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Abstract Liquid-liquid phase separation (LLPS) of RNA-protein complexes plays a major role in the cellular function of membraneless organelles (MLOs). MLOs are sensitive to changes in cellular conditions, such as fluctuations in cytoplasmic ion concentrations. To investigate the effect of these changes on MLOs, we studied the influence of divalent cations on the physical and chemical properties of RNA coacervates. Using a model system comprised of an arginine-rich peptide and RNA, we predicted and observed that variations in signaling cations exert interaction-dependent effects on RNA LLPS. Changing the ionic environment has opposing effects on the propensity for heterotypic peptide-RNA and homotypic RNA LLPS, which results in a switch between coacervate types. Furthermore, divalent ion variations continuously tune the microenvironments and fluid properties of heterotypic and homotypic droplets. Our results may provide a general mechanism for modulating the biochemical environment of RNA coacervates in a cellular context.

Abstract The EWS-FLI1 chimeric protein uniquely expressed in Ewing’s sarcoma has an obligate role in its aetiology. In our previous report we showed that ectopic expression of the DNA sequences form the junction region (a.a 251–280) can inhibit Ewing’s sarcoma cell growth. In the present report, we introduced a peptide (TAT/NLS/EWS-PEP) comprising of thirty amino acids spanning the junction in conjunction with HIV-1-trans-activating (TAT) and nuclear localization signal sequence (NLS). Peptide uptake and localization studies revealed presence of peptide in ~99% of transduced cells and in the nucleus. Peptide transfection induced cytotoxicity relative to untreated and TAT-NLS peptide treated Ewing’s sarcoma cells. The peptide inhibited clonogenicity, cell cycle, bromo-deoxy uridine (BrdU) uptake and invasion capacity of treated cells. The treatment also affected epithelial to mesenchymal transition (EMT) markers and EWS-FLI1 target gene expression levels. Co-immunoprecipitation experiments involving ectopically expressed full-length EWS-FLI1 protein and the peptide revealed an interaction. Additionally, we found that peptide interaction also occurs with the protein-GGAA microsatellite sequences complex known to contain EWS-FLI1. Further, in the pull-down assay, the peptide was found to interact with proteins known to potentially interact with EWS-FLI1. Based on these results we conclude that peptide could be applied in targeting EWS-FLI1 protein.

Abstract Despite the highly oxidative environment of the phagosomal lumen, the need for maintaining redox homeostasis is a critical aspect of mycobacterial biology. The pathogens are equipped with the sophisticated thioredoxin- (Trx) and peroxiredoxin system, including TrxC and the alkyl hydroperoxide reductase subunit C (AhpC), whereby TrxC is one of the reducing partners of AhpC. Here we visualize the redox modulated dodecamer ring formation of AhpC from Mycobacterium bovis (BCG strain; MbAhpC) using electron microscopy and present novel insights into the unique N-terminal epitope (40 residues) of mycobacterial AhpC. Truncations and amino acid substitutions of residues in the unique N-terminus of MbAhpC provide insights into their structural and enzymatic roles, and into the evolutionary divergence of mycobacterial AhpC versus that of other bacteria. These structural details shed light on the epitopes and residues of TrxC which contributes to its interaction with AhpC. Since human cells lack AhpC, the unique N-terminal epitope of mycobacterial AhpC as well as the MbAhpC-TrxC interface represent an ideal drug target.

Abstract During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa and 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. We conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.

Gallbladder carcinoma (GBC) is a major cancer of the gastrointestinal tract with poor prognosis. Reliable and affordable biomarker-based assays with high sensitivity and specificity for the detection of this cancer are a clinical need. With the aim of studying the potential of the plasma-derived extracellular vesicles (EVs), we carried out quantitative proteomic analysis of the EV proteins, using three types of controls and various stages of the disease, which led to the identification of 86 proteins with altered abundance. These include 29 proteins unique to early stage, 44 unique to the advanced stage and 13 proteins being common to both the stages. Many proteins are functionally relevant to the tumor condition or have been also known to be differentially expressed in GBC tissues. Several of them are also present in the plasma in free state. Clinical verification of three tumor-associated proteins with elevated levels in comparison to all the three control types-5'-nucleotidase isoform 2 (NT5E), aminopeptidase N (ANPEP) and neprilysin (MME) was carried out using individual plasma samples from early or advanced stage GBC. Sensitivity and specificity assessment based on receiver operating characteristic (ROC) analysis indicated a significant association of NT5E and ANPEP with advanced stage GBC and MME with early stage GBC. These and other proteins identified in the study may be potentially useful for developing new diagnostics for GBC., (© 2021. The Author(s).)

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z' value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.

The ATP-dependent chromatin remodeling factors regulate gene expression. However, it is not known whether these factors regulate each other. Given the ability of these factors to regulate the accessibility of DNA to transcription factors, we postulate that one ATP-dependent chromatin remodeling factor should be able to regulate the transcription of another ATP-dependent chromatin remodeling factor. In this paper, we show that BRG1 and SMARCAL1, both members of the ATP-dependent chromatin remodeling protein family, regulate each other. BRG1 binds to the SMARCAL1 promoter, while SMARCAL1 binds to the brg1 promoter. During DNA damage, the occupancy of SMARCAL1 on the brg1 promoter increases coinciding with an increase in BRG1 occupancy on the SMARCAL1 promoter, leading to increased brg1 and SMARCAL1 transcripts respectively. This is the first report of two ATP-dependent chromatin remodeling factors regulating each other.