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In this paper we outline a simplified protocol for the electrophoretic mobility shift assay utilizing TreviGel 500, a nontoxic alternative to polyacrylamide. The TreviGel 500 matrix combines the strength and resolution of polyacrylamide with the simplicity and flexibility of agarose in the casting of gels. Therefore, this method provides a simple, rapid and nontoxic alternative to current protocols for the investigation of protein: DNA interactions.
We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch-ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets.
Highly purified nuclear protein is required when using an electrophoretic mobility shift assay (EMSA) to study transcription factors, e.g. nuclear factor-κB (NF-κB), a major transcription factor that regulates both innate and adaptive immune responses following infection. Although many protocols have been developed for nuclear protein extraction, they are not necessarily optimized for use in EMSA, often require a large number of cells and long processing times, and do not always result in complete separation of the nuclear and cytoplasmic fractions.
Covalent modification of functionally important cell proteins by lipid oxidation products (LOPs) is a known mechanism initiating pathological consequences of oxidative stress. Identification of new proteins covalently modified by electrophilic lipids can be performed by a combination of chemical, immunological, and mass spectrometry-based methods, but requires prior knowledge either on the exact molecular structure of LOPs (e.g., 4-hydroxynonenal) or candidate protein targets. However, under the conditions of oxidative stress in vivo, a complex mixture of proteins (e.g., cytosolic proteome) reacts with a complex mixture of LOPs. Here we describe a method for detection of lipid-modified proteins that does not require an a priori knowledge on the chemical structure of LOPs or identity of target proteins. The method is based on the change of electrophoretic mobility of lipid-modified proteins, which is induced by conformational changes and cross-linking with other proteins. Abnormally migrating proteins are detected by mass spectrometry-based protein peptide sequencing. We applied this method to study effects of oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) on endothelial cells. Several known, but also many new, OxPAPC-binding proteins were identified. We expect that this technically relatively simple method can be widely applied for label-free analysis of lipid-protein interactions in complex protein samples treated with different LOPs.
Calcitriol (1alpha, 25-dihydroxyvitamin D(3)) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA. Antibodies used for WEMSA often provide less ambiguous signals than those used in EMSA, and these do not have to recognize epitopes under native conditions. In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.
High mobility group box 1 (HMGB1) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. Three HMGB1 orthologs were predicted in the genome of Toxoplasma gondii, an obligate intracellular protozoan pathogen, termed TgHMGB1a, b and c. Phylogenetic and bioinformatic analyses indicated that these proteins all contain a single HMG box and which shared in three genotypes. We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-α, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA). Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular. There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites. Our findings demonstrated that TgHMGB1a is indeed a nuclear protein that maintains HMG box architectural functions and is a potential proinflammatory factor during the T.gondii infection. Further studies that clarify the functions of TgHMGB1s will increase our knowledge of transcriptional regulation and parasite virulence, and might provide new insight into host-parasite interactions for T. gondii infection.
Endogenous host-derived molecules named damage-associated molecular patterns (DAMPs) can induce excessive non-sterile inflammatory responses on recognition of specific membrane-tethered receptors. Here in this study, we aimed to explore the role of DAMP molecule HMGB1 in astrocyte-mediated sterile neuroinflammation and the resultant influences on neurons. In vitro cultured astrocytes were challenged with rHMGB1 and then harvested at 6 h, 12 h, 24 h, 36 h, and 48 h, respectively. The astrocytic CD24 expression was determined by quantitative real-time polymerase chain reaction (qPCR), Western blot analysis and immunofluorescence, nuclear factor kappa B (NF-κB) binding activity was detected by electrophoretic mobility shift assay (EMSA), and the proinflammatory factors, tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β), were measured by qPCR. The neuronal morphology was assessed with phase-contrast microscopy. The results showed that astrocytic mRNA and protein CD24 expression began to rise at 24 h, peaked at 36 h, and remained elevated at 48 h after rHMGB1 stimulation, accompanied with enhanced NF-κB binding activity and augmented expression of TNF-α and IL-1β. Furthermore, rHMGB1 caused cocultured neuron damage and was aggregated upon CD24 knockdown. Taken together, these novel findings suggested that rHMGB1 could promote astrocytic CD24 expression, the inhibition of which could aggregate neuronal damage.
The growth pattern of filamentous fungi can switch between hyphal radial polar growth and non-polar yeast-like cell growth depending on the environmental conditions. Asexual conidiation after radial polar growth is called normal conidiation (NC), while yeast-like cell growth is called microcycle conidiation (MC). Previous research found that the disruption of MaH1 in Metarhizium acridum led to a conidiation shift from NC to MC. However, the regulation mechanism is not clear. Here, we found MaMsn2, an Msn2 homologous gene in M. acridum, was greatly downregulated when MaH1 was disrupted (ΔMaH1). Loss of MaMsn2 also caused a conidiation shift from NC to MC on a nutrient-rich medium. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that MaH1 could bind to the promoter region of the MaMsn2 gene. Disrupting the interaction between MaH1 and the promoter region of MaMsn2 significantly downregulated the transcription level of MaMsn2, and the overexpression of MaMsn2 in ΔMaH1 could restore NC from MC of ΔMaH1. Our findings demonstrated that MaMsn2 played a role in maintaining the NC pattern directly under the control of MaH1, which revealed the molecular mechanisms that regulated the conidiation pattern shift in filamentous fungi for the first time.
Dengue virus (DENV) is most prevalent arthropod-borne human pathogen belongs to Flaviviridae family causes thousands of deaths annually. HMGB1 is highly conserved, ubiquitously expressed, non-histone nuclear protein which plays important role in diseases like metabolic disorders, cancer, and viral infections. However, the importance of HMGB1 in DENV infection is understudied. In this study, we observed that DENV-2 induces cytoplasmic translocation and secretion of HMGB1. Interestingly, inhibition of HMGB1 secretion by ethyl pyruvate (EP) enhanced viral propagation while silencing of HMGB1 resulted in abrogated viral replication in DENV-2 infected A549 cells. RNA-Electrophoretic mobility shift assay and immunoprecipitation showed that HMGB1 interacts with 5'-3' UTRs of DENV-2 genome. This interaction further stimulates production of proinflammatory cytokines like TNF-α, IL-6 and IL-1β which have been implicated in pathogenesis of severe DENV disease. Together, our finding suggests that DENV-2 modulates HMGB1 translocation and HMGB1-DENV-2 UTRs RNA interaction further induces proinflammatory cytokines production in A549 cells. This study discloses HMGB1 as an important host factor contributing to disease pathogenesis and hence can be targeted as an alternative approach for antiviral development against DENV virus infection.
Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. METHODOLOGY/RESULT: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24 ± 0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX.
The ability to metastasize is a hallmark of malignant tumors, and metastasis is the principal cause of death of cancer patients. The High Mobility Group Box-1 (HMGB1) is a multifunction protein that serves as both a chromatin protein and an extracellular signaling molecule. Our current study demonstrated a novel mechanism of HMGB1 in the regulation of cancer cell actin polymerization, cell skeleton formation, cancer cell motility and metastasis. We found that knockdown of HMGB1 in human lung cancer A549 cells significantly increased cell β-actin polymerization, cell skeleton formation, cancer cell migration and invasion in vitro, as well as metastasis in vivo. And this increase could be inhibited by treatment of HMGB1 knockdown cells with recombinant human HMGB1. Further studies discovered that HMGB1 suppressed phosphorylation, nuclear translocation, and activation of CREB, by inhibiting nuclear translocation of PKA catalytic subunit. This reduces nWASP mRNA transcription and expression, further impairing cancer cell motility. Our findings on the novel mechanism underlying the HMGB1 anti-metastatic effect on cancer provides significant insight into the understanding of the nature of HMGB1 in cancer invasion and metastasis, further serving as key information for utilization of HMGB1 and its regulated downstream components as new targets for cancer therapy.
The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells.
We present supplementary data for the published article, "Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer" [1]. The assay described is designed to be performed at home or in a clinic without expensive instrumentation or professional training. SARS-CoV-2 is detected by an aptamer-based assay that targets the Nucleocapsid (N) or Spike (S) antigens. Binding of the N or S protein to their respective aptamer results in the competitive release of a complementary antisense-invertase enzyme complex. The released enzyme then catalyzes the conversion of sucrose to glucose that is measured by an off-the-shelf glucometer. The data presented here describe the optimization of the assay parameters and their contribution to developing this aptamer-based assay to detect SARS-CoV-2. The assay performance was checked in a standard buffer, contrived samples, and patient samples validated with well-established scientific methods. The resulting dataset can be used to further develop glucometer-based assays for diagnosing other communicable and non-communicable diseases.
The SARS-CoV-2 nucleocapsid protein (N) binds a single-stranded viral RNA genome to form a helical ribonucleoprotein complex that is packaged into virion particles. N is relatively conserved among coronaviruses and consists of the N-terminal domain (NTD) and C-terminal domain (CTD), which are flanked by three disorganized regions. N is highly immunogenic and has been widely used to develop a serological assay as a diagnostic tool for COVID-19 infection, although there is a concern that the natural propensity of N to associate with RNA might compromise the assay's specificity. We expressed and purified from bacterial cells two recombinant forms of SARS-CoV-2 N, one from the soluble fraction of bacterial cell lysates that is strongly associated with bacterial RNAs and the other that is completely devoid of RNAs. We showed that both forms of N can be used to develop enzyme-linked immunosorbent assays (ELISAs) for the specific detection of human and mouse anti-N monoclonal antibodies (mAb) as well as feline SARS-CoV-2 seropositive serum samples, but that the RNA-free form of N exhibits a slightly higher level of sensitivity than the RNA-bound form to react to anti-N mouse mAb. Using the electrophoretic mobility shift assay (EMSA), we also showed that N preferentially binds ssRNA in a sequence-independent manner and that both NTD and CTD of N contribute to RNA-binding activity. Collectively, our study describes methods to express, purify, and biochemically characterize the SARS-CoV-2 N protein and to use it for the development of serological assays to detect SARS-CoV-2 infection.
Pulmonary hypertension (PH) is a fatal disease characterized by remodeling processes such as increased migration and proliferation of pulmonary arterial smooth muscle cells (PASMC), enhanced matrix deposition, and dysregulation of cytoskeletal proteins. However, the contribution of cytoskeletal proteins in PH is still not fully understood. In this study, we have used a yeast two-hybrid screen to identify novel binding partners of the cytoskeletal adaptor protein four-and-a-half LIM domains 1 (Fhl-1). This identified paxillin as a new Fhl-1 interacting partner, and consequently we assessed its contribution to vascular remodeling processes. Native protein-protein binding was confirmed by co-immunoprecipitation studies in murine and human PASMC. Both proteins co-localized in PASMC in vitro and in vivo. In lung samples from idiopathic pulmonary arterial hypertension patients, paxillin expression was increased on mRNA and protein levels. Laser-microdissection of murine intrapulmonary arteries revealed elevated paxillin expression in hypoxia-induced PH. Furthermore, hypoxia-dependent upregulation of paxillin was HIF-1α dependent. Silencing of paxillin expression led to decreased PASMC adhesion, proliferation, and increased apoptosis. Regulation of these processes occurred via Akt and Erk1/2 kinases. In addition, adhesion of PASMC to the extracellular matrix protein fibronectin was critically dependent on paxillin expression. To summarize, we identified paxillin as a new regulator protein of PASMC growth.
Primary congenital hypothyroidism (CH) is a common neonatal endocrine disorder characterized by elevated concentrations of thyroid stimulating hormone (TSH) and low concentrations of free thyroxine (FT4). PAX8 and NKX2-1 are important transcription factors involved in thyroid development. In this study, we detected three novel variants in PAX8 (c.149A > C and c.329G > A) and NKX2-1 (c.706A > G) by whole exome sequencing (WES) in three unrelated CH patients with variable phenotypes. The results of Western blot and immunofluorescence analysis showed that the three variants had no effect on protein expression and subcellular localization. However, the results of the electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay suggested that the three variants in PAX8 and NKX2-1 both affected their DNA-binding ability and reduced their transactivation capacity. Moreover, a dominant-negative effect in K236E−NKX2-1 was identified by dual-luciferase reporter assay. To sum up, our findings extend our knowledge of the current mutation spectrum of PAX8 and NKX2-1 and provide important information for diagnosing, treating, and preventing CH in these families.
Germline mutations in the coding sequence of the tumour suppressor APC gene give rise to familial adenomatous polyposis (which leads to colorectal cancer) and are associated with many other oncopathologies. The loss of APC function because of deletion of putative promoter 1A or 1B also results in the development of colorectal cancer. Since the regions of promoters 1A and 1B contain many single nucleotide polymorphisms (SNPs), the aim of this study was to perform functional analysis of some of these SNPs by means of an electrophoretic mobility shift assay (EMSA) and a luciferase reporter assay.
Transcription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator gp33, and a universal component of cellular DNA replication, the sliding clamp gp45. Although genetic and biochemical studies have elucidated many aspects of T4 late gene transcription, no precise structure of the transcription machinery in the process is available. Here, we report the cryo-EM structures of a gp55-dependent RNAP-promoter open complex and an intact gp45-dependent transcription activation complex. The structures reveal the interactions between gp55 and the promoter DNA that mediate the recognition of T4 late promoters. In addition to the σR2 homology domain, gp55 has a helix-loop-helix motif that chaperons the template-strand single-stranded DNA of the transcription bubble. Gp33 contacts both RNAP and the upstream double-stranded DNA. Gp45 encircles the DNA and tethers RNAP to it, supporting the idea that gp45 switches the promoter search from three-dimensional diffusion mode to one-dimensional scanning mode.
NFAT involvement in adipocyte physiological processes was examined by treatment with CsA and/or GSK3beta inhibitors (Li(+) or TZDZ-8), which prevent or increase NFAT nuclear translocation, respectively. CsA treatment reduced basal and TNFalpha-induced rates of lipolysis by 50%. Adipocytes preincubated with Li(+) or TZDZ-8 prior to CsA and/or TNFalpha, exhibited enhanced basal rates of lipolysis and complete inhibition of CsA-mediated decreased rates of lipolysis. CsA treatment dramatically reduced the mRNA levels of adipocyte-specific genes (aP2, HSL, PPARgamma, ACS and Adn), compared with control or TNFalpha-treatment, whereas Li(+) pretreatment blocked the inhibitory effects of CsA, and mRNA levels of aP2, HSL, PPARgamma, and ACS were found at or above control levels. NFAT nuclear localization, assessed by EMSA, confirmed that CsA or Li(+) treatments inhibited or increased NFAT nuclear translocation, respectively. These results show that NFAT proteins in mature adipocytes participate in the transcriptional control of genes involved in adipocyte metabolism and lipolysis.
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