Protein Synthesis Assay Research Articles

EIF4G2 Promotes Hepatocellular Carcinoma Progression via IRES-dependent PLEKHA1 Translation Regulation.

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and proteomic studies have shown increased protein diversity and abundance in HCC tissues, whereas the role of protein translation has not been extensively explored in HCC. Our research focused on key molecules in the translation process to identify a potential contributor in HCC. We discovered that EIF4G2, a crucial translation initiation factor, is significantly upregulated in HCC tissues and associated with poor prognosis. This study uniquely highlights the impact of EIF4G2 deletion, which suppresses tumor growth and metastasis both in vitro and in vivo. Furthermore, polysome analysis and nascent protein synthesis assays revealed EIF4G2's role in regulating protein translation, specifically identifying PLEKHA1 as a key translational product. This represents a novel mechanistic insight into HCC malignancy. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assays further revealed that EIF4G2 facilitates PLEKHA1 translation via an IRES-dependent manner. Importantly, the synergistic effects of EIF4G2 depletion and PLEKHA1 reduction in inhibiting cell migration and invasion underscore the therapeutic potential of targeting this axis. This study not only advances our understanding of translational regulation in HCC but also identifies the EIF4G2-PLEKHA1 axis as a promising therapeutic target, offering new avenues for intervention in HCC treatment.

Cancer Antibody Engineering: Comparison of Mammalian, Yeast, Bacterial, Plants, Cell-free and Hybridoma Expression Systems.

Cancer is a significant issue worldwide. Generally, commercially available treatments, such as surgery, radiotherapy, and chemotherapy, are associated with undesirable complications. Hence, immunotherapy serves as a crucial alternative to those treatment options. This modality is aimed to boost the immune system through the application of engineered antibodies, which can be produced using recombinant DNA technology. The discussion of the technologies leads to an introduction of the single-chain variable fragment (scFv). Thereafter, the advantages, disadvantages, and challenges associated with different expression systems, such as mammalian cells, yeast cells, bacterial cells, plant cells, and phage display were discussed comprehensively. Furthermore, conventional approaches such as hybridoma and modern approaches such as cell-free protein synthesis (CFPS) and simple colony assays are included. In short, this article has compiled evidence relating to each display system and may serve as a reference for those who aim to explore antibody engineering using one of the methods listed in this article.

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

Abstract PO3-26-10: Small molecule inhibitor of PELP1 exhibit cytotoxic effects on breast cancer cells by blocking ribosome biogenesis

Abstract Introduction: Breast cancer (BC) is the second-leading cause of cancer-related deaths in women. Despite responsive to endocrine therapy, a sizeable portion of estrogen receptor positive (ER+) BC goes on to develop therapy-resistant breast cancer (TR-BC). Furthermore, triple-negative breast cancer (TNBC) has a more aggressive clinical course with fewer targeted therapeutic options. The development of more effective therapies for TR-BC or TNBC represent an unmet need. Excessive ribosome biogenesis has recently been linked to BC progression, and resistance to therapy. Oncogene PELP1, which is required for ribosome biogenesis, is frequently dysregulated in BC and serves as a prognostic factor for poor BC survival and therapy resistance. We recently identified SMIP34 as a PELP1 small molecule inhibitor of its kind and it is effective against both TR-BC and TNBC. The objective of this study is to determine the mechanisms by which SMIP34 exhibit cytotoxic effects on BC cells. Methods: We have used multiple ER+, therapy resistant and TNBC models in cell viability, and apoptosis assays. Modelling studies were done based on Cryo-EM structure of the WDR18/PELP1 Rix1 complex (PDB code 7UWF). Biochemical assays include immune precipitation, reporter assays, GST pulldown, and Western Blotting. SMIP34 effect on protein synthesis was measured by using Western blot of puromycin treated total lysates for puromycinylated protein content. SMIP34 effect on protein synthesis assessed using Cayman’s Protein Synthesis Assay Kit. Ribosome biogenesis in SMIP34 treated cells was monitored using confocal microscopy using rpS6 antibody. Results: In in vitro cell based assays, SMIP34 was highly effective in reducing the cell viability, reducing colony formation and inducing apoptosis of ER+, endocrine therapy resistant, and TNBC model cells. PELP1 homodimer has recently been predicated to act as the core of the WDR18/PELP1 assembly. Our biochemical assay results suggest that SMIP34 binds to the same region involved in the PELP1 homodimerization and disrupts the formation of PELP1 homodimer and subsequently blocks the formation of WDR18/PELP1 Rix1 complex. Modeling studies predicted that altering the conformation of PELP1 aa 696-720 loop by SMIP34 binding may influence the conformation of the following C-terminal region, which may disturb the interaction of other PELP1 partners such as SENP3 and TEX10. Accordingly, the results from immunoprecipitation experiments demonstrated decreased association of Rix1 complex proteins WDR18, TEX10, and SENP3 with PELP1 under conditions of SMIP34 treatment. Further, Western blotting analysis confirmed that SMIP34 treatment decreased levels of PELP1 and its complex proteins, such as WDR18, TEX10, LAS1L, and SENP3. SMIP34 significantly reduced ribosomal biogenesis in a dose dependent manner. Additionally, puromycin labelling experiments confirmed a dose dependent decrease in new protein synthesis upon SMIP34 administration. Data from the DepMap database analysis revealed that PELP1 is required for cancer cell survival. Additionally, analysis of TCGA breast cancer datasets revealed that PELP1 expression is positively correlated with WDR18, TEX10, LAS1L, and SENP3. In in vivo xenograft assays and ex vivo explant assays using tumor tissue, SMIP34 strongly inhibited the growth of ER+, endocrine treatment resistant, and TNBC model cells. Conclusion: These findings provide solid evidence that the Rix1 complex, which is required for ribosomes production, is disturbed by SMIP34 binding to PELP1. SMIP34 represents a new therapeutic for the treatment of both ER+ and TNBC breast cancer. Supported by VA grant 1 I01 BX004545-01A1. Citation Format: Xue Yang, Khaled Mohamed Nassar, Uday Pratap, Rahul Gopalam, Behnam Ebrahimi, Durga Meenakshi Panneerdoss, Xiaonan Li, Yaxia Yuan, Daohong Zhou, Gangadhara R. Sareddy, Rajeshwar R. Tekmal, Manjeet Rao, Suryavathi Viswanadhapalli, Ratna Vadlamudi. Small molecule inhibitor of PELP1 exhibit cytotoxic effects on breast cancer cells by blocking ribosome biogenesis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-10.

Macula densa mTORC1 is an important regulator of cellular and aging-dependent kidney functions

Macula densa (MD) cells are major regulators of nephron, kidney, and whole body functions, including the control of renal blood flow, glomerular filtration rate (GFR), renin release and thus the maintenance of body fluid and electrolyte balance. Our recent studies identified the high rate of MD protein synthesis regulated by the mTORC1 complex of the mechanistic target of rapamycin (mTOR) as a novel component of MD cell and glomerular hemodynamic regulatory functions. Raptor is an essential player in mTORC1’s key roles to regulate cell energy homeostasis, growth, protein synthesis, mitochondrial function, apoptosis, and autophagy via phosphorylation of substrates including ribosomal protein S6 kinase (S6K) and eukaryotic translation-initiation factor 4E-binding proteins (4E-BPs). mTORC1 signaling controls the aging process, and deficient mTORC1 activity extends health and lifespan. Therefore, we hypothesized that MD cell-specific mTOR loss of function (lof) prevents aging-related kidney function decline. The present study aimed to demonstrate the key importance of MD cell mTOR activity in renal physiology and aging by characterizing the phenotype of a newly generated genetic MD-mTOR lof mouse model. MD-mTOR lof mice were generated by crossing Nos1-CreERT2 and Raptor floxed mice. Tamoxifen induction (at 3 weeks of age) of Nos1-CreERT2+/- Raptor−/− mice resulted in conditional and inducible Raptor deletion in cells of the Nos1 lineage, which in the kidney is entirely specific to MD cells. Kidney structure/function phenotyping was performed using intravital multiphoton microscopy (MPM), the Medibeacon transdermal GFR measurement system, and tissue harvest, immunohistochemistry, and the OPP incorporation assay of protein synthesis. High and MD-specific Raptor, pS6K, and p4EBP1 immunofluorescence labeling was observed in WT kidney sections. The immunolabeling of these mTOR component and targets were absent specifically in MD cells in MD-mTOR lof mouse kidneys, validating the MD cell-specific genetic approach. The intensity of incorporated OPP labeling was highest in MD cells among all renal cell types in WT. In contrast, MD OPP labeling was significantly reduced in MD-mTOR lof mice indicating diminished, mTORC1-dependent global MD cell protein synthesis. Importantly, in 15-month old aged mice the GFR was significantly higher (preserved) in MD-mTOR lof (1794±197 uL/min/100g BW) compared to WT (1117±61) and MD-mTOR gof (929±61) mice (n=6-18 mice in each group). Altogether, these studies identified mTORC1 as an essential regulator of MD cell biology, and the importance of MD cells in aging-related kidney function decline. MD cells may be important new targets in future anti-aging therapies. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antiviral screening of natural, anti-inflammatory compound library against African swine fever virus

BackgroundAfrican swine fever virus (ASFV) is a major threat to pig production and the lack of effective vaccines underscores the need to develop robust antiviral countermeasures. Pathologically, a significant elevation in pro-inflammatory cytokine production is associated with ASFV infection in pigs and there is high interest in identifying dual-acting natural compounds that exhibit antiviral and anti-inflammatory activities.MethodsUsing the laboratory-adapted ASFV BA71V strain, we screened a library of 297 natural, anti-inflammatory compounds to identify promising candidates that protected Vero cells against virus-induced cytopathic effect (CPE). Virus yield reduction, virucidal, and cell cytotoxicity experiments were performed on positive hits and two lead compounds were further characterized in dose-dependent assays along with time-of-addition, time-of-removal, virus entry, and viral protein synthesis assays. The antiviral effects of the two lead compounds on mitigating virulent ASFV infection in porcine macrophages (PAMs) were also tested using similar methods, and the ability to inhibit pro-inflammatory cytokine production during virulent ASFV infection was assessed by enzyme-linked immunosorbent assay (ELISA).ResultsThe screen identified five compounds that inhibited ASFV-induced CPE by greater than 50% and virus yield reduction experiments showed that two of these compounds, tetrandrine and berbamine, exhibited particularly high levels of anti-ASFV activity. Mechanistic analysis confirmed that both compounds potently inhibited early stages of ASFV infection and that the compounds also inhibited infection of PAMs by the virulent ASFV Arm/07 isolate. Importantly, during ASFV infection in PAM cells, both compounds markedly reduced the production of pro-inflammatory cytokines involved in disease pathogenesis while tetrandrine had a greater and more sustained anti-inflammatory effect than berbamine.ConclusionsTogether, these findings support that dual-acting natural compounds with antiviral and anti-inflammatory properties hold promise as preventative and therapeutic agents to combat ASFV infection by simultaneously inhibiting viral replication and reducing virus-induced cytokine production.

DP7-C/mir-26a system promotes bone regeneration by remodeling the osteogenic immune microenvironment.

This study investigates the DP7-C/miR-26a complex as a stable entity resulting from the combination of miR-26a with the immunomodulatory peptide DP7-C. Our focus is on utilizing DP7-C loaded with miR-26a to modulate the immune microenvironment in bone and facilitate osteogenesis. The DP7-C/miR-26a complex was characterized through transmission electron microscopy, agarose electrophoresis, and nanoparticle size potentiometer analysis. Transfection efficiency and cytotoxicity of DP7-C were assessed using flow cytometry and the CCK-8 assay. We validated the effects of DP7-C/miR-26a on bone marrow mesenchymal stem cells (BMSCs) and macrophages RAW 264.7 through gene expression and protein synthesis assays. A comprehensive evaluation of appositional bone formation involved micro-CT imaging, histologic analysis, and immunohistochemical staining. DP7-C/miR-26a, a nanoscale, and low-toxic cationic complex, demonstrated the ability to enter BMSCs and RAW 264.7 via distinct pathways. The treatment with DP7-C/miR-26a significantly increased the synthesis of multiple osteogenesis-related factors in BMSCs, facilitating calcium nodule formation invitro. Furthermore, DP7-C/miR-26a promoted M1 macrophage polarization toward M2 while suppressing the release of inflammatory factors. Coculture studies corroborated these findings, indicating significant repair of rat skull defects following treatment with DP7-C/miR-26a. The DP7-C/miR-26a system offers a safer, more efficient, and feasible technical means for treating bone defects.

Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.

Assessment of translation rate in leukemic cells and immune cells of the microenvironment by OPP protein synthesis assay.

Despite being tightly regulated, messenger RNA (mRNA) translation, a manner in which cells control expression of genes and rapidly respond to stimuli, is highly dysfunctional and plastic in pathologies including cancer. Conversely, the investigation of molecular mechanisms whereby mRNA translation becomes aberrant in cancer, as well as inhibition thereof, become critical in developing novel therapeutic approaches. More specifically, in malignancies such as chronic lymphocytic leukemia in which aberrant global and transcript specific translation has been linked with poorer patient outcomes, targeting translation is a relevant approach, with various translation inhibitors under development. Here we elaborate on a protein synthesis assay by flow cytometry, O-propargyl-puromycin, demonstrating global mRNA translation rate with a variety of different applications including cell lines, primary cells or co-culture systems in vitro. This method provides a comprehensive tool in quantifying the rate of global mRNA translation in cancer cells, as well as that of the tumor microenvironment cells, or in response to inhibitory therapeutic agents while offering the possibility to simultaneously assess other cellular markers.

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems.

The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.

Cell-free transcription-translation system: a dual read-out assay to characterize riboswitch function.

Cell-free protein synthesis assays have become a valuable tool to understand transcriptional and translational processes. Here, we established a fluorescence-based coupled in vitro transcription-translation assay as a read-out system to simultaneously quantify mRNA and protein levels. We utilized the well-established quantification of the expression of shifted green fluorescent protein (sGFP) as a read-out of protein levels. In addition, we determined mRNA quantities using a fluorogenic Mango-(IV) RNA aptamer that becomes fluorescent upon binding to the fluorophore thiazole orange (TO). We utilized a Mango-(IV) RNA aptamer system comprising four subsequent Mango-(IV) RNA aptamer elements with improved sensitivity by building Mango arrays. The design of this reporter assay resulted in a sensitive read-out with a high signal-to-noise ratio, allowing us to monitor transcription and translation time courses in cell-free assays with continuous monitoring of fluorescence changes as well as snapshots of the reaction. Furthermore, we applied this dual read-out assay to investigate the function of thiamine-sensing riboswitches thiM and thiC from Escherichia coliand the adenine-sensing riboswitch ASW from Vibrio vulnificusand pbuE from Bacillus subtilis, which represent transcriptional and translational on- and off-riboswitches, respectively. This approach enabled a microplate-based application, a valuable addition to the toolbox for high-throughput screening of riboswitch function.

Liraglutide Protects Against Diastolic Dysfunction and Improves Ventricular Protein Translation.

Diastolic dysfunction is an increasingly common cardiac pathology linked to heart failure with preserved ejection fraction. Previous studies have implicated glucagon-like peptide 1 (GLP-1) receptor agonists as potential therapies for improving diastolic dysfunction. In this study, we investigate the physiologic and metabolic changes in a mouse model of angiotensin II (AngII)-mediated diastolic dysfunction with and without the GLP-1 receptor agonist liraglutide (Lira). Mice were divided into sham, AngII, or AngII+Lira therapy for 4 weeks. Mice were monitored for cardiac function, weight change, and blood pressure at baseline and after 4 weeks of treatment. After 4 weeks of treatment, tissue was collected for histology, protein analysis, targeted metabolomics, and protein synthesis assays. AngII treatment causes diastolic dysfunction when compared to sham mice. Lira partially prevents this dysfunction. The improvement in function in Lira mice is associated with dramatic changes in amino acid accumulation in the heart. Lira mice also have improved markers of protein translation by Western blot and increased protein synthesis by puromycin assay, suggesting that increased protein turnover protects against fibrotic remodeling and diastolic dysfunction seen in the AngII cohort. Lira mice also lost lean muscle mass compared to the AngII cohort, raising concerns about peripheral muscle scavenging as a source of the increased amino acids in the heart. Lira therapy protects against AngII-mediated diastolic dysfunction, at least in part by promoting amino acid uptake and protein turnover in the heart. Liraglutide therapy is associated with loss of mean muscle mass, and long-term studies are warranted to investigate sarcopenia and frailty with liraglutide therapy in the setting of diastolic disease.

SPSB1-mediated inhibition of TGF-β receptor-II impairs myogenesis in inflammation.

Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-β) is involved in both processes. We uncovered an increased expression of the TGF-β receptor II (TβRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TβRII signalling impairs myogenic differentiation in response to inflammation. We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-β/TβRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses. SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1β (IL-1β), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1β-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TβRII, resulting in TβRII ubiquitination and destabilization. SPSB1 impaired TβRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice. Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TβRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.

A Sporulation-Independent Way of Life for Bacillus thuringiensis in the Late Stages of an Infection.

The formation of endospores has been considered the unique survival and transmission mode of sporulating Firmicutes due to the exceptional resistance and persistence of this bacterial form. However, nonsporulated bacteria (Spo-) were reported at the early stages following the death of a host infected with Bacillus thuringiensis, an entomopathogenic sporulating bacterium. Here, we investigated the characteristics of the bacterial population in the late stages of an infection in the B. thuringiensis/Galleria mellonella infection model. Using fluorescent reporters and molecular markers coupled to flow cytometry, we demonstrated that the Spo- cells persist and constitute about half of the population 2 weeks post-infection (p.i.). Protein synthesis and growth recovery assays indicated that they are in a metabolically slowed-down state. These bacteria were extremely resistant to the insect cadaver environment, which did not support growth of in vitro-grown vegetative cells and spores. A transcriptomic analysis of this subpopulation at 7 days p.i. revealed a signature profile of this state, and the expression analysis of individual genes at the cell level showed that more bacteria mount an oxidative stress response as their survival time increases, in agreement with the increase of the free radical level in the host cadaver and in the number of reactive oxygen species (ROS)-producing bacteria. Altogether, these data show for the first time that nonsporulated bacteria are able to survive for a prolonged period of time in the context of an infection and indicate that they engage in a profound adaptation process that leads to their persistence in the host cadaver. IMPORTANCE Bacillus thuringiensis is an entomopathogenic bacterium widely used as a biopesticide. It belongs to the Bacillus cereus group, comprising the foodborne pathogen B. cereus sensu stricto and the anthrax agent Bacillus anthracis. Like other Firmicutes when they encounter harsh conditions, these Gram-positive bacteria can form dormant cells called spores. Due to its highly resistant nature, the spore was considered the unique mode of long-term survival, eclipsing any other form of persistence. Breaking this paradigm, we observed that B. thuringiensis was able to persist in its host cadaver in a nonsporulated form for at least 14 days. Our results show that these bacteria survived in the cadaver environment, which proved hostile for actively growing bacteria by engaging in a profound adaptation process. Studying this facet of the life cycle of a sporulating bacterium provides new fundamental knowledge and might lead to the development of strategies to combat sporulating pathogenic species.

Toosendanin targeting eEF2 impedes Topoisomerase I & II protein translation to suppress esophageal squamous cell carcinoma growth

BackgroundAlthough molecular targets such as HER2, TP53 and PIK3CA have been widely studied in esophageal cancer, few of them were successfully applied for clinical treatment. Therefore, it is urgent to discover novel actionable targets and inhibitors. Eukaryotic translational elongation factor 2 (eEF2) is reported to be highly expressed in various cancers. However, its contribution to the maintenance and progression of cancer has not been fully clarified.MethodsIn the present study, we utilized tissue array to evaluate eEF2 protein expression and clinical significance in esophageal squamous cell carcinoma (ESCC). Next, we performed knockdown, overexpression, RNA-binding protein immunoprecipitation (RIP) sequence, and nascent protein synthesis assays to explore the molecular function of eEF2. Furthermore, we utilized compound screening, Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) assay, cell proliferation and Patient derived xenograft (PDX) mouse model assays to discover an eEF2 inhibitor and assess its effects on ESCC growth.ResultsWe found that eEF2 were highly expressed in ESCC and negatively associated with the prognosis of ESCC patients. Knocking down of eEF2 suppressed the cell proliferation and colony formation of ESCC. eEF2 bond with the mRNA of Topoisomerase II (TOP1) and Topoisomerase II (TOP2) and enhanced the protein biosynthesis of TOP1 and TOP2. We also identified Toosendanin was a novel inhibitor of eEF2 and Toosendanin inhibited the growth of ESCC in vitro and in vivo.ConclusionsOur findings show that Toosendanin treatment suppresses ESCC growth through targeting eEF2 and regulating downstream TOP1 and TOP2 biosynthesis. eEF2 could be supplied as a potential therapeutic target in the further clinical studies.

Inhibition of YTHDF1 prevents hypoxia-induced pulmonary artery smooth muscle cell proliferation by regulating Foxm1 translation in an m6A-dependent manner

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by pulmonary vascular remodeling. It refers to the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), and hypoxia is an important risk factor for this progression. The present study aims to investigate the role of YTHDF1 in the regulation of hypoxic PASMC proliferation and the underlying mechanism. Human PASMCs were transfected with si-YTHDF1/2/3 followed by treatment of hypoxia, and the PASMC proliferation and Foxm1 expression were detected. Through RNA pull-down, RNA immunoprecipitation, and protein synthesis assay, the mechanism of YTHDF1 regulating Foxm1 was explored. Next, Foxm1 was inhibited by thiostrepton, and cell proliferation was detected. In vivo, mice received a tail vein injection of adenovirus containing si-YTHDF1 and were exposed to hypoxia treatment. Pulmonary vascular changes, right ventricular systolic pressure (RVSP), and genes involving proliferation were analyzed. YTHDF1 silencing reduced more hypoxic PASMC proliferation and Foxm1 protein level than YTHDF2/3 silencing. Mechanical results showed that YTHDF1 interacted with Foxm1 mRNA and up-regulated Foxm1 protein level by enhancing the translation efficiency in an m6A-dependent manner. Furthermore, YTHDF1 facilitated hypoxic PASMC proliferation and proliferation marker expressions through up-regulation of Foxm1 in an m6A-dependent manner. In vivo, the YTHDF1 silencing alleviated pulmonary vascular changes and fibrosis, reduced RVSP, inhibited the interaction of YTHDF1 and Foxm1, and reduced proliferation marker levels, as compared to the PAH group. In conclusion, YTHDF1 silencing inhibits hypoxic PASMC proliferation by regulating Foxm1 translation in an m6A-dependent manner.

Disrupted RNA‐networks in Alzheimer’s disease

AbstractBackgroundSynapse loss is well‐documented and underlies cognitive decline in Alzheimer’s disease (AD) and other comorbid neuropsychiatric disorders such as depression. To date, few molecular targets and mechanisms are identified to restore/maintain healthy synapses in AD. AD and depression both have disrupted mechanistic/mammalian target of rapamycin complex 1 (mTORC1) activity. Since mTORC1 is involved in protein synthesis, disruptions in mTORC1 activity creates an imbalance between mRNA translation and repression at synapses. We have demonstrated that mTORC1 signaling is overactive in the prefrontal cortex (PFC) of AD patients and animal models. RNA binding proteins (RBP) are significant regulators of mRNA translation. A single RBP can bind to multiple mRNAs to either enhance or repress their translation. Dysregulated expression of RBPs can thus lead to imbalanced mRNA translation and protein synthesis.MethodComputational methods, combined with the literature, have identified putative DJ‐1 mRNA binding proteins. DJ‐1 RNA‐immunoprecipitation were used to verify FMR1 mRNA as a target. A new protein synthesis assay was used to test if DJ‐1 expression affects FMRP expression. Network analyses was performed to identify new targets shared and unique to these two RNA binding proteins.ResultWe recently identified the mTORC1 regulated RBP, DJ‐1 (Park7) as a translational regulator whose expression can impact the expression and/or function of multiple proteins. We have found that Fmr1 mRNA, which is translated to the RBP, fragile X messenger ribonucleoprotein 1 (FMRP), is a target of DJ‐1. We have shown that FMRP functions to suppress the translation of various synaptic mRNAs to maintain proper transsynaptic signaling which is essential in the formation, stability, and remodeling of synapses. Notably, depression is often comorbid with AD. Furthermore, we have demonstrated that FMRP is required for antidepressant efficacy. Herein, we provide data that supports disrupted DJ‐1/FMRP expression at the synapse in preclinical models of AD. Network analysis of how these two RNA binding proteins interact will be discussed and their impact on synapse stability and behaviors in AD.ConclusionDJ‐1 and FMRP RNA networks are disrupted in Alzheimer’s disease. Network analyses linking these two proteins may reveal novel targets involved in synapse stability and comorbid neuropsychiatric disorders.

Effects of Psychotropic Drugs on Ribosomal Genes and Protein Synthesis.

Altered protein synthesis has been implicated in the pathophysiology of several neuropsychiatric disorders, particularly schizophrenia. Ribosomes are the machinery responsible for protein synthesis. However, there remains little information on whether current psychotropic drugs affect ribosomes and contribute to their therapeutic effects. We treated human neuronal-like (NT2-N) cells with amisulpride (10 µM), aripiprazole (0.1 µM), clozapine (10 µM), lamotrigine (50 µM), lithium (2.5 mM), quetiapine (50 µM), risperidone (0.1 µM), valproate (0.5 mM) or vehicle control for 24 h. Transcriptomic and gene set enrichment analysis (GSEA) identified that the ribosomal pathway was altered by these drugs. We found that three of the eight drugs tested significantly decreased ribosomal gene expression, whilst one increased it. Most changes were observed in the components of cytosolic ribosomes and not mitochondrial ribosomes. Protein synthesis assays revealed that aripiprazole, clozapine and lithium all decreased protein synthesis. Several currently prescribed psychotropic drugs seem to impact ribosomal gene expression and protein synthesis. This suggests the possibility of using protein synthesis inhibitors as novel therapeutic agents for neuropsychiatric disorders.

Galunisertib Exerts Antifibrotic Effects on TGF-β-Induced Fibroproliferative Dermal Fibroblasts.

Dermal fibroblasts in pathological scars secrete constitutively elevated levels of TGF-β, signaling the transcription of fibrotic genes via activin-like kinase 5 (ALK5). In the present study, we examine the antifibrotic effects of galunisertib, a small-molecule inhibitor of ALK5, on fibroproliferative dermal fibroblasts in an in vitro model of wound healing. We induced fibrosis in human dermal fibroblasts with exogenous TGF-β and performed cellular proliferation assays after treatment with varying concentrations of galunisertib. Dermal fibroblast proliferation was diminished to homeostatic levels without cytotoxicity at concentrations as high as 10 μM. An in vitro scratch assay revealed that galunisertib significantly enhanced cellular migration and in vitro wound closure beginning 24 h post-injury. A gene expression analysis demonstrated a significant attenuation of fibrotic gene expression, including collagen-1a, alpha-smooth muscle actin, fibronectin, and connective tissue growth factor, with increased expression of the antifibrotic genes MMP1 and decorin. Protein synthesis assays confirmed drug activity and corroborated the transcription findings. In summary, galunisertib simultaneously exerts antifibrotic effects on dermal fibroblasts while enhancing rates of in vitro wound closure. Galunisertib has already completed phase II clinical trials for cancer therapy with minimal adverse effects and is a promising candidate for the treatment and prevention of pathological cutaneous scars.

Antifungal Activity of NP20 Derived from Amphioxus Midkine/Pleiotrophin Homolog Against Aspergillus niger and Aspergillus fumigatus.

With the emergence of antifungal resistance, systematic infections with Aspergillus are becoming the major cause of the clinical morbidity. The development of novel antifungal agents with high efficacy, low drug tolerance, and few side effects is urgent. In response to that need, we have identified NP20. Here we demonstrate clearly that NP20 has antifungal activity, capable of killing the spores of Aspergillus niger and Aspergillus fumigatus as well as causing direct damage to the surface, membrane, cytoplasm, organelle, and nucleus of the fungal spores. Interestingly, NP20 is active under temperature stress and a wide range of pH. Subsequently, MTT assay, assay for binding of NP20 to fungal cell wall components, membrane depolarization assay, confocal microscopy, ROS assay, DNA replication, and protein synthesis assay are performed to clarify the mechanisms underlying NP20 against Aspergillus. The results show that NP20 can bind with and pass through the fungal cell wall, and then interfere with the lipid membrane. Moreover, NP20 can induce intracellular ROS production, DNA fragmentation, and protein synthesis inhibition of the fungal cells. These together indicate that NP20 is a novel antifungal peptide, which has considerable potential for future development as novel peptide antibiotics against Aspergillus.