Control Of Protein Translation Research Articles

ProSAAS is preferentially up-regulated during homeostatic scaling and reduces amyloid plaque burden in the 5xFAD mouse hippocampus.

The accumulation of β-amyloid in Alzheimer's disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti-aggregant behavior against alpha-synuclein and β-amyloid fibrillation invitro and is up-regulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti-aggregant role in Alzheimer's progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up- and down-regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control and/or altered protein degradation. ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild-type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Importantly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS-encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimer's disease progression.

FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury

The engineered clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.

The Odds of Protein Translation Control Under Stress.

Physical or chemical stress is commonly known to inhibit protein translation at the cellular level. Since the process of protein translation requires catalysis by a multi-component machinery containing eukaryotic initiation factors (eIFs) and ribosomes in a sequence of reactions, how the process fails to proceed and whether certain genes can escape such blockade have provoked research efforts. Lines of evidence have demonstrated that phosphorylation of eIF4E or dephosphorylation of 4E-binding proteins (4E-BPs) prevents the formation of the eukaryotic translation initiation factor 4F (eIF4F) complex, whereas phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) due to activation of heme-regulated inhibitor (HRI), general control nonderepressible 2 (GCN2), protein kinase RNA-like endoplasmic reticulum kinase (PERK), or protein kinase R (PKR) by a diverse array of stressors prevents eIF2-GTP-tRNAiMet ternary complex assembly. These signal the abandonment of translation initiation via 5'-7-methylguanine (m7G) cap recognition by eIF4E. Stress can promote cleavage of tRNAs, impediment of rRNA processing, changes in the epitranscriptomic landscape, ribosome stalling or collision, activation of ribosomal surveillance systems, and assembly of the stress granules. Although these events contribute to the general inhibition of protein translation, a few proteins can bypass such negativity and become translated selectively. Such selective protein translation is primarily m7G cap independent through the integrated stress response or Internal Ribosomal Entry Site (IRES). The newly synthesized proteins often influence cell fate, facilitate cell survival, and build endogenous defense. Insights into the general inhibition of protein translation and selective translation of specific proteins will advance our understanding of the etiology or progression of human diseases involving cellular stress from viral infection or inflammation to myocardial infarction, stroke, or neurodegenerative disease. Antioxid. Redox Signal. 40, 943-947.

Abstract 3174: Crosstalk between protein translation and DNA replication during stress response and cancer therapy

Abstract Tumor microenvironment, which includes both various types of cells (tumor cells, stromal cells, immune cells, etc.) and an interstitial fluid system, plays a key role in tumorigenesis, therapy response, and disease outcome. While the cell component has been heavily studied in cancer treatment, the interstitial fluid system has not. A key feature of the fluid system is constant oscillation of osmolytes such as salt, glucose, and proteins due to unstable blood supply and tumor metabolism, resulting in cycles of osmotic stress and recovery. Yet, how this osmotic stress - recovery cycle mediates tumorigenesis and cancer therapy remains poorly understood. Our unpublished results reveal sequential activation of molecular events during osmotic stress and recovery. In the stress stage, protein translation, transcription, and DNA replication are inhibited. Following recovery, protein translation resumes, gene transcription is greatly upregulated, but DNA replication is not immediately recovered. It turns out that a DNA replication checkpoint is activated during recovery; as a result, DNA replication is halted. Defects in activating protein translation inhibition during stress fail to activate the replication checkpoint during recovery, indicating a causative link from protein translation inhibition to DNA replication checkpoint activation. Many genes involved in this crosstalk are altered in a wide range of human tumors. Ovarian cancer cells that are resistant to standard of care (for instance, cisplatin) demonstrate defects in this crosstalk; however, they were also more sensitive to a combination of osmotic stress followed by the presence of replication checkpoint inhibitors during recovery than parental cancer cells. These findings highlight a unique role of the interstitial fluid system in tumor development and therapy response. They also suggest a new anticancer strategy by combining osmotic stress with checkpoint inhibitors. Key words: Tumor microenvironment; osmotic stress; protein translation control; DNA replication checkpoint; cancer therapy Citation Format: Fangfang Wang. Crosstalk between protein translation and DNA replication during stress response and cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3174.

In utero pulse injection of isotopic amino acids quantifies protein turnover rates during murine fetal development

Protein translational control is critical for ensuring that the fetus develops correctly and that necessary organsand tissues are formed and functional. We developed an in utero method to quantify tissue-specific protein dynamics by monitoring amino acid incorporation into the proteome after pulse injection. Fetuses of pregnant mice were injected with isotopically labeled lysine and arginine via the vitelline vein at various embyonic days, and organs and tissues were harvested. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates were calculated between 3.81E-5 and 0.424 h-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver vs. brain); however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates, which correlated with known physiological changes during mouse development.

Copper‐Free Click Chemistry‐Mediated Assembly of Single Quantum Dot Nanosensor for Accurately Monitoring Locus‐Specific m6A in Cancer Cells†

Comprehensive SummaryN6‐methyladenosine (m6A) plays an important role in embryogenesis, nuclear export, transcription splicing, and protein translation control. Herein, we demonstrate a copper‐free click chemistry‐mediated assembly of single quantum dot (QD) nanosensor for accurately monitoring locus‐specific m6A in cancer cells. The m6A‐sensitive endoribonuclease MazF can digest the unmethylated A‐RNA, and the intact m6A‐RNA then hybridizes with DNA probes a and b to produce a sandwich hybrid, initiating the click chemistry to generate probe a–b ligation product via first tandem ligation detection reaction (LDR) cycle. Subsequently, DNA probes c and d can hybridize with the probe a–b ligation product to generate the probe c–d ligation product via second LDR cycle. Both LDR cycles can be repeated through denaturation and annealing reaction to generate abundant biotin‐/fluorophore‐modified probe c–d ligation products that can easily assemble on the QD surface to induce distinct fluorescence resonance energy transfer (FRET) between QD and Cy5. This assay can be homogenously performed without the involvement of copper catalyst, m6A‐specific antibody, radioactive labeling, ligase enzyme, enzymatic reverse transcription, and next‐generation sequencing. Moreover, it can discriminate even 0.01% m6A level in complex samples and accurately measure cellular m6A‐RNA expression, providing a promising avenue for clinical diagnostics and biomedical research.

MicroRNAs Function in Dental Stem Cells as a Promising Biomarker and Therapeutic Target for Dental Diseases.

Undifferentiated, highly proliferative, clonogenic, and self-renewing dental stem cells have paved the way for novel approaches to mending cleft palates, rebuilding lost jawbone and periodontal tissue, and, most significantly, recreating lost teeth. New treatment techniques may be guided by a better understanding of these cells and their potential in terms of the specificity of theregenerative response. MicroRNAs have been recognized as an essential component in stem cell biology due to their role as epigenetic regulators of the processes that determine stem cell destiny. MicroRNAs have been proven to be crucial in a wide variety of molecular and biological processes, including apoptosis, cell proliferation, migration, and necrocytosis. MicroRNAs have been recognized to control protein translation, messenger RNA stability, and transcription and have been reported to play essential roles in dental stem cell biology, including the differentiation of dental stem cells, the immunological response, apoptosis, and the inflammation of the dental pulp. Because microRNAs increase dental stem cell differentiation, they may be used in regenerative medicine to either preserve the stem cell phenotype or to aid in the development of tooth tissue. The development of novel biomarkers and therapies for dental illnesses relies heavily on progress made in our knowledge of the roles played by microRNAs in regulating dental stem cells. In this article, we discuss how dental stem cells and their associated microRNAs may be used to cure dental illness.

Ribonucleic Acid-Mediated Control of Protein Translation Under Stress.

Significance: The need of cells to constantly respond to endogenous and exogenous stress has necessitated the evolution of pathways to counter the deleterious effects of stress and to restore cellular homeostasis. The inability to activate a timely and adequate response can lead to disease and is a hallmark of aging. Besides protein-coding genes, cells contain a plethora of noncoding regulatory elements that allow cells to respond rapidly and efficiently to external stimuli by activating highly specific and tightly controlled mechanisms. Many of these programs converge on the regulation of translation, one of the most energy-consuming processes in cells. Recent Advances: The noncoding dimension of translational regulation includes short and long noncoding ribonucleic acids (ncRNAs), as well as messenger RNA features, such as the sequence and modification status of the 5' and 3' untranslated regions (UTRs), that do not change the amino acid sequence of the produced protein. Critical Issues: In this review, we discuss the regulatory role of the nonprotein-coding components of translation under stress, particularly oxidative stress. We conclude that the regulation of translation through ncRNAs, UTRs, and nucleotide modifications is emerging as a critical component of the stress response. Future Directions: Further areas of study using long-read sequencing technologies will be discussed. Antioxid. Redox Signal. 39, 374-389.

Differential Participation of Plant Ribosomal Proteins from the Small Ribosomal Subunit in Protein Translation under Stress.

Upon exposure to biotic and abiotic stress, plants have developed strategies to adapt to the challenges imposed by these unfavorable conditions. The energetically demanding translation process is one of the main elements regulated to reduce energy consumption and to selectively synthesize proteins involved in the establishment of an adequate response. Emerging data have shown that ribosomes remodel to adapt to stresses. In Arabidopsis thaliana, ribosomes consist of approximately eighty-one distinct ribosomal proteins (RPs), each of which is encoded by two to seven genes. Recent research has revealed that a mutation in a given single RP in plants can not only affect the functions of the RP itself but can also influence the properties of the ribosome, which could bring about changes in the translation to varying degrees. However, a pending question is whether some RPs enable ribosomes to preferentially translate specific mRNAs. To reveal the role of ribosomal proteins from the small subunit (RPS) in a specific translation, we developed a novel approach to visualize the effect of RPS silencing on the translation of a reporter mRNA (GFP) combined to the 5'UTR of different housekeeping and defense genes. The silencing of genes encoding for NbRPSaA, NbRPS5A, and NbRPS24A in Nicotiana benthamiana decreased the translation of defense genes. The NbRACK1A-silenced plant showed compromised translations of specific antioxidant enzymes. However, the translations of all tested genes were affected in NbRPS27D-silenced plants. These findings suggest that some RPS may be potentially involved in the control of protein translation.

Electron transport chain and mTOR inhibition synergistically decrease CD40 signaling and counteract venetoclax resistance in chronic lymphocytic leukemia.

CD40 signaling upregulates BCL-XL and MCL-1 expression in the chronic lymphocytic leukemia (CLL) lymph node microenvironment, affording resistance to the BCL-2 inhibitor, venetoclax. Venetoclax resistance in the therapeutic setting and after long-term laboratory selection has been linked to metabolic alterations, but the underlying mechanism(s) are unknown. We aimed here to discover how CD40 stimulation as a model for tumor microenvironment-mediated metabolic changes, affects venetoclax sensitivity/resistance. CD40 stimulation increased oxidative phosphorylation and glycolysis, but only inhibition of oxidative phosphorylation countered venetoclax resistance. Furthermore, blocking mitochondrial import of pyruvate, glutamine or fatty acids affected CLL metabolism, but did not prevent CD40-mediated resistance to venetoclax. In contrast, inhibition of the electron transport chain (ETC) at complex I, III or V attenuated CLL activation and ATP production, and downregulated MCL-1 and BCL-XL, correlating with reduced CD40 surface expression. Moreover, ETC inhibition equaled mTOR1/2 but not mTOR1 inhibition alone for venetoclax resistance, and all three pathways were linked to control of general protein translation. In line with this, ETC plus mTOR inhibition synergistically counteracted venetoclax resistance. These findings link oxidative CLL metabolism to CD40 expression and cellular signaling, and may hold clinical potential.

The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S] cluster in the archaeum Methanococcus maripaludis

Thiolation of uridine 34 in the anticodon loop of several tRNAs is conserved in the three domains of life and guarantees fidelity of protein translation. U34-tRNA thiolation is catalyzed by a complex of two proteins in the eukaryotic cytosol (named Ctu1/Ctu2 in humans), but by a single NcsA enzyme in archaea. We report here spectroscopic and biochemical experiments showing that NcsA from Methanococcus maripaludis (MmNcsA) is a dimer that binds a [4Fe-4S] cluster, which is required for catalysis. Moreover, the crystal structure of MmNcsA at 2.8 Å resolution shows that the [4Fe-4S] cluster is coordinated by three conserved cysteines only, in each monomer. Extra electron density on the fourth nonprotein-bonded iron most likely locates the binding site for a hydrogenosulfide ligand, in agreement with the [4Fe-4S] cluster being used to bind and activate the sulfur atom of the sulfur donor. Comparison of the crystal structure of MmNcsA with the AlphaFold model of the human Ctu1/Ctu2 complex shows a very close superposition of the catalytic site residues, including the cysteines that coordinate the [4Fe-4S] cluster in MmNcsA. We thus propose that the same mechanism for U34-tRNA thiolation, mediated by a [4Fe-4S]-dependent enzyme, operates in archaea and eukaryotes.

Celastrol attenuates hepatitis C virus translation and inflammatory response in mice by suppressing heat shock protein 90β.

Hepatitis C virus (HCV) infection is one of the major factors to trigger a sustained hepatic inflammatory response and hence hepatocellular carcinoma (HCC), but direct-acting-antiviral (DAAs) was not efficient to suppress HCC development. Heat shock protein 90 kDa (HSP90) is highly abundant in different types of cancers, and especially controls protein translation, endoplasmic reticulum stress, and viral replication. In this study we investigated the correlation between the expression levels of HSP90 isoforms and inflammatory response marker NLRP3 in different types of HCC patients as well as the effect of a natural product celastrol in suppression of HCV translation and associated inflammatory response in vivo. We identified that the expression level of HSP90β isoform was correlated with that of NLRP3 in the liver tissues of HCV positive HCC patients (R2 = 0.3867, P < 0.0101), but not in hepatitis B virus-associated HCC or cirrhosis patients. We demonstrated that celastrol (3, 10, 30 μM) dose-dependently suppressed the ATPase activity of both HSP90α and HSP90β, while its anti-HCV activity was dependent on the Ala47 residue in the ATPase pocket of HSP90β. Celastrol (200 nM) halted HCV internal ribosomal entry site (IRES)-mediated translation at the initial step by disrupting the association between HSP90β and 4EBP1. The inhibitory activity of celastrol on HCV RNA-dependent RNA polymerase (RdRp)-triggered inflammatory response also depended on the Ala47 residue of HSP90β. Intravenous injection of adenovirus expressing HCV NS5B (pAde-NS5B) in mice induced severe hepatic inflammatory response characterized by significantly increased infiltration of immune cells and hepatic expression level of Nlrp3, which was dose-dependently ameliorated by pretreatment with celastrol (0.2, 0.5 mg/kg, i.p.). This study reveals a fundamental role of HSP90β in governing HCV IRES-mediated translation as well as hepatic inflammation, and celastrol as a novel inhibitor of HCV translation and associated inflammation by specifically targeting HSP90β, which could be developed as a lead for the treatment of HSP90β positive HCV-associated HCC.

Network biology analysis of P23H rhodopsin interactome identifies protein and mRNA quality control mechanisms

Rhodopsin is essential for phototransduction, and many rhodopsin mutations cause heritable retinal degenerations. The P23H rhodopsin variant generates a misfolded rhodopsin protein that photoreceptors quickly target for degradation by mechanisms that are incompletely understood. To gain insight into how P23H rhodopsin is removed from rods, we used mass spectrometry to identify protein interaction partners of P23H rhodopsin immunopurified from RhoP23H/P23H mice and compared them with protein interaction partners of wild-type rhodopsin from Rho+/+ mice. We identified 286 proteins associated with P23H rhodopsin and 276 proteins associated with wild-type rhodopsin. 113 proteins were shared between wild-type and mutant rhodopsin protein interactomes. In the P23H rhodopsin protein interactome, we saw loss of phototransduction, retinal cycle, and rhodopsin protein trafficking proteins but gain of ubiquitin-related proteins when compared with the wild-type rhodopsin protein interactome. In the P23H rhodopsin protein interactome, we saw enrichment of gene ontology terms related to ER-associated protein degradation, ER stress, and translation. Protein–protein interaction network analysis revealed that translational and ribosomal quality control proteins were significant regulators in the P23H rhodopsin protein interactome. The protein partners identified in our study may provide new insights into how photoreceptors recognize and clear mutant rhodopsin, offering possible novel targets involved in retinal degeneration pathogenesis.

Dynamic Regulation of the Nexus Between Stress Granules, Roquin, and Regnase-1 Underlies the Molecular Pathogenesis of Warfare Vesicants.

The use of chemical warfare agents is prohibited but they have been used in recent Middle Eastern conflicts. Their accidental exposure (e.g. arsenical lewisite) is also known and causes extensive painful cutaneous injury. However, their molecular pathogenesis is not understood. Here, we demonstrate that a nexus of stress granules (SGs), integrated stress, and RNA binding proteins (RBPs) Roquin and Reganse-1 play a key role. Lewisite and its prototype phenylarsine oxide (PAO) induce SG assembly in skin keratinocytes soon after exposure, which associate with various RBPs and translation-related proteins. SG disassembly was detected several hours after exposure. The dynamics of SG assembly-disassembly associates with the chemical insult and cell damage. Enhanced Roquin and Regnase-1 expression occurs when Roquin was recruited to SGs and Regnase-1 to the ribosome while in the disassembling SGs their expression is decreased with consequent induction of inflammatory mediators. SG-targeted protein translational control is regulated by the phosphorylation-dependent activation of eukaryotic initiation factors 2α (eIF2α). Treatment with integrated stress response inhibitor (ISRIB), which blocks eIF2α phosphorylation, impacted SG assembly dynamics. Topical application of ISRIB attenuated the inflammation and tissue disruption in PAO-challenged mice. Thus, the dynamic regulation of these pathways provides underpinning to cutaneous injury and identify translational therapeutic approach for these and similar debilitating chemicals.

Effects of tumor protein translation control antisense RNA1 on radiosensitivity, proliferation, migration and invasion of hepatocellular carcinoma cells by targeting miR-30c-5p

Objective: To investigate the effects of tumor protein translation control antisense RNA1 (TPT1-AS1) on the radiosensitivity, cell proliferation, migration and invasion of hepatocellular carcinoma cells by targeting microRNA-30c-5p (miR-30c-5p). Methods: Thirty-four cases of liver cancer tissues and adjacent normal tissues were derived from liver cancer patients who were admitted to Shanxi Provincial People's Hospital from March 2016 to March 2018. Liver cancer HepG2 cell was transfected with negative control siRNA (si-NC group), si-TPT1-AS1 (si-TPT1-AS1 group), pcDNA3.1 (pcDNA3.1 group), pcDNA3.1-TPT1-AS1 (pcDNA3.1-TPT1-AS1 group), si-TPT1-AS1 and anti-miR-NC (si-TPT1-AS1+ anti-miR-NC group), si-TPT1-AS1 and anti-miR-30c-5p (si-TPT1-AS1+ anti-miR-30c-5p group), respectively. Real-time quantitative reverse transcription polymerase chain reaction (qPCR) was used to detect the transcription levels of TPT1-AS1 and miR-30c-5p in normal tissues adjacent to cancer and liver cancer tissues, the clone formation test was used to test the radiosensitivity of HepG2 cells, and the Methyl Thiazolyl Tetrazolium (MTT) test was used to test the proliferation of HepG2 cells. Cell cycle distribution was detected by flow cytometry, Transwell array was used to detect the migration and invasion ability of HepG2 cells, dual luciferase reporter array was used to verify the targeting relationship of TPT1-AS1 and miR-30c-5p, western blot was used to detect the expressions of proliferation, migration and invasion-related proteins. Results: The expression levels of TPT1-AS1 and miR-30c-5p in liver cancer tissues were 0.84±0.08 and 0.13±0.01, statistically different from 0.31±0.03 and 0.50±0.05 in normal tissues adjacent to cancer (P<0.05). When the cells were treated with 2, 4, 6, 8 Gy irradiation, the cell survival scores of the si-TPT1-AS1 group were 0.280±0.040, 0.069±0.011, 0.020±0.003 and 0.005±0.001, respectively, lower than 0.648±0.070, 0.348±0.080, 0.130±0.020 and 0.060±0.009 of the si-NC group (P<0.05), the radiosensitization ratio of the si-TPT1-AS1 group was 1.672. The number of cell migration and invasion in the si-TPT1-AS1 group were (50.00±4.36) and (44.00±4.03), respectively, which were lower than (109.00±8.68) and (94.00±7.49) in the si-NC group (P<0.05), the cell absorbance (A) values at 24, 48 and 72 hours were 0.28±0.03, 0.43±0.04 and 0.68±0.07, respectively, lower than 0.46±0.04, 0.87±0.08 and 1.35±0.13 of the si-NC group (P<0.05), the protein expression levels of Cyclin D1, p21, E-cadherin and MMP-2 were 0.25±0.02, 0.65±0.06, 0.68±0.07 and 0.27±0.03, respectively, statistically different from 0.88±0.08, 0.17±0.02, 0.14±0.01 and 0.89±0.09 of si-NC group (P<0.05), the proportions of S phase and G(2) phase in the si-TPT1-AS1 group were (17.82±1.03)% and (34.15±2.29)%, respectively, significantly different from (35.14±2.61)% and (16.84±1.21)% in the si-NC group (P<0.05). The luciferase activity of cells in the WT-TPT1-AS1+ miR-30c-5p group was 0.26±0.02, lower than 0.92±0.09 in the WT-TPT1-AS1+ miR-NC group (P<0.05). The cell survival scores in the si-TPT1-AS1+ anti-miR-30c-5p group were 0.450±0.081, 0.200+ 0.045, 0.070±0.010, 0.026±0.004 after treatment with 2, 4, 6, 8 Gy irradiation, higher than 0.285±0.043, 0.075±0.014, 0.028±0.004, 0.006±0.001 of si-TPT1-AS1+ anti-miR-NC group (P<0.05). The radiosensitization ratio of the si-TPT1-AS1+ anti-miR-30c-5p group was 0.694. The number of migration and invasion in the si-TPT1-AS1+ anti-miR-30c-5p group were 79.00±6.65 and 68.00±6.33, higher than (52.00±4.41) and (46.00±4.06) of si-TPT1-AS1+ anti-miR-NC Group (P<0.05), the A values at 24, 48 and 72 hours were 0.37±0.03, 0.64±0.06 and 0.96±0.09, respectively, higher than 0.26±0.03, 0.41±0.04, and 0.65±0.06 of si-TPT1-AS1+ anti-miR-NC group (P<0.05), the expression levels of Cyclin D1, p21, E-cadherin and MMP-2 protein were 0.57±0.06, 0.43±0.04, 0.43±0.04 and 0.64±0.06, statistically different from 0.24±0.02, 0.66±0.06, 0.65±0.06 and 0.28±0.03 of the si-TPT1-AS1+ anti-miR-NC group (P<0.05). Conclusions: The expression of TPT1-AS1 up-regulates in the liver cancer tissues. TPT1-AS1 may down-regulate miR-30c-5p expression, reduce the radiosensitivity of liver cancer cells, and promote the proliferation, migration and invasion of liver cancer cells.

Monomethyl branched-chain fatty acid mediates amino acid sensing upstream of mTORC1

Animals have developed various nutrient-sensing mechanisms for survival under fluctuating environmental conditions. Although extensive cell-culture-based analyses have identified diverse mediators of amino acid sensing upstream of mTOR, studies using animal models to examine intestine-initiated amino acid sensing mechanisms under specific physiological conditions are lacking. Here, we developed a Caenorhabditis elegans model to examine the impact of amino acid deficiency on development. We discovered a leucine-derived monomethyl branched-chain fatty acid and its downstream metabolite, glycosphingolipid, which critically mediates the overall amino acid sensing by intestinal and neuronal mTORC1, which in turn regulates postembryonic development at least partly by controlling protein translation and ribosomal biogenesis. Additional data suggest that a similar mechanism may operate in mammals. This study uncovers an amino-acid-sensing mechanism mediated by a lipid biosynthesis pathway.

Cytoplasmic TDP-43 is involved in cell fate during stress recovery.

Transactive response DNA binding protein 43 (TDP-43) is an RNA processing protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear TDP-43 mislocalizes in patients to the cytoplasm, where it forms ubiquitin-positive inclusions in affected neurons and glia. Physiologically, cytoplasmic TDP-43 is associated with stress granules (SGs). Here, we explored TDP-43 cytoplasmic accumulation and stress granule formation following osmotic and oxidative stress. We show that sorbitol drives TDP-43 redistribution to the cytoplasm, while arsenite induces the recruitment of cytoplasmic TDP-43 to TIA-1 positive SGs. We demonstrate that inducing acute oxidative stress after TDP-43 cytoplasmic relocalization by osmotic shock induces poly (ADP-ribose) polymerase (PARP) cleavage, which triggers cellular toxicity. Recruitment of cytoplasmic TDP-43 to polyribosomes occurs in an SH-SY5Y cellular stress model and is observed in FTD brain lysate. Moreover, the processing body (P-body) marker DCP1a is detected in TDP-43 granules during recovery from stress. Overall, this study supports a central role for cytoplasmic TDP-43 in controlling protein translation in stressed cells.

Schizophrenia risk ZNF804A interacts with its associated proteins to modulate dendritic morphology and synaptic development

Schizophrenia (SZ) is a devastating brain disease that affects about 1% of world population. Among the top genetic associations, zinc finger protein 804A (ZNF804A) gene encodes a zinc finger protein, associated with SZ and biolar disorder (BD). Copy number variants (CNVs) of ZNF804A have been observed in patients with autism spectrum disorders (ASDs), anxiety disorder, and BD, suggesting that ZNF804A is a dosage sensitive gene for brain development. However, its molecular functions have not been fully determined. Our previous interactomic study revealed that ZNF804A interacts with multiple proteins to control protein translation and neural development. ZNF804A is localized in the cytoplasm and neurites in the human cortex and is expressed in various types of neurons, including pyramidal, dopaminergic, GABAergic, and Purkinje neurons in mouse brain. To further examine the effect of gene dosage of ZNF804A on neurite morphology, both knockdown and overexpression of ZNF804A in primary neuronal cells significantly attenuate dendritic complex and spine formation. To determine the factors mediating these phenotypes, interestingly, three binding proteins of ZNF804A, galectin 1 (LGALS1), fasciculation and elongation protein zeta 1 (FEZ1) and ribosomal protein SA (RPSA), show different effects on reversing the deficits. LGALS1 and FEZ1 stimulate neurite outgrowth at basal level but RPSA shows no effect. Intriguingly, LGALS1 but not FEZ1, reverses the neurite outgrowth deficits induced by ZNF804A knockdown. However, FEZ1 and RPSA but not LGALS1, can ameliorate ZNF804A overexpression-mediated dendritic abnormalities. Thus, our results uncover a critical post-mitotic role of ZNF804A in neurite and synaptic development relevant to neurodevelopmental pathologies.

Control of the Anterior Pituitary Cell Lineage Regulator POU1F1 by the Stem Cell Determinant Musashi.

The adipokine leptin regulates energy homeostasis through ubiquitously expressed leptin receptors. Leptin has a number of major signaling targets in the brain, including cells of the anterior pituitary (AP). We have previously reported that mice lacking leptin receptors in AP somatotropes display growth hormone (GH) deficiency, metabolic dysfunction, and adult-onset obesity. Among other targets, leptin signaling promotes increased levels of the pituitary transcription factor POU1F1, which in turn regulates the specification of somatotrope, lactotrope, and thyrotrope cell lineages within the AP. Leptin's mechanism of action on somatotropes is sex dependent, with females demonstrating posttranscriptional control of Pou1f1 messenger RNA (mRNA) translation. Here, we report that the stem cell marker and mRNA translational control protein, Musashi1, exerts repression of the Pou1f1 mRNA. In female somatotropes, Msi1 mRNA and protein levels are increased in the mouse model that lacks leptin signaling (Gh-CRE Lepr-null), coincident with lack of POU1f1 protein, despite normal levels of Pou1f1 mRNA. Single-cell RNA sequencing of pituitary cells from control female animals indicates that both Msi1 and Pou1f1 mRNAs are expressed in Gh-expressing somatotropes, and immunocytochemistry confirms that Musashi1 protein is present in the somatotrope cell population. We demonstrate that Musashi interacts directly with the Pou1f1 mRNA 3' untranslated region and exerts translational repression of a Pou1f1 mRNA translation reporter in a leptin-sensitive manner. Musashi immunoprecipitation from whole pituitary reveals coassociated Pou1f1 mRNA. These findings suggest a mechanism in which leptin stimulation is required to reverse Musashi-mediated Pou1f1 mRNA translational control to coordinate AP somatotrope function with metabolic status.

The role of cap-dependent translation in aged-related changes in neuroimmunity and affective behaviors

Translation regulation in the context of aged-associated inflammation and behavioral impairments is not well characterized. Aged individuals experience lower life quality due to behavioral impairments. In this study, we used young and aged transgenic mice that are unable to activate the cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E) to examine the role of protein translation control in aging, memory, depression, and anxiety. To determine how products of cap-dependent translation play a permissive role in aged-associated inflammation, we assessed levels of pro-inflammatory cytokines in various brain regions involved in the above-mentioned behaviors. We found that functional eIF4E is not necessary for age-related deficits in spatial and short-term memory but is important for depressive and anxiety-like behavior and this is correlated with pro-inflammatory cytokines in discrete brain regions. Thus, we have begun to elucidate a role for eIF4E phosphorylation in the context of aged-related behavioral impairments and chronic low-grade inflammation that may help identify novel immune modulators for therapeutic targets and decrease the burden of self-care among the geriatric population.