Global Inhibition Research Articles

Abstract 3860: Discovery and characterization of novel anti-cancer small molecule inhibitors of Sec61

Abstract Secreted and membrane proteins play key roles in malignant transformation and tumor growth, including autocrine growth factor expression, receptor oncogene signal transduction pathways, metastasis, and immune system evasion. The biogenesis of most secreted and transmembrane proteins involves cotranslational translocation of nascent polypeptides into the endoplasmic reticulum through the Sec61 translocon. Broad inhibition of protein secretion can be mediated by natural product-derived Sec61 inhibitors that also possess anti-tumor activity, suggesting that Sec61 represents an untapped therapeutic target for cancer treatment. Here we describe a process for discovering and evaluating novel Sec61 inhibitors with varying degrees of selectivity for therapeutically relevant targets in oncology. We used HEK293 reporter cell lines stably transfected to express secreted and transmembrane proteins of interest fused to luciferase to enable high-throughput screening of potential inhibitors of Sec61-dependent protein secretion. As expected, target selectivity was largely dependent on respective signal sequences. However, full-length protein constructs were found to be 5-10 fold less sensitive to inhibition compared to reporters containing only the signal sequence plus 10 amino acids, suggesting that additional interactions beyond the signal sequence are involved in promoting cotranslational translocation. To verify that novel compounds specifically targeted Sec61, we found that compounds were unable to inhibit the activity of luciferase localized to the cytosol and had reduced potency in reporter cells expressing a Sec61 mutant known to confer resistance to published inhibitors such as cotransin, mycolactone, and apratoxin. We assessed compound selectivity using a library of 37 transiently expressed Sec61-client proteins of therapeutic relevance, revealing 4 distinct chemical scaffolds differentiated by target selectivity profile. Consistent with data from published compounds, broad inhibitors of protein secretion had anti-tumor activity. Of particular interest were compounds able to block expression of multiple immune checkpoints including PD-1, PD-L1, CTLA-4, and CD96. Activity in primary cells, such as receptor expression on activated T-cells and cytokine release from stimulated PBMC, was assessed to verify specificity. Compounds with adequate in vitro metabolic stability were tested in mice for pharmacokinetics as well as inhibition of target protein secretion and global inhibition of Sec61 activity. Taken together, our data suggest that Sec61 is a novel therapeutic target which represents a unique opportunity to both selectively block expression of multiple proteins involved in tumor growth and globally modulate protein homeostasis, resulting in tumor cell death. Citation Format: Eric Lowe, Janet L. Anderl, Andrea R. Fan, Ying Fang, Jing Jiang, Henry W. Johnson, Christopher J. Kirk, Dustin McMinn, Tony Muchamuel, Meera Rao, Phillip P. Sharp, Jack Taunton, Jinhai Wang, Jennifer A. Whang. Discovery and characterization of novel anti-cancer small molecule inhibitors of Sec61 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3860.

Abstract 923: FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells

Abstract Androgen receptor (AR) plays a critical function in driving prostate cancer (PCa) development and androgen deprivation therapy (ADT) is standard of care in treating PCa patients. Although patients generally response well, the cancer eventually relapse and progress to the castration-resistant stage of PCa (called CRPC), which can be further treated with more aggressive ADT. However, the tumors still develop resistance within a year and restored AR activity is found in the majority of relapsed tumors, indicating a pressing need for developing novel AR-targeted therapies. In PCa cells, AR activity is dependent on the chromatin binding of the pioneer factor, FOXA1. In particular, FOXA1 recognizes the H3K4me1,2 marked enhancers and its binding opens the chromatin for subsequent AR recruitment. Although the role of FOXA1 on AR signaling has been intensively studied, the mechanisms regulating FOXA1 binding to chromatin remain elusive. LSD1/KDM1A was initially identified as a transcription repressor through demethylation of H3K4me1,2. In contrast to its well-established corepressor function, LSD1 has been found to coactivate several transcription factors including AR. This AR coactivator function has been attributed to the phosphorylation of histone 3 on threonine 6 and threonine 11, which may switch LSD1 substrate specificity from H3K4me1,2 to the repressive mark H3K9me1,2. However, we have recently reported that the H3K4 demethylase activity of LSD1 persists at AR-regulated enhancer sites, including sites marked by H3T6ph, arguing against the coactivator function of LSD1 being generally controlled by this mechanism. Importantly, we have shown in a recent study that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 silencing impaired FOXA1 binding at AR-regulated enhancers, suggesting a role of LSD1 in regulating enhancer availability to AR. In the current study, we found that LSD1 inhibition globally disrupts FOXA1 chromatin binding prior to androgen stimulation and thus impairs further AR recruitment, resulting in the global inhibition of AR transcriptome. Using immunoprecipitation and mass-spectrometry assays, we discovered that LSD1 maintains FOXA1 binding by demethylating lysine K270, adjacent to its DNA binding domain. We further found that the expression of a methylation-deficient FOXA1 mutant (K270R) stabilizes AR chromatin binding and allows PCa cells resistant to enzalutamide treatment. Using a clinical tested LSD1 inhibitor, we have shown in vivothat LSD1 inhibition dramatically decreases PCa growth alone and in synergy with enzalutamide in AR/AR variants-positive CRPC models. These findings provide fundamental new insights into FOXA1, LSD1, and mechanisms that regulate the activity of AR in PCa cells. As LSD1 inhibitors are moving into the clinic, these new insights into LSD1 function strongly support LSD1 as a therapeutic target in CRPC. Citation Format: Shuai Gao, Sujun Chen, Dong Han, Wanting Han, Jude N. Owiredu, Steven P. Balk, Housheng H. He, Changmeng Cai. FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 923.

CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.

Non-Hermitian quasilocalization and ring attractor neural networks.

Eigenmodes of a broad class of "sparse" random matrices, with interactions concentrated near the diagonal, exponentially localize in space, as initially discovered in 1957 by Anderson for quantum systems. Anderson localization plays ubiquitous roles in varieties of problems from electrons in solids to mechanical and optical systems. However, its implications in neuroscience (where the connections can be strongly asymmetric) have been largely unexplored, mainly because synaptic connectivity matrices of neural systems are often "dense," which makes the eigenmodes spatially extended. Here we explore roles that Anderson localization could be playing in neural networks by focusing on "spatially structured" disorder in synaptic connectivity matrices. Recently neuroscientists have experimentally confirmed that the local excitation and global inhibition (LEGI) ring attractor model can functionally represent head direction cells in Drosophila melanogaster central brain. We first study a non-Hermitian (i.e., asymmetric) tight-binding model with disorder and then establish a connection to the LEGI ring attractor model. We discover that (1) principal eigenvectors of the LEGI ring attractor networks with structured nearest-neighbor disorder are "quasilocalized," even with fully dense inhibitory connections; and (2) the quasilocalized eigenvectors play dominant roles in the early time neural dynamics, and the location of the principal quasilocalized eigenvectors predicts an initial location of the "bump of activity" representing, for example, a head direction of an insect. Our investigations open up venues for explorations at the intersection between the theory of Anderson localization and neural networks with spatially structured disorder.

A Novel Sex-Dependent Target for the Treatment of Postoperative Pain: The NLRP3 Inflammasome.

In recent years the innate immune system has been shown to be crucial for the pathogenesis of postoperative pain. The mediators released by innate immune cells drive the sensitization of sensory neurons following injury by directly acting on peripheral nerve terminals at the injury site. The predominate sensitization signaling pathway involves the proinflammatory cytokine interleukin-1β (IL-1β). IL-1β is known to cause pain by directly acting on sensory neurons. Evidence demonstrates that blockade of IL-1β signaling decreases postoperative pain, however complete blockade of IL-1β signaling increases the risk of infection and decreases effective wound healing. IL-1β requires activation by an inflammasome; inflammasomes are cytosolic receptors of the innate immune system. NOD-like receptor protein 3 (NLRP3) is the predominant inflammasome activated by endogenous molecules that are released by tissue injury such as that which occurs during neuropathic and inflammatory pain disorders. Given that selective inhibition of NLRP3 alleviates postoperative mechanical pain, its selective targeting may be a novel and effective strategy for the treatment of pain that would avoid complications of global IL-1β inhibition. Moreover, NLRP3 is activated in pain in a sex-dependent and cell type-dependent manner. Sex differences in the innate immune system have been shown to drive pain and sensitization through different mechanisms in inflammatory and neuropathic pain disorders, indicating that it is imperative that both sexes are studied when researchers investigate and identify new targets for pain therapeutics. This review will highlight the roles of the innate immune response, the NLRP3 inflammasome, and sex differences in neuropathic and inflammatory pain.

Targeted Notch1 inhibition with a Notch1 antibody, OMP-A2G1, decreases tumor growth in two murine models of prostate cancer in association with differing patterns of DNA damage response gene expression.

Notch plays a protumorigenic role in many cancers including prostate cancer (PCa). Global notch inhibition of multiple Notch family members using γ-secretase inhibitors has shown efficacy in suppressing PCa growth in murine models. However, global Notch inhibition is associated with marked toxicity due to the widespread function of many different Notch family members in normal cell physiology. Accordingly, in the current study, we explored if specific inhibition of Notch1 would effectively inhibit PCa growth in a murine model. The androgen-dependent VCaP and androgen-independent DU145 cell lines were injected subcutaneously into mice. The mice were treated with either control antibody 1B7.11, anti-Notch1 antibody (OMP-A2G1), docetaxel or the combination of OMP-A2G1 and docetaxel. Tumor growth was measured using calipers. At the end of the study, tumors were assessed for proliferative response, apoptotic response, Notch target gene expression, and DNA damage response (DDR) expression. OMP-A2G1 alone inhibited tumor growth of both PCa cell lines to a greater extent than docetaxel alone. There was no additive or synergistic effect of OMP-A2G1 and docetaxel. The primary toxicity was weight loss that was controlled with dietary supplementation. Proliferation and apoptosis were affected differentially in the two cell lines. OMP-A2G1 increased expression of the DDR gene GADD45α in VCaP cells but downregulated GADD45α in Du145 cells. Taken together, these data show that Notch1 inhibition decreases PCa xenograft growth but does so through different mechanisms in the androgen-dependent VCaP cell line vs the androgen-independent DU145 cell line. These results provide a rationale for further exploration of targeted Notch inhibition for therapy of PCa.

Sialyltransferase inhibition leads to inhibition of tumor cell interactions with E-selectin, VCAM1, and MADCAM1, and improves survival in a human multiple myeloma mouse model.

Aberrant glycosylation resulting from altered expression of sialyltransferases, such as ST3 β-galactoside α2-3-sialyltransferase 6, plays an important role in disease progression in multiple myeloma (MM). Hypersialylation can lead to increased immune evasion, drug resistance, tumor invasiveness, and disseminated disease. In this study, we explore the in vitro and in vivo effects of global sialyltransferase inhibition on myeloma cells using the pan-sialyltransferase inhibitor 3Fax-Neu5Ac delivered as a per-acetylated methyl ester pro-drug. Specifically, we show in vivo that 3Fax-Neu5Ac improves survival by enhancing bortezomib sensitivity in an aggressive mouse model of MM. However, 3Fax-Neu5Ac treatment of MM cells in vitro did not reverse bortezomib resistance conferred by bone marrow (BM) stromal cells. Instead, 3Fax-Neu5Ac significantly reduced interactions of myeloma cells with E-selectin, MADCAM1 and VCAM1, suggesting that reduced sialylation impairs extravasation and retention of myeloma cells in the BM. Finally, we showed that 3Fax-Neu5Ac alters the post-translational modification of the α4 integrin, which may explain the reduced affinity of α4β1/α4β7 integrins for their counter-receptors. We propose that inhibiting sialylation may represent a valuable strategy to restrict myeloma cells from entering the protective BM microenvironment, a niche in which they are normally protected from chemotherapeutic agents such as bortezomib. Thus, our work demonstrates that targeting sialylation to increase the ratio of circulating to BM-resident MM cells represents a new avenue that could increase the efficacy of other anti-myeloma therapies and holds great promise for future clinical applications.

Executive Functions in Girls With and Without Childhood ADHD Followed Through Emerging Adulthood: Developmental Trajectories

Using an all-female sample, we examined trajectories of executive functioning (EF) performance from childhood through emerging adulthood—and their prediction of key emerging-adult outcomes. One hundred forty girls carefully diagnosed with attention deficit/hyperactivity disorder (ADHD) and 88 matched comparison girls were administered EF measurements assessing global EF, response inhibition, and verbal working memory during childhood (M age = 9.5 years), adolescence (M age = 14.1 years), the earliest years of adulthood (M age = 19.6 years), and the end of emerging adulthood (M age = 25.6 years). Retention rates were excellent. Hierarchical linear modeling was used to estimate growth curves for each EF measure. The linear EF slopes were then used to explore how changes in EF interacted with each participant’s persistence/remission of ADHD over time to influence behavioral, emotional, and academic impairment in emerging adulthood. Although all women experienced absolute improvements in EF performance across time, women with histories of ADHD consistently lagged behind comparison women, even if their ADHD symptoms had remitted by early adulthood. However, EF performance over time did not significantly influence the link between ADHD status and (a) maternal reports of associated behavioral and emotional impairment or (b) objective measures of academic achievement. These findings indicate that EF deficits should be considered when developing and implementing treatments for ADHD through emerging adulthood. Future research should be aimed at understanding the mechanisms behind these observed trajectory differences.

Control of excitatory hierarchical circuits by parvalbumin-FS basket cells in layer 5 of the frontal cortex: insights for cortical oscillations.

The cortex contains multiple neuron types with specific connectivity and functions. Recent progress has provided a better understanding of the interactions of these neuron types as well as their output organization particularly for the frontal cortex, with implications for the circuit mechanisms underlying cortical oscillations that have cognitive functions. Layer 5 pyramidal cells (PCs) in the frontal cortex comprise two major subtypes: crossed-corticostriatal (CCS) and corticopontine (CPn) cells. Functionally, CCS and CPn cells exhibit similar phase-dependent firing during gamma waves but participate in two distinct subnetworks that are linked unidirectionally from CCS to CPn cells. GABAergic parvalbumin-expressing fast-spiking (PV-FS) cells, necessary for gamma oscillation, innervate PCs, with stronger and global inhibition to somata and weaker and localized inhibitions to dendritic shafts/spines. While PV-FS cells form reciprocal connections with both CCS and CPn cells, the excitation from CPn to PV-FS cells exhibits short-term synaptic dynamics conducive for oscillation induction. The electrical coupling between PV-FS cells facilitates spike synchronization among PV-FS cells receiving common excitatory inputs from local PCs and inhibits other PV-FS cells via electrically communicated spike afterhyperpolarizations. These connectivity characteristics can promote synchronous firing in the local networks of CPn cells and firing of some CCS cells by anode-break excitation. Thus subsets of L5 CCS and CPn cells within different levels of connection hierarchy exhibit coordinated activity via their common connections with PV-FS cells, and the resulting PC output drives diverse neuronal targets in cortical layer 1 and the striatum with specific temporal precision, expanding the computational power of the cortical network.

Novel insights into gene expression regulation during meiosis revealed by translation elongation dynamics

The ability to dynamically control mRNA translation has a great impact on many intracellular processes. Whereas it is believed that translational control in eukaryotes occurs mainly at initiation, the condition-specific changes at the elongation level and their potential regulatory role remain unclear. Using computational approaches applied to ribosome profiling data, we show that elongation rate is dynamic and can change considerably during the yeast meiosis to facilitate the selective translation of stage-specific transcripts. We observed unique elongation changes during meiosis II, including a global inhibition of translation elongation at the onset of anaphase II accompanied by a sharp shift toward increased elongation for genes required at this meiotic stage. We also show that ribosomal proteins counteract the global decreased elongation by maintaining high initiation rates. Our findings provide new insights into gene expression regulation during meiosis and demonstrate that codon usage evolved, among others, to optimize timely translation.

Peptidyl arginine deiminase-4 exacerbates ischemic AKI by finding NEMO.

Peptidyl arginine deiminase-4 (PAD4) catalyzes the conversion of peptidylarginine residues to peptidylcitrulline. We have previously shown that kidney ischemia-reperfusion (I/R) injury increases renal proximal tubular PAD4 expression and activity. Furthermore, kidney PAD4 plays a critical role in ischemic acute kidney injury (AKI) by promoting renal tubular inflammation, neutrophil infiltration, and NF-κB activation. However, the mechanisms of PAD4-mediated renal tubular inflammation and NF-κB activation after I/R remain unclear. Here, we show that recombinant PAD4 preferentially citrullinates recombinant IKKγ [also called NF-κB essential modulator (NEMO)] over recombinant IKKα or IKKβ. Consistent with this finding, PAD4 citrullinated renal proximal tubular cell IKKγ and promoted NF-κB activation via IκBα phosphorylation in vitro. NEMO inhibition with a selective NEMO-binding peptide attenuated PAD4-mediated proinflammatory cytokine mRNA induction in HK-2 cells. Moreover, NEMO inhibition did not affect proximal tubular cell survival, proliferation, or apoptosis, unlike global NF-κB inhibition. In vivo, NEMO-binding peptide treatment protected against ischemic AKI. Finally, NEMO-binding peptide attenuated recombinant PAD4-mediated exacerbation of ischemic AKI, renal tubular inflammation, and apoptosis. Taken together, our results show that PAD4 exacerbates ischemic AKI and inflammation by promoting renal tubular NF-κB activity and inflammation via NEMO citrullination. Targeting NEMO activation may serve as a potential therapy for this devastating clinical problem.

Global translation inhibition yields condition-dependent de-repression of ribosome biogenesis mRNAs.

Ribosome biogenesis (RiBi) is an extremely energy intensive process that is critical for gene expression. It is thus highly regulated, including through the tightly coordinated expression of over 200 RiBi genes by positive and negative transcriptional regulators. We investigated RiBi regulation as cells initiated meiosis in budding yeast and noted early transcriptional activation of RiBi genes, followed by their apparent translational repression 1 hour (h) after stimulation to enter meiosis. Surprisingly, in the representative genes examined, measured translational repression depended on their promoters rather than mRNA regions. Further investigation revealed that the signature of this regulation in our data depended on pre-treating cells with the translation inhibitor, cycloheximide (CHX). This treatment, at 1 h in meiosis, but not earlier, rapidly resulted in accumulation of RiBi mRNAs that were not translated. This effect was also seen in with CHX pre-treatment of cells grown in media lacking amino acids. For NSR1, this effect depended on the –150 to –101 region of the promoter, as well as the RiBi transcriptional repressors Dot6 and Tod6. Condition-specific RiBi mRNA accumulation was also seen with translation inhibitors that are dissimilar from CHX, suggesting that this phenomenon might represent a feedback response to global translation inhibition.

Unsupervised learning by competing hidden units

It is widely believed that end-to-end training with the backpropagation algorithm is essential for learning good feature detectors in early layers of artificial neural networks, so that these detectors are useful for the task performed by the higher layers of that neural network. At the same time, the traditional form of backpropagation is biologically implausible. In the present paper we propose an unusual learning rule, which has a degree of biological plausibility and which is motivated by Hebb's idea that change of the synapse strength should be local-i.e., should depend only on the activities of the pre- and postsynaptic neurons. We design a learning algorithm that utilizes global inhibition in the hidden layer and is capable of learning early feature detectors in a completely unsupervised way. These learned lower-layer feature detectors can be used to train higher-layer weights in a usual supervised way so that the performance of the full network is comparable to the performance of standard feedforward networks trained end-to-end with a backpropagation algorithm on simple tasks.

Widespread PERK-dependent repression of ER targets in response to ER stress

The UPR (Unfolded Protein Response) is a well-orchestrated response to ER protein folding and processing overload, integrating both transcriptional and translational outputs. Its three arms in mammalian cells, the PERK translational response arm, together with the ATF6 and IRE1-XBP1-mediated transcriptional arms, have been thoroughly investigated. Using ribosome footprint profiling, we performed a deep characterization of gene expression programs involved in the early and late ER stress responses, within WT or PERK −/− Mouse Embryonic Fibroblasts (MEFs). We found that both repression and activation gene expression programs, affecting hundreds of genes, are significantly hampered in the absence of PERK. Specifically, PERK −/− cells do not show global translational inhibition, nor do they specifically activate early gene expression programs upon short exposure to ER stress. Furthermore, while PERK −/− cells do activate/repress late ER-stress response genes, the response is substantially weaker. Importantly, we highlight a widespread PERK-dependent repression program, consisting of ER targeted proteins, including transmembrane proteins, glycoproteins, and proteins with disulfide bonds. This phenomenon occurs in various different cell types, and has a major translational regulatory component. Moreover, we revealed a novel interplay between PERK and the XBP1-ATF6 arms of the UPR, whereby PERK attenuates the expression of a specific subset of XBP1-ATF6 targets, further illuminating the complexity of the integrated ER stress response.

A Legionella pneumophila Kinase Phosphorylates the Hsp70 Chaperone Family to Inhibit Eukaryotic Protein Synthesis

Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's abilityto inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.

By-Products of Heparin Production Provide a Diverse Source of Heparin-like and Heparan Sulfate Glycosaminoglycans

Global production of pharmaceutical heparin (Hp) is increasing, and the production process from raw mucosal material results in large amounts of waste by-products. These contain lower sulfated Hp-like and heparan sulfate (HS), as well as other glycosaminoglycans, which are bioactive entities with pharmaceutical potential. Here we describe the first purification, structural and functional characterisation of Hp-like and HS polysaccharides from the four major by-product fractions of standard heparin production. Analysis of the by-products by disaccharide composition analysis and NMR demonstrated a range of structural characteristics which differentiate them from Hp (particularly reduced sulfation and sulfated disaccharide content), and that they are each distinct. Functional properties of the purified by-products varied, each displaying distinct anticoagulant profiles in different assays, and all exhibiting significantly lower global and specific inhibition of the coagulation pathway than Hp. The by-products retained the ability to promote cell proliferation via fibroblast growth factor receptor signalling, with only minor differences between them. These collective analyses indicate that they represent an untapped and economical source of structurally-diverse Hp-like and HS polysaccharides with the potential for enhancing future structure-activity studies and uncovering new biomedical applications of these important natural products.

Ionizing radiation affects miRNA composition in both young and old mice

Purpose: Humans are exposed to both natural (e.g. soil, cosmic rays) and human-made radiation sources (e.g. medical devices, nuclear energy production) on a daily basis. The use of medical radiation sources such as Computed Tomography (CT) scans and X-ray has increased rapidly, especially in the treatment of older populations. Micro Ribonucleic Acids (miRNAs) are the major regulators of multiple low-dose radiation-induced biological processes through post-translational inhibition. As a result, understanding age-related changes of miRNA profiles that may compromise the population after low dose radiation exposure has become increasingly important.Materials and methods: In this study, we irradiated both young (2 months) and old (26 months) C57BL/6J mice with low dose radiation (10 mGy and 100 mGy at 1 mGy/min using an open beam 60Co gamma source) and checked the miRNA expression profiles.Results: The global miRNA expression of old mice was significantly reduced compared to that of young mice. Low dose radiation at 10 mGy significantly increased the global miRNA expression in both old and young mice one week following irradiation, which suggests that 10 mGy low dose radiation may reverse the global inhibition effects of aging on miRNA expression. Higher 100 mGy radiation slightly reduced the global expression of miRNAs. We also identified several miRNAs that were elevated or reduced in all of the radiation treatment groups; these can be further explored as candidates for the radiation-induced bio-markers.Conclusions: The results of our study demonstrate that both radiation and aging can influence the global expression of miRNAs, while low dose radiation modulates the expression of miRNAs in a dose-, time-, and age-dependent manner.

YAP and TAZ limit cytoskeletal and focal adhesion maturation to enable persistent cell motility.

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

Novel Mutations in nsP2 Abolish Chikungunya Virus-Induced Transcriptional Shutoff and Make the Virus Less Cytopathic without Affecting Its Replication Rates.

Alphavirus infections are characterized by global inhibition of cellular transcription and rapid induction of a cytopathic effect (CPE) in cells of vertebrate origin. Transcriptional shutoff impedes the cellular response to alphavirus replication and prevents establishment of an antiviral state. Chikungunya virus (CHIKV) is a highly pathogenic alphavirus representative, and its nonstructural protein 2 (nsP2) plays critical roles in both inhibition of transcription and CPE development. Previously, we have identified a small peptide in Sindbis virus (SINV) nsP2 (VLoop) that determined the protein's transcriptional inhibition function. It is located in the surface-exposed loop of the carboxy-terminal domain of nsP2 and exhibits high variability between members of different alphavirus serocomplexes. In this study, we found that SINV-specific mutations could not be directly applied to CHIKV. However, by using a new selection approach, we identified a variety of new VLoop variants that made CHIKV and its replicons incapable of inhibiting cellular transcription and dramatically less cytopathic. Importantly, the mutations had no negative effect on RNA and viral replication rates. In contrast to parental CHIKV, the developed VLoop mutants were unable to block induction of type I interferon. Consequently, they were cleared from interferon (IFN)-competent cells without CPE development. Alternatively, in murine cells that have defects in type I IFN production or signaling, the VLoop mutants established persistent, noncytopathic replication. The mutations in nsP2 VLoop may be used for development of new vaccine candidates against alphavirus infections and vectors for expression of heterologous proteins.IMPORTANCE Chikungunya virus is an important human pathogen which now circulates in both the Old and New Worlds. As in the case of other Old World alphaviruses, CHIKV nsP2 not only has enzymatic functions in viral RNA replication but also is a critical inhibitor of the antiviral response and one of the determinants of CHIKV pathogenesis. In this study, we have applied a new strategy to select a variety of CHIKV nsP2 mutants that no longer exhibited transcription-inhibitory functions. The designed CHIKV variants became potent type I interferon inducers and acquired a less cytopathic phenotype. Importantly, they demonstrated the same replication rates as the parental CHIKV. Mutations in the same identified peptide of nsP2 proteins derived from other Old World alphaviruses also abolished their nuclear functions. Such mutations can be further exploited for development of new attenuated alphaviruses.

A Non-canonical Pathway with Potential for Safer Modulation of Transforming Growth Factor-β1 in Steroid-Resistant Airway Diseases

SummaryImpaired therapeutic responses to anti-inflammatory glucocorticoids (GC) in chronic respiratory diseases are partly attributable to interleukins and transforming growth factor β1 (TGF-β1). However, previous efforts to prevent induction of GC insensitivity by targeting established canonical and non-canonical TGF-β1 pathways have been unsuccessful. Here we elucidate a TGF-β1 signaling pathway modulating GC activity that involves LIM domain kinase 2-mediated phosphorylation of cofilin1. Severe, steroid-resistant asthmatic airway epithelium showed increased levels of immunoreactive phospho-cofilin1. Phospho-cofilin1 was implicated in the activation of phospholipase D (PLD) to generate the effector(s) (lyso)phosphatidic acid, which mimics the TGF-β1-induced GC insensitivity. TGF-β1 induction of the nuclear hormone receptor corepressor, SMRT (NCOR2), was dependent on cofilin1 and PLD activities. Depletion of SMRT prevented GC insensitivity. This pathway for GC insensitivity offers several promising drug targets that potentially enable a safer approach to the modulation of TGF-β1 in chronic inflammatory diseases than is afforded by global TGF-β1 inhibition.