.</p>Dissertatio

Herpes Simplex Virus Proteins ICP27 and UL47 Associate with Polyadenylate-Binding Protein and Control Its Subcellular Distribution▿

Human pathogenic viruses manipulate host cell translation machinery to ensure efficient expression of viral genes and to thwart host cell protein synthesis. Viral strategies include cleaving translation factors, manipulating translation factor abundance and recruitment into translation initiation complexes, or expressing viral translation factor analogs. Analyzing translation factors in herpes simplex virus type 1 (HSV-1)-infected HeLa cells, we found diminished association of the polyadenylate-binding protein (PABP) with the cap-binding complex. Although total PABP levels were unchanged, HSV-1 infection prompted accumulation of cytoplasmic PABPC1, but not its physiologic binding partner PABP-interacting protein 2 (Paip2), in the nucleus. Using glutathione S-transferase-PABP pull-down and proteomic analyses, we identified several viral proteins interacting with PABPC1 including tegument protein UL47 and infected-cell protein ICP27. Transient expression of ICP27 and UL47 in HeLa cells suggested that ICP27 and UL47 jointly displace Paip2 from PABP. ICP27 expression alone was sufficient to cause PABPC1 redistribution to the nucleus. ICP27 and UL47 did not alter translation efficiency of transfected reporter RNAs but modulated transcript abundance and expression of reporter cDNAs in transfected cells. This indicates that redistribution of PABPC1 may be involved in co- and posttranscriptional regulation of mRNA processing and/or nuclear export by HSV-1 gene regulatory proteins

Regulation of Eukaryotic Initiation Factor 4E (eIF4E) Phosphorylation by Mitogen-Activated Protein Kinase Occurs through Modulation of Mnk1-eIF4G Interaction▿

The m7G cap binding protein eukaryotic initiation factor 4E (eIF4E) is a rate-limiting determinant of protein synthesis. Elevated eIF4E levels, commonly associated with neoplasia, promote oncogenesis, and phosphorylation of eIF4E at Ser209 is critical for its tumorigenic potential. eIF4E phosphorylation is catalyzed by mitogen-activated protein kinase (MAPK)-interacting serine/threonine kinase (Mnk), a substrate of Erk1/2 and p38 MAPKs. Interaction with the scaffolding protein eIF4G, which also binds eIF4E, brings Mnk and its substrate into physical proximity. Thus, Mnk-eIF4G interaction is important for eIF4E phosphorylation. Through coimmunoprecipitation assays, we showed that MAPK-mediated phosphorylation of the Mnk1 active site controls eIF4G binding. Utilizing a naturally occurring splice variant, we demonstrated that the C-terminal domain of Mnk1 restricts its interaction with eIF4G, preventing eIF4E phosphorylation in the absence of MAPK signaling. Furthermore, using a small-molecule Mnk1 inhibitor and kinase-dead mutant, we established that Mnk1 autoregulates its interaction with eIF4G, releasing itself from the scaffold after phosphorylation of its substrate. Our findings indicate tight control of eIF4E phosphorylation through modulation of Mnk1-eIF4G interaction

Poly(A)-binding protein is differentially required for translation mediated by viral internal ribosome entry sites

The 3′ poly(A) tail present on the majority of mature eukaryotic mRNAs is an important regulator of protein synthesis and mRNA stability. The poly(A) tail improves the efficiency of translation initiation through recruitment of PABP, enabling its interaction with eIF4F located at the mRNA 5′-end. Recent evidence has also implicated a possible role for PABP and the poly(A) tract in translation control at steps beyond the initiation phase. Similar to conventional mRNAs, plus-strand RNA virus genomes that utilize internal ribosome entry sites (IRESes) to promote cap-independent translation are influenced by PABP and poly(A) status. However, the relative contribution of these factors to translation initiation mediated by distinct IRESes is unclear. We have investigated cis- and trans-acting effects of poly(A) and PABP, respectively, on RNAs harboring IRESes from three diverse viruses: encephalomyocarditis virus (EMCV), hepatitis C virus (HCV), and coxsackievirus B3 (CBV3). A 3′ poly(A) tract enhanced translation of both capped and IRES-containing reporter RNAs. However, only CBV3 and capped transcripts were stabilized as a result of polyadenylation. Correspondingly, translation of polyadenylated CBV3 and capped RNAs displayed heightened sensitivity to the PABP inhibitor Paip2 compared with EMCV and HCV. Sucrose density gradient analyses suggested a stimulatory role for PABP and 3′ poly(A) in the CBV3 initiation phase, while assembly of HCV and EMCV RNAs into ribosomal complexes was little affected by either factor. Collectively, the observed differential effects of PABP and poly(A) on translation imply mechanistic differences between viral IRES elements and suggest modulating roles for PABP and the poly(A) tail at multiple phases of translation

LIN28B Impairs the Transition of hESC-Derived β Cells from the Juvenile to Adult State.

Differentiation of human embryonic stem cells into pancreatic β cells holds great promise for the treatment of diabetes. Recent advances have led to the production of glucose-responsive insulin-secreting cells in&nbsp;vitro, but resulting cells remain less mature than their adult primary β cell counterparts. The barrier(s) to in&nbsp;vitro β cell maturation are unclear. Here, we evaluated a potential role for microRNAs. MicroRNA profiling showed high expression of let-7 family microRNAs in&nbsp;vivo, but not in in&nbsp;vitro differentiated β cells. Reduced levels of let-7 in&nbsp;vitro were associated with increased levels of the RNA binding protein LIN28B, a negative regulator of let-7 biogenesis. Ablation of LIN28B during human embryonic stem cell (hESC) differentiation toward β cells led to a more mature glucose-stimulated insulin secretion profile and the suppression of juvenile-specific genes. However, let-7 overexpression had little effect. These results uncover LIN28B as a modulator of β cell maturation in&nbsp;vitro