Role of symmetry in 3,3-diphenyl-1,3-dihydroindol-2-one derivatives as inhibitors of translation initiation.

The ternary complex (eIF2·GTP·Met-tRNAiMet) and the eIF4F complex assembly are two major regulatory steps in the eukaryotic translation initiation. Inhibition of the ternary complex assembly is therefore a promising target for the development of novel anti-cancer therapeutics. Building on the finding that clotrimazole (CLT), a molecular probe that depletes intracellular Ca2+ stores and subsequently induce eIF2α phosphorylation, inhibit translation initiation, and reduce preferentially the expression of oncoproteins over "housekeeping" ones,1-3 we undertook structure activity relationship (SAR) studies that identified 3,3-diarylindoline-2-one #1181 as an interesting scaffold. Compound #1181 also induce phosphorylation of eIF2α thereby reducing the availability of the ternary complex, which leads to inhibition of translation initiation.4 Our subsequent efforts focused on understanding SAR iterative lead optimization to enhance potency and improve bioavailability. Herein, we report a complementing study focusing on heavily substituted symmetric and asymmetric 3,3-(o,m-disubstituted)diarylindoline-2-ones. These compounds were evaluated by the dual luciferase reporter ternary complex assay that recapitualates phosphorylation of eIF2α in a quantitative manner. We also evaluated all compounds by sulforhodamine B assay, which measures the overall effect of compounds on cell proliferations and/or viability.

Versatile cell ablation tools and their applications to study loss of cell functions.

Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.

The eIF2α Kinase Heme-Regulated Inhibitor Protects the Host from Infection by Regulating Intracellular Pathogen Trafficking.

The host employs both cell-autonomous and system-level responses to limit pathogen replication in the initial stages of infection. Previously, we reported that the eukaryotic initiation factor 2α (eIF2α) kinases heme-regulated inhibitor (HRI) and protein kinase R (PKR) control distinct cellular and immune-related activities in response to diverse bacterial pathogens. Specifically for Listeria monocytogenes, there was reduced translocation of the pathogen to the cytosolic compartment in HRI-deficient cells and consequently reduced loading of pathogen-derived antigens on major histocompatibility complex class I (MHC-I) complexes. Here we show that Hri-/- mice, as well as wild-type mice treated with an HRI inhibitor, are more susceptible to listeriosis. In the first few hours of L. monocytogenes infection, there was much greater pathogen proliferation in the liver of Hri-/- mice than in the liver of Hri+/+ mice. Further, there was a rapid increase of serum interleukin-6 (IL-6) levels in Hri+/+ mice in the first few hours of infection whereas the increase in IL-6 levels in Hri-/- mice was notably delayed. Consistent with these in vivo findings, the rate of listeriolysin O (LLO)-dependent pathogen efflux from infected Hri-/- macrophages and fibroblasts was significantly higher than the rate seen with infected Hri+/+ cells. Treatment of cells with an eIF2α kinase activator enhanced both the HRI-dependent and PKR-dependent infection phenotypes, further indicating the pharmacologically malleability of this signaling pathway. Collectively, these results suggest that HRI mediates the cellular confinement and killing of virulent L. monocytogenes in addition to promoting a system-level cytokine response and that both are required to limit pathogen replication during the first few hours of infection.

Restriction of drug transport by the tumor environment.

As in the systemic treatment of any disease, it is crucial for anti-cancer drugs to reach their target at a sufficient that is a therapeutically effective dose. However, unlike normal organs, solid tumors have a tendency to be undersupplied and hypoxic. This not only leads to insufficient supply of oxygen and nutrients but also to inefficient transport of drugs into tumors. As a consequence, administered doses have to be raised, resulting in increased side effects and often premature termination of treatment. A better understanding of the mechanisms that hamper transport of drugs into tumors could lead to the development of auxiliary strategies aimed at increasing tumor drug delivery and accumulation and thereby improving the efficacy of anti-cancer drugs at our disposal. The tumor microenvironment (TME), i.e., its vasculature, stroma, extracellular matrix and immune environment affect the transport of drugs to the tumor and their distribution within the tumor tissue in various ways. In this review we will highlight the current research regarding the cellular and molecular mechanisms that remain as an obstacle towards an effective cancer therapy, and also focus on the various strategies to alter the TME to increase tumor drug exposure and thereby treatment efficacy.

IL-24 Promotes Apoptosis through cAMP-Dependent PKA Pathways in Human Breast Cancer Cells.

Interleukin 24 (IL-24) is a tumor-suppressing protein, which inhibits angiogenesis and induces cancer cell-specific apoptosis. We have shown that IL-24 regulates apoptosis through phosphorylated eukaryotic initiation factor 2 alpha (eIF2α) during endoplasmic reticulum (ER) stress in cancer. Although multiple stresses converge on eIF2α phosphorylation, the cellular outcome is not always the same. In particular, ER stress-induced apoptosis is primarily regulated through the extent of eIF2α phosphorylation and activating transcription factor 4 (ATF4) action. Our studies show for the first time that cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation is required for IL-24-induced cell death in a variety of breast cancer cell lines and this event increases ATF4 activity. We demonstrate an undocumented role for PKA in regulating IL-24-induced cell death, whereby PKA stimulates phosphorylation of p38 mitogen-activated protein kinase and upregulates extrinsic apoptotic factors of the Fas/FasL signaling pathway and death receptor 4 expression. We also demonstrate that phosphorylation and nuclear import of tumor suppressor TP53 occurs downstream of IL-24-mediated PKA activation. These discoveries provide the first mechanistic insights into the function of PKA as a key regulator of the extrinsic pathway, ER stress, and TP53 activation triggered by IL-24.

The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X-box binding protein 1 transcription factor.

Endoplasmic reticulum (ER) stress triggers the integrated ER-stress response (IERSR) that ensures cellular survival of ER stress and represents a primordial form of innate immunity. We investigated the role of IERSR duringLeishmania amazonensisinfection. Treatment of RAW 264.7 infected macrophages with the ER stress-inducing agent thapsigargin (TG; 1 µM) increasedL. amazonensisinfectivity in an IFN1-α receptor (IFNAR)-dependent manner. In Western blot assays, we showed thatL. amazonensisactivates the inositol-requiring enzyme (IRE1)/ X-box binding protein (XBP)-1-splicing arms of the IERSR in host cells. In chromatin immunoprecipitation (ChIP) assays, we showed an increased occupancy of enhancer and promoter sequences for theIfnbgene by XBP1 in infected RAW 264.7 cells. Knocking down XBP1 expression by transducing RAW 264.7 cells with the short hairpin XBP1 lentiviral vector significantly reduced the parasite proliferation associated with impaired translocation of phosphorylated IFN regulatory transcription factor (IRF)-3 to the nucleus and a decrease in IFN1-ß expression. Knocking down XBP1 expression also increased NO concentration, as determined by Griess reaction and reduced the expression of antioxidant genes, such as heme oxygenase (HO)-1, that protect parasites from oxidative stress. We conclude thatL. amazonensisactivation of XBP1 plays a critical role in infection by protecting the parasites from oxidative stress and increasing IFN1-ß expression.-Dias-Teixeira, K. L., Calegari-Silva, T. C., Dos Santos, G. R. R. M., Vitorino dos Santos, J., Lima, C., Medina, J. M., Aktas, B. H., Lopes, U. G. The integrated endoplasmic reticulum stress response inLeishmania amazonensismacrophage infection: the role of X-box binding protein 1 transcription factor.

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between ß-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

3-substituted indazoles as configurationally locked 4EGI-1 mimetics and inhibitors of the eIF4E/eIF4G interaction.

4EGI-1, the prototypic inhibitor of eIF4E/eIF4G interaction, was identified in a high-throughput screening of small-molecule libraries with the aid of a fluorescence polarization assay that measures inhibition of binding of an eIF4G-derived peptide to recombinant eIF4E. As such, the molecular probe 4EGI-1 has potential for the study of molecular mechanisms involved in human disorders characterized by loss of physiological restraints on translation initiation. A hit-to-lead optimization campaign was carried out to overcome the configurational instability in 4EGI-1, which stems from the E-to-Z isomerization of the hydrazone function. We identified compound 1 a, in which the labile hydrazone was incorporated into a rigid indazole scaffold, as a promising rigidified 4EGI-1 mimetic lead. In a structure-activity relationship study directed towards probing the structural latitude of this new chemotype as an inhibitor of eIF4E/eIF4G interaction and translation initiation we identified 1 d, an indazole-based 4EGI-1 mimetic, as a new and improved lead inhibitor of eIF4E/eIF4G interaction and a promising molecular probe candidate for elucidation of the role of cap-dependent translation initiation in a host of pathophysiological states.

Activity of the Di-Substituted Urea-Derived Compound I-17 in Leishmania In Vitro Infections.

Protein synthesis has been a very rich target for developing drugs to control prokaryotic and eukaryotic pathogens. Despite the development of new drug formulations, treating human cutaneous and visceral Leishmaniasis still needs significant improvements due to the considerable side effects and low adherence associated with the current treatment regimen. In this work, we show that the di-substituted urea-derived compounds I-17 and 3m are effective in inhibiting the promastigote growth of different Leishmania species and reducing the macrophage intracellular load of amastigotes of the Leishmania (L.) amazonensis and L. major species, in addition to exhibiting low macrophage cytotoxicity. We also show a potential immunomodulatory effect of I-17 and 3m in infected macrophages, which exhibited increased expression of inducible Nitric Oxide Synthase (NOS2) and production of Nitric Oxide (NO). Our data indicate that I-17, 3m, and their analogs may be helpful in developing new drugs for treating leishmaniasis.

Dual activators of protein kinase R (PKR) and protein kinase R-like kinase PERK identify common and divergent catalytic targets.

Chemical genetics has evolved into a powerful tool for studying gene function in normal and pathobiology. PKR and PERK, two eukaryotic translation initiation factor 2 alpha (eIF2α) kinases, play critical roles in the maintenance of cellular hemostasis, metabolic stability, and anti-viral defenses. Both kinases interact with and phosphorylate additional substrates including tumor suppressor p53 and nuclear protein 90. Loss of function of both kinases has been studied by reverse genetics and with recently identified inhibitors. In contrast, no activating probes for studying the catalytic activity of these kinases are available. We identified 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHBDC) as a specific dual activator of PKR and PERK by screening a chemical library of 20 000 small molecules in a dual luciferase surrogate eIF2α phosphorylation assay. We present here extensive biological characterization and a preliminary structure-activity relationship of DHBDC, which phosphorylates eIF2α by activating PKR and PERK but no other eIF2α kinases. These agents also activate downstream effectors of eIF2α phosphorylation by inducing CEBP homologue protein, suppressing cyclin D1 expression, and inhibiting cancer cell proliferation, all in a manner dependent on PKR and PERK. Consistent with the role of eIF2α phosphorylation in viral infection, DHBDC inhibits the proliferation of human hepatitis C virus. Finally, DHBDC induces the phosphorylation of IκBα and activates the NF-κB pathway. Surprisingly, activation of the NF-κB pathway is dependent on PERK but independent of PKR activity. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal and pathobiology.

Chemical genetics identify eIF2α kinase heme-regulated inhibitor as an anticancer target.

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2-GTP-tRNA(i)(Met) translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2-GTP-tRNA(i)(Met) ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N'-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N'-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2α and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N'-diarylureas are potent and specific tools for studying the role of eIF2-GTP-tRNA(i)(Met) ternary complex in the pathobiology of human disorders.

In vitro inhibition of translation initiation by N,N'-diarylureas--potential anti-cancer agents.

Symmetrical N,N'-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP·tRNA(i)(Met) ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N'-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.

Embryonic lethal abnormal vision-like HuR-dependent mRNA stability regulates post-transcriptional expression of cyclin-dependent kinase inhibitor p27Kip1.

The cyclin-dependent kinase inhibitor p27(Kip1) plays a critical role in regulating entry into and exit from the cell cycle. Post-transcriptional regulation of p27(Kip1) expression is of significant interest. The embryonic lethal abnormal vision (ELAV)-like RNA-binding protein HuR is thought be important for the translation of p27(Kip1), however, different reports attributed diametrically opposite roles to HuR. We report here an alternative mechanism wherein HuR regulates stability of the p27(Kip1) mRNA. Specifically, human and mouse p27(Kip1) mRNAs interact with HuR protein through multiple U-rich elements in both 5' and 3' untranslated regions (UTR). These interactions, which occur in vitro and in vivo, stabilize p27(Kip1) mRNA and play a critical role in its accumulation. Deleting HuR binding sites or knocking down HuR expression destabilizes p27(Kip1) mRNA and reduces its accumulation. We also identified a CT repeat in the 5' UTR of full-length p27(Kip1) mRNA isoforms that interact with a approximately 41-kDa protein and represses p27(Kip1) expression. This CT-rich element and diffuse elements in the 3' UTR regulate post-transcriptional expression of p27(Kip1) at the level of translation. This is the first demonstration that HuR-dependent mRNA stability and HuR-independent mRNA translation plays a critical role in the regulation of post-transcriptional p27(Kip1) expression.

Endothelial cell infection and dysfunction, immune activation in severe COVID-19.

Rationale: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. Methods: To address these questions, we studied SARS-CoV-2-infected K18-hACE2 (K18) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. Results: We show that SARS-CoV-2-infected K18 mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in K18 mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. Conclusion: Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.

SARS-CoV-2 Infects Endothelial Cells In Vivo and In Vitro.

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.

New activators of eIF2α Kinase Heme-Regulated Inhibitor (HRI) with improved biophysical properties.

Heme-regulated inhibitor (HRI), a eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, is critically important for coupling protein synthesis to heme availability in reticulocytes and adaptation to various environmental stressors in all cells. HRI modifies the severity of several hemoglobin misfolding disorders including ß-thalassemia. Small molecule activators of HRI are essential for studying normal- and patho-biology of this kinase as well as for the treatment of various human disorders for which activation of HRI or phosphorylation of eIF2α may be beneficial. We previously reported development of 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific HRI activators and demonstrated their potential as molecular probes for studying HRI biology and as lead compounds for treatment of various human disorders. To develop more druglike cHAUs for in vivo studies and drug development and to expand the chemical space, we undertook bioassay guided structure-activity relationship studies replacing cyclohexyl ring with various 4-6-membered rings and explored further substitutions on the N-phenyl ring. We tested all analogs in the surrogate eIF2α phosphorylation and cell proliferation assays, and a subset of analogs in secondary mechanistic assays that included endogenous eIF2α phosphorylation and expression of C/EBP homologous protein (CHOP), a downstream effector. Finally, we determined specificity of these compounds for HRI by testing their anti-proliferative activity in cells transfected with siRNA targeting HRI or mock. These compounds have significantly improved cLogPs with no loss of potencies, making them excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.

Distinct fate, dynamics and niches of renal macrophages of bone marrow or embryonic origins.

Renal macrophages (RMs) participate in tissue homeostasis, inflammation and repair. RMs consist of embryo-derived (EMRMs) and bone marrow-derived RMs (BMRMs), but the fate, dynamics, replenishment, functions and metabolic states of these two RM populations remain unclear. Here we investigate and characterize RMs at different ages by conditionally labeling and ablating RMs populations in several transgenic lines. We find that RMs expand and mature in parallel with renal growth after birth, and are mainly derived from fetal liver monocytes before birth, but self-maintain through adulthood with contribution from peripheral monocytes. Moreover, after the RMs niche is emptied, peripheral monocytes rapidly differentiate into BMRMs, with the CX3CR1/CX3CL1 signaling axis being essential for the maintenance and regeneration of both EMRMs and BMRMs. Lastly, we show that EMRMs have a higher capacity for scavenging immune complex, and are more sensitive to immune challenge than BMRMs, with this difference associated with their distinct glycolytic capacities.

The Best for the Most Important: Maintaining a Pristine Proteome in Stem and Progenitor Cells.

Pluripotent stem cells give rise to reproductively enabled offsprings by generating progressively lineage-restricted multipotent stem cells that would differentiate into lineage-committed stem and progenitor cells. These lineage-committed stem and progenitor cells give rise to all adult tissues and organs. Adult stem and progenitor cells are generated as part of the developmental program and play critical roles in tissue and organ maintenance and/or regeneration. The ability of pluripotent stem cells to self-renew, maintain pluripotency, and differentiate into a multicellular organism is highly dependent on sensing and integrating extracellular and extraorganismal cues. Proteins perform and integrate almost all cellular functions including signal transduction, regulation of gene expression, metabolism, and cell division and death. Therefore, maintenance of an appropriate mix of correctly folded proteins, a pristine proteome, is essential for proper stem cell function. The stem cells' proteome must be pristine because unfolded, misfolded, or otherwise damaged proteins would interfere with unlimited self-renewal, maintenance of pluripotency, differentiation into downstream lineages, and consequently with the development of properly functioning tissue and organs. Understanding how various stem cells generate and maintain a pristine proteome is therefore essential for exploiting their potential in regenerative medicine and possibly for the discovery of novel approaches for maintaining, propagating, and differentiating pluripotent, multipotent, and adult stem cells as well as induced pluripotent stem cells. In this review, we will summarize cellular networks used by various stem cells for generation and maintenance of a pristine proteome. We will also explore the coordination of these networks with one another and their integration with the gene regulatory and signaling networks.

Eukaryotic Translation Initiation Factor 4A Down-Regulation Mediates Interleukin-24-Induced Apoptosis through Inhibition of Translation.

Dysregulated activity of helicase eIF4A drives transformation to and maintenance of cancer cell phenotype by reprogramming cellular translation. Interleukin 24 (IL-24) is a tumor-suppressing protein, which has the ability to inhibit angiogenesis, sensitize cancer cells to chemotherapy, and induce cancer cell-specific apoptosis. In this study, we found that eIF4A is inhibited by IL-24. Consequently, selective reduction of translation was observed for mRNAs harboring strong secondary structures in their 5'-untranslated regions (5'UTRs). These mRNAs encode proteins, which function in cell survival and proliferation. Consistently, overexpression of eIF4A conferred cancer cells with resistance to IL-24-induced cell death. It has been established that inhibition of eIF4A triggers mitochondrial-mediated apoptosis. We showed that IL-24 induces eIF4A-dependent mitochondrial depolarization. We also showed that IL-24 induces Sigma 1 Receptor-dependent eIF4A down-regulation and mitochondrial depolarization. Thus, the progress of apoptosis triggered by IL-24 is characterized by a complex program of changes in regulation of several initiation factors, including the eIF4A.