Endonucleases are enzymes that cleave internal phosphodiester bonds within double-stranded DNA or RNA and are essential for biological functions. Herein, we use genetic code expansion to create an ...unnatural endonuclease that cleaves non-coding RNAs including short interfering RNA (siRNA) and microRNAs (miRNAs), a function that does not exist in nature. We introduce a metal-chelating unnatural amino acid, (2,2'-bipyridin-5-yl)alanine (BpyAla) to impart endonuclease activity to the viral suppressor of RNA silencing protein p19. Upon binding of copper, the mutant p19-T111BpyAla displays catalytic site-specific cleavage of siRNA and human miRNAs. Catalysis is confirmed using fluorescence polarization and fluorescence turn-on. Global miRNA profiling reveals that the engineered enzyme cleaves miRNAs in a human cell line. The therapeutic potential is demonstrated by targeting miR-122, a critical host factor for the hepatitis C virus (HCV). Unnatural endonuclease function is shown to deplete miR-122 levels with similar effects to an antagomir that reduces HCV levels therapeutically.
Insulin-like growth factor (IGF)-II is a hormone with mitogenic activity for many cell types and tissues. We demonstrate that its intracellular processing and secretion strictly depend on the ...endoplasmic reticulum chaperone glucose-regulated protein (GRP) 94. GRP94 interacts physically and transiently with pro-IGF-II intermediates, and its activity is essential for secretion of active IGF-II, thus establishing IGF-II as a client of GRP94. Embryonic stem (ES) cells that lack GRP94 are hypersensitive to stress conditions such as serum deprivation and die by apoptosis because they cannot respond to the stress by producing active IGF-II. This chaperone-client interaction may explain the previously documented antiapoptotic activity of GRP94 in a number of stress responses.
With the emergence of the novel betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there has been an urgent need for the development of fast-acting antivirals, particularly ...in dealing with different variants of concern (VOC). SARS-CoV-2, like other RNA viruses, depends on host cell machinery to propagate and misregulate metabolic pathways to its advantage. Herein, we discovered that the immunometabolic microRNA-185 (miR-185) restricts SARS-CoV-2 propagation by affecting its entry and infectivity. The antiviral effects of miR-185 were studied in SARS-CoV-2 Spike protein pseudotyped virus, surrogate virus (HCoV-229E), as well as live SARS-CoV-2 virus in Huh7, A549, and Calu-3 cells. In each model, we consistently observed microRNA-induced reduction in lipid metabolism pathways-associated genes including SREBP2, SQLE, PPARG, AGPAT3, and SCARB1. Interestingly, we also observed changes in angiotensin-converting enzyme 2 (ACE2) levels, the entry receptor for SARS-CoV-2. Taken together, these data show that miR-185 significantly restricts host metabolic and other pathways that appear to be essential to SAR-CoV-2 replication and propagation. Overall, this study highlights an important link between non-coding RNAs, immunometabolic pathways, and viral infection. miR-185 mimics alone or in combination with other antiviral therapeutics represent possible future fast-acting antiviral strategies that are likely to be broadly antiviral against multiple variants as well as different virus types of potential pandemics.
Because only few of its client proteins are known, the physiological roles of the endoplasmic reticulum chaperone glucose-regulated protein 94 (GRP94) are poorly understood. Using targeted disruption ...of the murine GRP94 gene, we show that it has essential functions in embryonic development. grp94-/- embryos die on day 7 of gestation, fail to develop mesoderm, primitive streak, or proamniotic cavity. grp94-/- ES cells grow in culture and are capable of differentiation into cells representing all three germ layers. However, these cells do not differentiate into cardiac, smooth, or skeletal muscle. Differentiation cultures of mutant ES cells are deficient in secretion of insulin-like growth factor II and their defect can be complemented with exogenous insulin-like growth factors I or II. The data identify insulin-like growth factor II as one developmentally important protein whose production depends on the activity of GRP94.
Meiotic development (sporulation) in the yeast Saccharomyces cerevisiae is induced by nutritional deprivation. Smk1 is a meiosis-specific MAP kinase homolog that controls spore morphogenesis after ...the meiotic divisions have taken place. In this study, recessive mutants that suppress the sporulation defect of a smk1-2 temperature-sensitive hypomorph were isolated. The suppressors are partial function alleles of CDC25 and CYR1, which encode the Ras GDP/GTP exchange factor and adenyl cyclase, respectively, and MDS3, which encodes a kelch-domain protein previously implicated in Ras/cAMP signaling. Deletion of PMD1, which encodes a Mds3 paralog, also suppressed the smk1-2 phenotype, and a mds3-Delta pmd1-Delta double mutant was a more potent suppressor than either single mutant. The mds3-Delta, pmd1-Delta, and mds3-Delta pmd1-Delta mutants also exhibited mitotic Ras/cAMP phenotypes in the same rank order. The effect of Ras/cAMP pathway mutations on the smk1-2 phenotype required the presence of low levels of glucose. Ime2 is a meiosis-specific CDK-like kinase that is inhibited by low levels of glucose via its carboxy-terminal regulatory domain. IME2-DeltaC241, which removes the carboxy-terminal domain of Ime2, exacerbated the smk1-2 spore formation phenotype and prevented cyr1 mutations from suppressing smk1-2. Inhibition of Ime2 in meiotic cells shortly after Smk1 is expressed revealed that Ime2 promotes phosphorylation of Smk1's activation loop. These findings demonstrate that nutrients can negatively regulate Smk1 through the Ras/cAMP pathway and that Ime2 is a key activator of Smk1 signaling.
Recently, microRNAs (miRNAs), as endogenous noncoding RNAs that inhibit mRNA translation, have been identified to broadly possess functional roles in regulating cellular signaling and metabolic ...processes due to their chemical and biological properties. In addition, they have emerged to be of critical importance in modulating host–virus interactions, especially for RNA viruses. Herein, we discovered that miR-383-5p targets certain lipid and cholesterol biosynthetic pathways and restricts Dengue virus (DENV) infection in hepatic cells. Global transcriptomics analysis of Huh7 human hepatoma cells overexpressing miR-383-5p revealed enrichment of lipid and cholesterol metabolic processes. Bioinformatics analysis of genes repressed in miR-383-5p overexpressing cells divulged the repression of a key target PLA2G4A, a pro-viral host factor essential for the production of infectious DENV particles. Our study demonstrated the effectiveness of miRNA mimics as tools to study cellular signaling pathways that contribute to viral pathogenesis. Overall, our study identifies miR-383-5p as an interesting host factor during DENV propagation and highlights a potential therapeutic role in the regulation of hepatic lipid metabolism and an antiviral response to DENV.
Abstract only Primary cardiac tumors are rare entity, and the most common benign cardiac neoplasms include myxomas, lipomas and papillary fibroelastomas (PFE) with PFE accounting for 8% of the ...tumors. A 53-year-old female presented with chronic chest pain and exertional shortness of breath. She first sought evaluation four years ago, when her pulmonologist referred her to thoracic surgery after an incidental finding of a 3.9 cm anterior mediastinal mass on CT-scan (Figure 1A-B). The patient described the pain as a constant pressure and squeezing sensation in her chest and worsening shortness of breath while laying on her sides, relieved only when a pillow was propped up under her chest. The pain did not significantly reduce her mobility or capacity to work. Initial evaluation, including transthoracic echocardiogram, stress testing, carotid ultrasound, and pulmonary function testing - was performed and found to be unremarkable. Further evaluations revealed a hiatal hernia, leading her symptoms to be attributed to GERD as causing her persistent non-exertional chest pain. The patient remained in good health despite her symptoms persisting. She later re-sought attention for chest pain on exertion and hoarseness three years later. She again underwent a full cardiopulmonary workup, yielding benign findings on EKG, troponins, and echocardiogram. Due to no alarming symptoms at the time, she was subsequently discharged. The patient continued to endorse chest pain and a follow-up MRI revealed a now 5 cm anterior mediastinal mass, thought to be a thymic cyst. Over the next few months, the patient continued to endorse worsening chest pain and exertional shortness of breath. She followed up with her cardiologist and a subsequent TEE was significant for a pulmonic valvular mass. She was admitted to the hospital for further investigation. During her hospital stay, the patient underwent surgery to resect the anterior mediastinal mass likely to be a thymic cyst and the pulmonary artery mass which was determined to be a pulmonary valve papillary fibroelastoma in the histopathology exam (Figure 2). The surgery was successful with no complications postoperatively. The patient was discharged with no further chest pain and with improvement of the exertional shortness of breath.
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