Characterization of dsRNA Binding Proteins through Proteome Integral Solubility Alteration Analysis Uncovers ZNF385A as a Regulator of dsRNA Homeostasis
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PROVIDER: PRJNA1224849 | ENA |
REPOSITORIES: ENA
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Project description:Double-stranded RNA (dsRNA) binding proteins (dsRBPs) play crucial roles in various cellular processes, especially in the innate immune response. Comprehensive characterization of dsRBPs is essential to understand the intricate mechanisms for dsRNA sensing and response. Traditional methods have predominantly relied on affinity purification, favoring the isolation of strong dsRNA binders. Here, we adopted the proteome integral solubility alteration (PISA) workflow for characterizing dsRBPs, resulting in the observation of 18 known dsRBPs and the identification of 200 novel potential dsRBPs. Next, we focused on zinc finger protein 385A (ZNF385A) and discovered that its knockout activated the transcription of interferon-β in the absence of immunogenic stimuli. The knockout of ZNF385A elevated the level of endogenous dsRNAs, especially transcripts associated with retroelements, such as short interspersed nuclear element (SINE), long interspersed nuclear element (LINE), and long terminal repeat (LTR). Moreover, loss of ZNF385A enhanced the bioactivity of 5-Aza-2'-deoxycytidine (5-AZA-CdR) and tumor-killing effect of NK cells. Our findings greatly expand the dsRBP reservoir and contribute to the understanding of cellular dsRNA homeostasis.
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Project description:Most drugs used in the clinic and drug candidates target multiple proteins, and thus detailed characterization of their efficacy targets is required. While current methods rely on quantitative measurements at thermodynamic equilibrium, kinetic parameters such as the residence time of a drug on its target provide a better proxy for efficacy in vivo. Here, we present a new Residence Time Proteome Integral Solubility Alteration (ResT-PISA) assay which provides monitoring temporal protein solubility profiles after drug removal in either cell lysate or intact cells, quantifying the lifetime of the drug-target interaction. A compressed version of the assay measures the integral under the off-curve and enables the multiplexing of binding affinity proxy measurements with residence time assessment into a single proteomic analysis. We introduce a combined scoring system for three parametric dimensions to improve prioritization of targets. By providing complementary information to other characteristics of drug-target interaction, ResT-PISA approach can become a useful tool in drug development and precision medicine.
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Project description:The thermal shift assay is a robust method of discovering protein-ligand interactions by measuring the alterations in protein thermal stability under various conditions. Several thermal shift assays have been developed and their throughput has been advanced greatly by the rapid progress in tandem mass tag-based quantitative proteomics. A recent paper by Gaetani et al. (J Proteome Res 2019, 18 (11), 4027-4037) introduced proteome integral solubility alteration (PISA) assay, further increasing throughput and simplifying the data analysis. Yet, it remains unclear how fold changes (integral treated samples versus integral control samples) perform in this assay. We show that fold changes have compromised linearity with ΔTm (shift in melting points) by simulation, and the magnitudes of the fold changes are inherently small in PISA assay, which is a challenge for quantitation. Both simulation and experimental results show that the selection of heating temperatures can tackle the small fold change problem and improve the sensitivity and specificity of the PISA assay.
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Project description:Double-stranded RNA (dsRNA) binding proteins (dsRBPs) play crucial roles in various cellular processes, especially in the innate immune response. Comprehensive characterization of dsRBPs is essential to understand the intricate mechanisms for dsRNA sensing and response. Traditional methods have predominantly relied on affinity purification, favoring the isolation of strong dsRNA binders. Here, we adopt the proteome integral solubility alteration (PISA) workflow for characterizing dsRBPs, resulting in the observation of 18 known dsRBPs and the identification of 200 potential dsRBPs. Next, we focus on zinc finger protein 385A (ZNF385A) and discover that its knockout activates the transcription of interferon-βin the absence of immunogenic stimuli. The knockout of ZNF385A elevates the level of endogenous dsRNAs, especially transcripts associated with retroelements, such as short interspersed nuclear element (SINE), long interspersed nuclear element (LINE), and long terminal repeat (LTR). Moreover, loss of ZNF385A enhances the bioactivity of 5-Aza-2'-deoxycytidine (5-AZA-CdR) and tumor-killing effect of NK cells. Our findings greatly expand the dsRBP reservoir and contribute to the understanding of cellular dsRNA homeostasis.
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Project description:To simplify chemical risk assessment, the adverse outcome pathway (AOP) method has arisen as a framework to predict the impact of chemical exposure on human and environmental health. The development of this predictive tool requires knowledge of the molecular interaction between chemicals and protein targets. Here, we demonstrate that the identification and selection of a target candidate to develop adverse outcome pathways can be obtained by applying high-throughput identification of chemical targets by proteome integral solubility alteration (PISA) assay, followed by selecting the priority target candidate by an analytical hierarchy process. From 8 identified protein targets for TCDD from a soluble proteome extracted from hepatocytes, we selected the priority target for developing AOPs. Our combined methodologies provide identification of a molecular initiating event, essential knowledge to link molecular interaction to impact on health. The identification of chemical targets following a systematic analysis of chemical protein interaction by proteomics increases the high-throughput and reduces the traditional bias. The target selection eliminates conflicting criteria by applying a multiple-criteria decision-making analysis.
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Project description:The One Health initiative connects human and animal health with their shared environment. The development of novel methodology to unbiased identification of mechanisms of action of chemicals and chemicals mixtures would address one of today´s challenges of environmental and human toxicology. Zebrafish embryos offer a unique opportunity to explore the proteome integral solubility alteration (PISA) assay to study the proteins that increase or decrease their solubility as protein targets of the bioactive compounds in an organism at an developmental stage. We presented here 4 different applications of the PISA method in zebrafish embryo with different types of compounds of environmental and health concern: i) endocrine disrupting chemical, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to outline the pathways compromised under exposure; ii) complex chemical mixtures composed by TCDD + alpha-endosulfan + bisphenol A (BPA) previously studied in vitro cells lines to evaluate single chemical versus mixture; iii) novel compounds from blue-green circular economy to map the organismal response, and a iv) novel drug directed to anti-cancer activity in lymphoma models to discharge side-effects at non-target organs. Our results demonstrated the unique opportunities of PISA assay in zebrafish for toxicology. We remark some differences to its application for drug target deconvolution. First, we evaluate both targets arising from an increase in solubility and in those decreasing, as both would be involved in modulation the cellular functions. Second, the method could be applicable to bioactive compound before chemical characterization or a chemical mixture of unknown stoichiometry. Third, the results from zebrafish embryo could provide a first glimpse of unspecific interactions with proteins in non-target tissues.The introduction of PISA assay in toxi- and ecotoxicoproteomics offers high-resolution and throughput in support with the 3R, and 3M principles.
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Project description:The physiological role of ubiquitous rhomboid proteases, membrane-integral proteins that cleave their substrates inside the lipid bilayer, is ill-defined in prokaryotes. The two rhomboid genes cg0049 and cg2767 of Corynebacterium glutamicum were deleted and consequences investigated in respect to growth phenotype, stress resistance, transcriptome, proteome and lipidome composition.
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