Calpain Inhibition Restores Autophagy and Prevents Mitochondrial Fragmentation in a Human iPSC Model of Diabetic Endotheliopathy

The relationship between diabetes and endothelial dysfunction remains unclear, particularly the association with pathological activation of calpain, an intracellular cysteine protease. Here, we used human induced pluripotent stem cells-derived endothelial cells (iPSC-ECs) to investigate the effects of diabetes on vascular health. Our results indicate that iPSC-ECs exposed to hyperglycemia had impaired autophagy, increased mitochondria fragmentation, and was associated with increased calpain activity. In addition, hyperglycemic iPSC-ECs had increased susceptibility to cell death when subjected to a secondary insult-simulated ischemia-reperfusion injury (sIRI). Importantly, calpain inhibition restored autophagy and reduced mitochondrial fragmentation, concurrent with maintenance of ATP production, normalized reactive oxygen species levels and reduced susceptibility to sIRI. Using a human iPSC model of diabetic endotheliopathy, we demonstrated that restoration of autophagy and prevention of mitochondrial fragmentation via calpain inhibition improves vascular integrity. Our human iPSC-EC model thus represents a valuable platform to explore biological mechanisms and new treatments for diabetes-induced endothelial dysfunction.Singapore Ministry of Health's National Medical Research Council Open Fund-Young Individual Research Grant [NMRC/OFYIRG/0021/2016]; Khoo Postdoctoral Fellowship Award [Duke-NUS-KPFA/2016/0010]; Hitachi Scholarship Research Support Grant from the Hitachi Global Foundation, Japan [RS-13, H-1]; American Heart Association Scientist Development Grant [16SDG27560003]; Stanford Diabetes Research Center under NIH [P30DK116074]; Frontier Research Grant 2017 from the Frontier Science Research Cluster (FSRC), Universiti Malaya, Malaysia [FG021-17AFR]; NIH [R01HL126516, R00HL130416]; Samsung Biomedical Research Institute [OTC 1180261]; National Research Foundation of Korea [NRF-2016R1A2B4008235]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

eIF2α phosphorylation controls thermal nociception

A response to environmental stress is critical to alleviate cellular injury and maintain cellular homeostasis. Eukaryotic initiation factor 2 (eIF2) is a key integrator of cellular stress responses and an important regulator of mRNA translation. Diverse stress signals lead to the phosphorylation of the α subunit of eIF2 (Ser51), resulting in inhibition of global protein synthesis while promoting expression of proteins that mediate cell adaptation to stress. Here we report that eIF2α is instrumental in the control of noxious heat sensation. Mice with decreased eIF2α phosphorylation (eIF2α(+/S51A)) exhibit reduced responses to noxious heat. Pharmacological attenuation of eIF2α phosphorylation decreases thermal, but not mechanical, pain sensitivity, whereas increasing eIF2α phosphorylation has the opposite effect on thermal nociception. The impact of eIF2α phosphorylation (p-eIF2α) on thermal thresholds is dependent on the transient receptor potential vanilloid 1. Moreover, we show that induction of eIF2α phosphorylation in primary sensory neurons in a chronic inflammation pain model contributes to thermal hypersensitivity. Our results demonstrate that the cellular stress response pathway, mediated via p-eIF2α, represents a mechanism that could be used to alleviate pathological heat sensation

Control of embryonic stem cells self-renewal and differentiation via coordinated splicing and translation of YY2

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Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis

Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.</div

Transcriptional and translational control in cell fate determination

Multi-cellular organisms generate functional and morphological diversity in their cells through spatiotemporal regulation of gene expression. Gene expression is regulated through multiple mechanisms at the levels of transcription, translation, and degradation. During my PhD studies, I examined transcription factor-induced somatic-to-induced pluripotent stem cell reprogramming. For this, I adopted two different strategies that are based on transcriptional regulation by post-translational modification and translational control by the translational repressor 4E-BP. Specifically, in the project described in Chapter III, I studied the role of sumoylation in controlling the transcriptional activity of KLF4, KLF2, and KLF5 during reprogramming and adipocyte differentiation. I also explored the role of Ubc9, the sole SUMO-conjugating enzyme, in reprogramming and adipogenesis. These results and a comprehensive review of related studies, presented in Chapter I, have helped me propose a model for the functional specificity of KLF family proteins. In the research project described in Chapter IV, I investigated the role of translational control in somatic cell reprogramming. This investigation uncovered a functional duality in 4E-BP-dependent translation during reprogramming that is controlled by the p53-p21 pathway. To introduce this chapter and to provide insights for future research, I summarize in Chapter II the current findings regarding the role of the PI3K/AKT/mTOR pathway--the main control of eIF4E-dependent translation--in regulating embryonic stem cell self-renewal and somatic-to-stem cell reprogramming. The results of this thesis may lead to a better understanding of the mechanism underlying cell type determination that has important application in regenerative medicine.Les organismes multicellulaires génèrent la diversité fonctionnelle et morphologique de leurs cellules par une régulation spatiotemporel de l'expression de leur gène. L'expression de ces gènes est régulée par de multiples mécanismes au niveau de la transcription, de la traduction et de la dégradation. Pendant mon PhD, j'ai étudié la reprogrammation par des facteurs de transcription de cellules somatiques en cellules souches pluripotentes. Pour ce faire, j'ai adopté deux stratégies basées sur le control de la transcription par des modifications post-traductionnelles et le control de la traduction par le répresseur 4E-BP. Spécifiquement, dans le projet décrit dans le Chapitre III, j'ai étudié le rôle de la sumoylation dans l'activité transcriptionnelle de KLF4, KLF2, et KLF5 pendant la reprogrammation et la pendant la différentiation des adipocytes. J'ai aussi exploré le rôle d'Ubc9, la seule enzyme capable de conjuguer un groupe SUMO à une protéine, dans la reprogrammation et l'adipogenèse. Les résultats de cette étude et la révision d'études connexes présentés dans le Chapitre I m'ont aidé à proposer un modèle pour la spécificité fonctionnelle de la famille de protéines KLF. Dans le projet de recherche présenté au chapitre IV, j'ai étudié le rôle du contrôle de la traduction dans la reprogrammation somatique. Cette étude m'a permis de découvrir la dualité fonctionnelle de la traduction eIF4E-dépendante durant la reprogrammation cellulaire contrôlée par la voie p53/p21. Pour introduire ce chapitre et pour discuter de recherches futures possibles, j'ai résumé dans le chapitre II les connaissances actuelles concernant le rôle de la voie de signalisation PI3K/AKT/mTOR—la principale voie qui contrôle la traduction eIF4E-dépendante--dans la régulation de la reprogrammation de cellules somatiques en cellules souches et dans la régulation de l'auto-renouvellement des cellules souches embryonnaires. Les résultats de cette thèse pourraient mener à une meilleure compréhension des mécanismes responsables de la détermination cellulaire qui aura d'importantes applications dans la médecine régénérative

Molecular Architecture of Quartet MOZ/MORF Histone Acetyltransferase Complexes▿ §

The monocytic leukemia zinc finger protein MOZ and the related factor MORF form tetrameric complexes with ING5 (inhibitor of growth 5), EAF6 (Esa1-associated factor 6 ortholog), and the bromodomain-PHD finger protein BRPF1, -2, or -3. To gain new insights into the structure, function, and regulation of these complexes, we reconstituted them and performed various molecular analyses. We found that BRPF proteins bridge the association of MOZ and MORF with ING5 and EAF6. An N-terminal region of BRPF1 interacts with the acetyltransferases; the enhancer of polycomb (EPc) homology domain in the middle part binds to ING5 and EAF6. The association of BRPF1 with EAF6 is weak, but ING5 increases the affinity. These three proteins form a trimeric core that is conserved from Drosophila melanogaster to humans, although authentic orthologs of MOZ and MORF are absent in invertebrates. Deletion mapping studies revealed that the acetyltransferase domain of MOZ/MORF is sufficient for BRPF1 interaction. At the functional level, complex formation with BRPF1 and ING5 drastically stimulates the activity of the acetyltransferase domain in acetylation of nucleosomal histone H3 and free histones H3 and H4. An unstructured 18-residue region at the C-terminal end of the catalytic domain is required for BRPF1 interaction and may function as an “activation lid.” Furthermore, BRPF1 enhances the transcriptional potential of MOZ and a leukemic MOZ-TIF2 fusion protein. These findings thus indicate that BRPF proteins play a key role in assembling and activating MOZ/MORF acetyltransferase complexes

RNA-based translation activators for targeted gene upregulation

Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present “translation-activating RNAs” (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research

Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells

Summary: The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/β) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network

Multifaceted Regulation of Somatic Cell Reprogramming by mRNA Translational Control

SummaryTranslational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process