Inhibition Of mRNA Translation Research Articles

Binding of SARS-CoV-2 Nonstructural Protein 1 to 40S Ribosome Inhibits mRNA Translation.

Experimental evidence has established that SARS-CoV-2 NSP1 acts as a factor that restricts cellular gene expression and impedes mRNA translation within the ribosome's 40S subunit. However, the precise molecular mechanisms underlying this phenomenon have remained elusive. To elucidate this issue, we employed a combination of all-atom steered molecular dynamics and coarse-grained alchemical simulations to explore the binding affinity of mRNA to the 40S ribosome, both in the presence and absence of SARS-CoV-2 NSP1. Our investigations revealed that the binding of SARS-CoV-2 NSP1 to the 40S ribosome leads to a significant enhancement in the binding affinity of mRNA. This observation, which aligns with experimental findings, strongly suggests that SARS-CoV-2 NSP1 has the capability to inhibit mRNA translation. Furthermore, we identified electrostatic interactions between mRNA and the 40S ribosome as the primary driving force behind mRNA translation. Notably, water molecules were found to play a pivotal role in stabilizing the mRNA-40S ribosome complex, underscoring their significance in this process. We successfully pinpointed the specific SARS-CoV-2 NSP1 residues that play a critical role in triggering the translation arrest.

KDM2B is required for ribosome biogenesis and its depletion unequally affects mRNA translation.

KDM2B is a JmjC domain lysine demethylase, which promotes cell immortalization, stem cell self-renewal and tumorigenesis. Here we employed a multi-omics strategy to address its role in ribosome biogenesis and mRNA translation. These processes are required to sustain cell proliferation, an important cancer hallmark. Contrary to earlier observations, KDM2B promotes ribosome biogenesis by stimulating the transcription of genes encoding ribosome biogenesis factors and ribosomal proteins, particularly those involved in the biogenesis of the 40S ribosomal subunits. Knockdown of KDM2B impaired the assembly of the small and large subunit processomes, as evidenced by specific defects in pre-ribosomal RNA processing. The final outcome was a decrease in the rate of ribosome assembly and in the abundance of ribosomes, and inhibition of mRNA translation. The inhibition of translation was distributed unequally among mRNAs with different features, suggesting that mRNA-embedded properties influence how mRNAs interpret ribosome abundance. This study identified a novel mechanism contributing to the regulation of translation and provided evidence for a rich biology elicited by a pathway that depends on KDM2B, and perhaps other regulators of translation.

Exploring the Impact of mRNA Modifications on Translation Efficiency and Immune Tolerance to Self-Antigens.

Therapeutic modified mRNAs are being developed for a broad range of human diseases. However, the impact of potential miscoding of modified mRNAs on self-tolerance remains unknown. Additionally, more studies are needed to explore the effects of nucleoside alkylation on translation. While all six tested modifications are tolerated as substrates by T7 RNA polymerase and inhibited mRNA immunogenicity, the translation efficiency varied significantly depending on the type of modification. In contrast to methylation, ethylation at the N1 position of pseudouridine (Ψ) hindered translation, suggesting that the C5-C1' glycosidic bond alone is not a critical element for high translation. Inhibition of mRNA translation was also observed with 5-methoxyuridine modification. However, this inhibition was partially alleviated through the optimization of mRNA coding sequences. BALB/c mice immunized with syngeneic ψ-modified mRNA encoding for Wilms' tumor antigen-1 (WT1) developed a low but significant level of anti-WT1 IgG antibodies compared to those immunized with either unmodified or N1-methyl ψ-modified mRNA. Overall, the data indicate that adding a simple ethyl group (-CH2CH3) at the N1 position of ψ has a major negative effect on translation despite its reduced immunogenicity. Additionally, mRNA containing Ψ may alter translation fidelity at certain codons, which could lead to a breakdown of immune tolerance to self-antigens. This concern should be taken into account during gene replacement therapies, although it could benefit mRNA-based vaccines by generating a diverse repertoire of antigens.

Cryo-EM structure of Influenza A virus NS1 and antiviral protein kinase PKR complex

Influenza A virus is the cause of a widespread human disease with high morbidity and mortality rates. The influenza virus encodes non-structural protein 1 (NS1), an exceedingly multifunctional virulence component. NS1 plays essential roles in viral replication and evasion of the cellular innate immune system. Protein kinase RNA-activated also known as protein kinase R (PKR) phosphorylates translation initiation factor eIF-2α on serine 51 to inhibit protein synthesis in virus-infected mammalian cells. Consequently, PKR activation inhibits mRNA translation, which results in the assert of both viral protein synthesis and cellular and possibly apoptosis in response to virus infection. Host signaling pathways are important in the replication of influenza virus, but the mechanisms involved remain to be characterized. Herein, the structure of NS1 and PKR complex was determined using Cryo-EM. We found the N91, E94, and G95 residues of PKR bind directly with N188, D125, and K126, respectively, of NS1. Furthermore, the study shows that PKR peptide offers a potential treatment for Influenza A virus infections.

FAM86A methylation of eEF2 links mRNA translation elongation to tumorigenesis

eEF2 post-translational modifications (PTMs) can profoundly affect mRNA translation dynamics. However, the physiologic function of eEF2K525 trimethylation (eEF2K525me3), a PTM catalyzed by the enzyme FAM86A, is unknown. Here, we find that FAM86A methylation of eEF2 regulates nascent elongation to promote protein synthesis and lung adenocarcinoma (LUAD) pathogenesis. The principal physiologic substrate of FAM86A is eEF2, with K525me3 modeled to facilitate productive eEF2-ribosome engagement during translocation. FAM86A depletion in LUAD cells causes 80S monosome accumulation and mRNA translation inhibition. FAM86A is overexpressed in LUAD and eEF2K525me3 levels increase through advancing LUAD disease stages. FAM86A knockdown attenuates LUAD cell proliferation and suppression of the FAM86A-eEF2K525me3 axis inhibits cancer cell and patient-derived LUAD xenograft growth invivo. Finally, FAM86A ablation strongly attenuates tumor growth and extends survival in KRASG12C-driven LUAD mouse models. Thus, our work uncovers an eEF2 methylation-mediated mRNA translation elongation regulatory node and nominates FAM86A as an etiologic agent in LUAD.

Phase separation of GRP7 facilitated by FERONIA-mediated phosphorylation inhibits mRNA translation to modulate plant temperature resilience

Changes in ambient temperature profoundly affect plant growth and performance. Therefore, the molecular basis of plant acclimation to temperature fluctuation is of great interest. In this study, we discovered that GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7) contributes to cold and heat tolerance in Arabidopsis thaliana. We found that exposure to a warm temperature rapidly induces GRP7 condensates in planta, which can be reversed by transfer to a lower temperature. Cell biology and biochemical assays revealed that GRP7 undergoes liquid–liquid phase separation (LLPS) in vivo and in vitro. LLPS of GRP7 in the cytoplasm contributes to the formation of stress granules that recruit RNA, along with the translation machinery component eukaryotic initiation factor 4E1 (eIF4E1) and the mRNA chaperones COLD SHOCK PROTEIN 1 (CSP1) and CSP3, to inhibit translation. Moreover, natural variations in GRP7 affecting the residue phosphorylated by the receptor kinase FERONIA alter its capacity to undergo LLPS and correlate with the adaptation of some Arabidopsis accessions to a wider temperature range. Taken together, our findings illustrate the role of translational control mediated by GRP7 LLPS to confer plants with temperature resilience.

A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

MiR-2765 involved in ammonia nitrogen stress via negative regulation of autophagy in shrimp

MicroRNA (miRNA) is a highly conserved non-coding tiny endogenous RNA molecule that regulates various cellular functions by inhibiting mRNA translation or promoting the degradation of proteins. In this study, we identified a specific miRNA (designed as Pva-miR-2765) from Penaeus vannamei, which widely distributed in different tissues of shrimp, with the highest concentration found in the intestine. Through fluorescence in situ hybridization (FISH), we observed that Pva-miR-2765 is primarily located in the cytoplasm. Interestingly, we found that the expression of Pva-miR-2765 significantly decreased in hemocytes, hepatopancreas and gill under ammonia nitrogen stress. Furthermore, when Pva-miR-2765 was silenced, the autophagy level in shrimp significantly increased. Additionally, Pva-miR-2765 was found to promote pathological damage in the hepatopancreas of shrimp. Subsequently, correlation analysis revealed a negative relationship between the expression of Pva-miR-2765 and PvTBC1D7. To confirm this interaction, we conducted a dual luciferase reporter gene assay, which demonstrated that Pva-miR-2765 inhibit the expression of PvTBC1D7 by interacting with its 3’UTR. And the expression level of PvTBC1D7 in shrimp decreased significantly under ammonia nitrogen stress in Pva-miR-2765 overexpressed. Our findings suggest that Pva-miR-2765 can reduce autophagy in P. vannamei by inhibiting the regulation of PvTBC1D7, thereby participating in the oxidative stress of shrimp caused by ammonia nitrogen stress.

Different dysregulations of CYFIP1 and CYFIP2 in distinct types of dementia

In humans, the cytoplasmic FMR1 interacting protein (CYFIP) family consists of two members, namely CYFIP1 and CYFIP2. Both CYFIP1 and CYFIP2 function in the WAVE regulatory complex (WRC), which regulates actin polymerization. Additionally, these two proteins form a posttranscriptional regulatory complex with the fragile X mental retardation protein (FMRP), which suppresses mRNA translation. Thus, CYFIP1 and CYFIP2 are important signalling regulators at synapses, and mutations in their genes are associated with neurodevelopmental and neuropsychiatric disorders, including intellectual disabilities. Moreover, dysregulation of the CYFIP protein family is involved in Alzheimer’s disease (AD). However, the relevance of the CYFIP family in other dementias is largely unknown. Here, we compared CYFIP1/2 protein levels in the post-mortem hippocampus from patients with AD, dementia with Lewy bodies (DLB), vascular dementia (VaD) and frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). Consistent with previous findings, CYFIP2 was reduced in AD hippocampus. In DLB and VaD hippocampus, the protein level of CYFIP2 and CYFIP1 was unaltered. Finally, an increase in the protein level of both CYFIP1 and CYFIP2 was noted in FTLD-TDP hippocampus. These findings reveal that the protein levels of the CYFIP family is distinct in different types of dementia, suggesting that the pathogenesis of these neurodegenerative disorders has divergent impacts on hippocampal synaptic function.

Detailed role of microRNA-mediated regulation of PI3K/AKT axis in human tumors.

The regulation of signal transmission and biological processes, such as cell proliferation, apoptosis, metabolism, migration, and angiogenesis are greatly influenced by the PI3K/AKT signaling pathway. Highly conserved endogenous non-protein-coding RNAs known as microRNAs (miRNAs) have the ability to regulate gene expression by inhibiting mRNA translation or mRNA degradation. MiRNAs serve key role in PI3K/AKT pathway as upstream or downstream target, and aberrant activation of this pathway contributes to the development of cancers. A growing body of research shows that miRNAs can control the PI3K/AKT pathway to control the biological processes within cells. The expression of genes linked to cancers can be controlled by the miRNA/PI3K/AKT axis, which in turn controls the development of cancer. There is also a strong correlation between the expression of miRNAs linked to the PI3K/AKT pathway and numerous clinical traits. Moreover, PI3K/AKT pathway-associated miRNAs are potential biomarkers for cancer diagnosis, therapy, and prognostic evaluation. The role and clinical applications of the PI3K/AKT pathway and miRNA/PI3K/AKT axis in the emergence of cancers are reviewed in this article.

Targeting eIF4A triggers an interferon response to synergize with chemotherapy and suppress triple-negative breast cancer.

Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A with zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages toward an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon (IFN) response uniformly across models. The induction of an IFN response is partially due to the inhibition of Sox4 translation by zotatifin. A similar induction of IFN-stimulated genes was observed in breast cancer patient biopsies following zotatifin treatment. Surprisingly, zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened IFN response, resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for zotatifin, and provide a rationale for new combination regimens consisting of zotatifin and chemotherapy or immunotherapy as treatments for TNBC.

Long-Acting E. coli-Derived Asparaginase Potentiates the Anti-Leukemic Effect of BCL2 Inhibition, but Not MCL1 Inhibition, in Preclinical Models of Acute Myeloid Leukemia

Background: Asparaginases are enzymes that deplete circulating asparagine and glutamine from plasma and can therefore be used therapeutically for cancers that are sensitive to amino acid depletion. Commercially available asparaginases are isolated fromeither E. coli or Erwinia chrysanthemi (also called crisantaspase), the latter having a higher glutaminase activity. Our previous studies have shown that pegcrisantaspase (PegC), a long-acting pegylated crisantaspase, synergizes with the BCL2 inhibitor, Venetoclax (Ven), to kill several AML cell lines and patient-derived primary AML cells with complex karyotype in vitro and in vivo in a xenograft mouse model. Whether long-acting E. coli-derived asparaginases will have similar anti-AML activity as a single agent or in combination with BCL-2 inhibition, and the potential of asparaginases to synergize with inhibitors of MCL1, a key resistance factor of BCL2 inhibition, has yet to be determined. Methods: Using a panel of 4 human AML cell lines (MOLM14, MV411, MonoMac6, HL60) as well as primary AML patient samples, weestablished the anti-leukemic activity of the BCL2 inhibitor S55746 and the MCL1 inhibitor S63845 alone and in combination with the long-acting E. coli asparaginase, calaspargase pegol-mknl (CalPegA). To determine the ability of S55746 or S63845 with or without CalPegA to inhibit mRNA translation, an established mechanism of Ven-PegC anti-leukemic activity, we measured the dissociation of the translation initiation factor eIF4E from 4EBP1, a negative regulator of cap-dependent mRNA translation, which then allows the formation of translation initiation complexes. The in vivo anti-leukemic efficacy of S55746 and S63845 alone and in combination with CalPegA was tested using an orthotopic AML xenograft mouse model with luciferase-expressing MV411 cells. Results: We found that CalPegA treatment resulted in a dose-dependent inhibition of AML cell proliferation, with IC 50 values ranging from 0.78 to 2.8 IU/mL. Single agent S55746 and S63845 treatment inhibited AML cell proliferation with IC 50 values ranging from 0.01 to 6.7 µM and 0.0001 to 0.08 µM, respectively. In 3 of the human AML cell lines (MOLM-14, MV4-11, and MonoMac6), the addition of a small dose (IC 20) of CalPegA potentiated the anti-leukemic effect of S55746, reducing the IC 50 by approximately 1- 4-fold, and by 10-fold in HL60 cells (Figure 1). This potentiation was also observed in 3 out of 4 primary AML patient samples tested, with the IC 50 of S55746 reduced by 4.5, 18, and 156-fold by the addition of CalPegA. The activity of S63845 was not substantially impacted by the addition of CalPegA in either AML cell lines (Figure 1) or primary samples. Immunoprecipitation of 4EBP1 revealed that treatment with a combination of either S63845 or S55746 with CalPegA increased the binding of eIF4E to 4EBP1 compared to single agent treatments, suggesting an inhibition of translational complex formation. In a MV411 tumor-bearing model, treatment withS55746-CalPegA combination reduced tumor burden over time significantly more than either single agent. S63845 treatment was highly effective at inhibiting leukemia growth over time and was not improved by the addition of CalPegA, as tumor burden in the S63845 and S63845 + CalPegA-treated groups was not significantly different over the course of the study (Figure 2). Conclusions: We report that the E. coli asparaginase, CalPegA, enhances the anti-leukemic effect of the BCL2 inhibitor, S55746, but does not impact the activity of the MCL1 inhibitor, S63845, in AML cell lines, patient derived primary AML samples, and in an AML xenograft mouse model. Ongoing studies are further investigating the anti-leukemic mechanism of S55476/S63845 + CalPegA and examining the efficacy and pharmacodynamic/pharmacokinetic effects of these agents in a patient derived xenograft model of AML.

MRNA Translation Inhibition Targets Bioenergetic Homeostasis in AML Cells in Vitro and In Vivo and Synergizes with Cytarabine and Venetoclax

Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. Recently, inhibition of BCL-2 with venetoclax in combination with hypomethylating agents has emerged as an attractive strategy for high-risk AML cases. Another promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1) (Oki et al, Nature Comm, 2021). However, clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. To curtail these drawbacks, we adopted a strategy of inhibiting an important effector of the mTORC1 signaling pathway controlling mRNA translation - the eukaryotic initiation factor 4A (eIF4A), subunit of the translation initiation complex eIF4F. Recent evidence suggests that translational programs mediated by the mTORC1/4E-BP/eIF4F axis can support resistance to therapy in various cancer models driven by oncogenic kinases, in part by allowing cellular metabolic plasticity (Hulea L et al, Cell Metab. 2018). In fact, metabolism, and specifically mitochondrial oxidative phosphorylation, has emerged as a central dependency of AML cells, sustaining resistance to therapy and recurrence. Using the MOLM-14 human AML cell line to model therapy-resistant disease, we previously demonstrated the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, both in vitro and in vivo (Fooks et al, J Exp Clin Cancer Res 2022). eIF4Ai affected cellular metabolism, by reducing mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Concomitantly, eIF4Ai decreased intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mitochondrial ROS. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Importantly, CR-1-31-B acted synergistically in vitro in combination with cytarabine or venetoclax. Recently, we have expanded our characterization of the eIF4Ai/venetoclax combination in MOML-14 cells, and showed that venetoclax potentiates the CR-1-31-B-induced inhibition of cellular respiration, glycolysis and ATP production (Seahorse). The combination provokes a robust apoptotic response with different temporal dynamics to single treatment, as measured using the Incucyte platform. In vivo, we have validated the strong anti-leukemic response induced by CR-1-31-B in a MOLM-14 transplantation model, after only 7 days of treatment, and showed that venetoclax slightly potentiates this effect. In addition, eIF4A inhibition reduces the levels of several metabolic proteins (GLS1, IDH1), which supports our previous observations of decreased levels of TCA cycle metabolites. We have confirmed the metabolic and pro-apoptotic effect of eIF4Ai in a second cellular model of AML, U937. Consistent with previous results, in U937 cells CR-1-31-B induces apoptosis at low nM concentrations, reduces cellular bioenergetics, ATP production, as well as levels of pro-proliferative (cyclin D3, CDK4) and anti-apoptotic (BCL-2, MCL-1) proteins. Our collective studies highlight (i) the importance of the crosstalk between mRNA translation and metabolic regulation and (ii) that direct inhibition of translation represents an appealing therapeutic strategy for clinical cases of therapy resistance that are dependent on the mTORC1/eIF4F axis. This is of interest as several translation inhibitors are currently tested in phase 1/2 clinical trials in solid malignancies. # co-corresponding authors

MicroRNA-34 Family in Cancers: Role, Mechanism, and Therapeutic Potential.

MicroRNA (miRNA) are small noncoding RNAs that play vital roles in post-transcriptional gene regulation by inhibiting mRNA translation or promoting mRNA degradation. The dysregulation of miRNA has been implicated in numerous human diseases, including cancers. miR-34 family members (miR-34s), including miR-34a, miR-34b, and miR-34c, have emerged as the most extensively studied tumor-suppressive miRNAs. In this comprehensive review, we aim to provide an overview of the major signaling pathways and gene networks regulated by miR-34s in various cancers and highlight the critical tumor suppressor role of miR-34s. Furthermore, we will discuss the potential of using miR-34 mimics as a novel therapeutic approach against cancer, while also addressing the challenges associated with their development and delivery. It is anticipated that gaining a deeper understanding of the functions and mechanisms of miR-34s in cancer will greatly contribute to the development of effective miR-34-based cancer therapeutics.

Non-Invasive Transdermal Delivery of Antisense Oligonucleotides with Biocompatible Ionic Liquids.

Nucleic acid drugs, including antisense oligonucleotides (ASOs), have received considerable attention as novel therapeutics for the treatment of intractable diseases. Despite their potential benefits, ASOs are currently administered via injection, which can negatively impact patient quality of life because of the prevalence of severe injection site reactions. Non-invasive transdermal administration of ASOs is desirable but highly challenging owing to the strong barrier imposed by the stratum corneum, which only permits the penetration of small molecules under 500 Da. For ASOs to exert their antisense effect, they must traverse the negatively charged cell membrane and reach the cytoplasm. In this study, we used the solid-in-oil (S/O) dispersion technology to facilitate the skin permeation of ASOs by coating the drug with a hydrophobic surfactant molecule, specifically lipid-based ionic liquid (IL) surfactants with high biocompatibility and transdermal penetration-enhancing properties. To induce the antisense effect, it was important to achieve simultaneous transdermal delivery and intracellular entrapment of ASOs. In vitro investigations indicated that the newly prepared IL-S/O enhanced the transdermal penetration and intracellular delivery of ASOs, thus inhibiting mRNA translation of the target TGF-β. In addition, in vivo investigations of tumor-bearing mice suggested that the anti-tumor effect of the IL-S/O was similar to that of injection. This study demonstrates the potential of non-invasive transdermal delivery carriers based on biocompatible ILs, which can be applied to a variety of nucleic acid drugs.

Biochemical and HDX Mass Spectral Characterization of the SARS-CoV-2 Nsp1 Protein.

A major challenge in defining the pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to better understand virally encoded multifunctional proteins and their interactions with host factors. Among the many proteins encoded by the positive-sense, single-stranded RNA genome, nonstructural protein 1 (Nsp1) stands out due to its impact on several stages of the viral replication cycle. Nsp1 is the major virulence factor that inhibits mRNA translation. Nsp1 also promotes host mRNA cleavage to modulate host and viral protein expression and to suppress host immune functions. To better define how this multifunctional protein can facilitate distinct functions, we characterize SARS-CoV-2 Nsp1 by using a combination of biophysical techniques, including light scattering, circular dichroism, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and temperature-dependent HDX-MS. Our results reveal that the SARS-CoV-2 Nsp1 N- and C-terminus are unstructured in solution, and in the absence of other proteins, the C-terminus has an increased propensity to adopt a helical conformation. In addition, our data indicate that a short helix exists near the C-terminus and adjoins the region that binds the ribosome. Together, these findings provide insights into the dynamic nature of Nsp1 that impacts its functions during infection. Furthermore, our results will inform efforts to understand SARS-CoV-2 infection and antiviral development.

MiR-3960 inhibits bladder cancer progression via targeting of DEXI

PurposeMicroRNAs (miRNAs) are dominant cargo in exosomes and act as master regulators of cell function, inhibiting mRNA translation and affecting gene silencing. Some aspects of tissue-specific miRNA transport in bladder cancer (BC) and its role in cancer progression are not fully understood. Materials and methodsA microarray was used to identify miRNAs in mouse bladder carcinoma cell line MB49 exosomes. Real-time reverse transcription polymerase chain reaction was used to examine the expression of miRNAs in BC and healthy donor serum. Western blotting and immunohistochemical staining were used to examine the expression of dexamethasone-induced protein (DEXI) in patients with BC. CRISPR-Cas 9 was used to knock out Dexi in MB49, and flow cytometry was performed to test cell proliferation ability and apoptosis under chemotherapy. Human BC organoid culture, miR-3960 transfection, and 293T-exosome-loaded miR-3960 delivery were used to analyze the effect of miR-3960 on BC progression. ResultsThe results showed that miR-3960 levels in BC tissue were positively correlated with patient survival time. Dexi was a major target of miR-3960. Dexi knockout inhibited MB49 cell proliferation and promoted cisplatin- and gemcitabine-induced apoptosis. Transfection of miR-3960 mimic inhibited DEXI expression and organoid growth. In parallel, 293T-exosome-loaded miR-3960 delivery and Dexi knockout significantly inhibited subcutaneous growth of MB49 cells in vivo. ConclusionOur results demonstrate the potential role of miR-3960-mediated inhibition of DEXI as a therapeutic strategy against BC.

FMRP phosphorylation and interactions with Cdh1 regulate association with dendritic RNA granules and MEF2-triggered synapse elimination

Fragile X Messenger Ribonucleoprotein (FMRP) is necessary for experience-dependent, developmental synapse elimination and the loss of this process may underlie the excess dendritic spines and hyperconnectivity of cortical neurons in Fragile X Syndrome, a common inherited form of intellectual disability and autism. Little is known of the signaling pathways that regulate synapse elimination and if or how FMRP is regulated during this process. We have characterized a model of synapse elimination in CA1 neurons of organotypic hippocampal slice cultures that is induced by expression of the active transcription factor Myocyte Enhancer Factor 2 (MEF2) and relies on postsynaptic FMRP. MEF2-induced synapse elimination is deficient in Fmr1 KO CA1 neurons, and is rescued by acute (24 h), postsynaptic and cell autonomous reexpression of FMRP in CA1 neurons. FMRP is an RNA binding protein that suppresses mRNA translation. Derepression is induced by posttranslational mechanisms downstream of metabotropic glutamate receptor signaling. Dephosphorylation of FMRP at S499 triggers ubiquitination and degradation of FMRP which then relieves translation suppression and promotes synthesis of proteins encoded by target mRNAs. Whether this mechanism functions in synapse elimination is not known. Here we demonstrate that phosphorylation and dephosphorylation of FMRP at S499 are both necessary for synapse elimination as well as interaction of FMRP with its E3 ligase for FMRP, APC/Cdh1. Using a bimolecular ubiquitin-mediated fluorescence complementation (UbFC) assay, we demonstrate that MEF2 promotes ubiquitination of FMRP in CA1 neurons that relies on activity and interaction with APC/Cdh1. Our results suggest a model where MEF2 regulates posttranslational modifications of FMRP via APC/Cdh1 to regulate translation of proteins necessary for synapse elimination.

Hydrogel armed with Bmp2 mRNA-enriched exosomes enhances bone regeneration

BackgroundSustained release of bioactive BMP2 (bone morphogenetic protein-2) is important for bone regeneration, while the intrinsic short half-life of BMP2 at protein level cannot meet the clinical need. In this study, we aimed to design Bmp2 mRNA-enriched engineered exosomes, which were then loaded into specific hydrogel to achieve sustained release for more efficient and safe bone regeneration.ResultsBmp2 mRNA was enriched into exosomes by selective inhibition of translation in donor cells, in which NoBody (non-annotated P-body dissociating polypeptide, a protein that inhibits mRNA translation) and modified engineered BMP2 plasmids were co-transfected. The derived exosomes were named ExoBMP2+NoBody. In vitro experiments confirmed that ExoBMP2+NoBody had higher abundance of Bmp2 mRNA and thus stronger osteogenic induction capacity. When loaded into GelMA hydrogel via ally-L-glycine modified CP05 linker, the exosomes could be slowly released and thus ensure prolonged effect of BMP2 when endocytosed by the recipient cells. In the in vivo calvarial defect model, ExoBMP2+NoBody-loaded GelMA displayed great capacity in promoting bone regeneration.ConclusionsTogether, the proposed ExoBMP2+NoBody-loaded GelMA can provide an efficient and innovative strategy for bone regeneration.

Abstract 2554: Phosphorylationdependent control of protein synthesis in response to inhibitors of mRNA translation and mitochondrial respiration in HeLa cancer cell line

Abstract Control of protein turnover via regulation of the rates of protein synthesis and degradation is one of the principal means by which cell signaling networks can be modulated. This regulation can also be achieved through alterations in post-translational modifications such as phosphorylation. The human genome encodes ~21,300 proteins, and over 95% of these are subjected to reversible phosphorylation at nearly a million sites by at least 568 protein kinases (PK) and about 156 protein phosphatases (PP). The aim of current study was to use differential treatments of cultured cells with diverse agents to uncover connectivities between protein kinases and their substrates to elucidate the architecture of signaling networks. Western blot analyses showed that the protein synthesis inhibitor anisomycin (Alomone labs, Israel) and the mitochondrial respiratory chain inhibitor arsenate (Sigma, Saint Louis, MO) both increased ribosomal proteins S6 (RPS6) S235+S236+S240 phosphorylation. There were similar patterns of activatory phosphorylations observed for upstream kinases of RPS6, including p70S6K at T255 and RSK1 at S380. Anisomycin and arsenate also enhanced activatory phosphorylation of the kinases p38α at T180+Y182 and CK2α at Y255. We explored whether or not the effect of anisomycin or arsenate on these protein kinases were dependent on mTOR signaling with its inhibitor rapamycin (Calbiochem, EMD Chemicals Inc.), which by itself slightly increased the RPS6 S235+S236+S240 phosphorylation. However, rapamycin completely blocked both anisomycin and arsenate induced RPS6 phosphorylation. Rapamycin did not block phosphorylation of p38α MAPK T180 and Y182 in response to anisomycin or arsenate. The results of CK2α Y255 phosphorylation were particularly interesting, because rapamycin treatment alone increased the phosphorylation at this site as did anisomycin alone, but the combination of these inhibitors blocked this phosphorylation. However, CK2α Y255 phosphorylation in response to arsenate alone and in combination with rapamycin showed that the arsenate induction of phosphorylation was not dependent on mTOR signaling. We hypothesized that this mTOR-independent signaling pathway involves the activation of protein-tyrosine kinases that may target both CK2a and p38 MAPK. Therefore, we designed an in vitro experiment and we found that some protein-tyrosine kinases like Blk, Bmx, Btk, EGFR, Fer, Fes, Frk, Fyn, LynA, Pyk2, Src, Syk, Tec, and TrkB can significantly phosphorylate p38 MAPK at Y182 and CK2α at Y255, whereas some other protein-tyrosine kinases such as Fgr, InsR, Jak2, Met, and Txk primarily targeted CK2α at Y255. We also demonstrated that p38 MAPK is able to directly phosphorylate CK2α near its C-terminus at T360 and S362, and this correlated with activation of its casein phosphotransferase activity. Citation Format: Hamidreza Galavi, Steven Pelech. Phosphorylationdependent control of protein synthesis in response to inhibitors of mRNA translation and mitochondrial respiration in HeLa cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2554.