Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium

Mining large-scale response networks reveals `topmost activities' in Mycobacterium tuberculosis infection

Mycobacterium tuberculosis owes its high pathogenic potential to its ability to evade host immune responses and thrive inside the macrophage. The outcome of infection is largely determined by the cellular response comprising a multitude of molecular events. The complexity and inter-relatedness in the processes makes it essential to adopt systems approaches to study them. In this work, we construct a comprehensive network of infection-related processes in a human macrophage comprising 1888 proteins and 14,016 interactions. We then compute response networks based on available gene expression profiles corresponding to states of health, disease and drug treatment. We use a novel formulation for mining response networks that has led to identifying highest activities in the cell. Highest activity paths provide mechanistic insights into pathogenesis and response to treatment. The approach used here serves as a generic framework for mining dynamic changes in genome-scale protein interaction networks

Current trends in modeling hostpathogen interactions

The rapid emergence of infectious diseases calls for immediate attention to determine practical solutions for intervention strategies. To this end, it becomes necessary to obtain a holistic view of the complex hostpathogen interactome. Advances in omics and related technology have resulted in massive generation of data for the interacting systems at unprecedented levels of detail. Systems-level studies with the aid of mathematical tools contribute to a deeper understanding of biological systems, where intuitive reasoning alone does not suffice. In this review, we discuss different aspects of hostpathogen interactions (HPIs) and the available data resources and tools used to study them. We discuss in detail models of HPIs at various levels of abstraction, along with their applications and limitations. We also enlist a few case studies, which incorporate different modeling approaches, providing significant insights into disease. (c) 2013 Wiley Periodicals, Inc

Analysis of the differential effect of n-Butanol, U0126, Wortmannin and LY294002 on reporter gene expression and phosphorylation of mTOR in transfected PC3 and 293T cells.

<p>Effect of n-butanol on expression of (<b>A</b>) GFP; (<b>B</b>) β-Gal, and (<b>C</b>) Inhibition of phosphorylation of mTOR by n-butanol; Effect of U0126 (U0), Wortmannin (Wort) and LY294002 (LY) on (<b>D</b>) β-Gal and (<b>E</b>) GFP expression. Proteins were separated by 12–14% SDS-PAGE and detected by immunoblotting using antibodies specific to GFP and β-Gal.</p

Analysis of RNA and protein levels derived from the transfected expression vectors of PDGF-B, GFP and β-Gal in PC3, PC3BM, HeLa, 293T and MA104 cells.

<p>(<b>A</b>) PDGF-B mRNA levels were determined by RNase protection Assay. The 144 nt protected band corresponds to PDGF-B and the 120 nt band represents that of β-Actin mRNA. (<b>B</b>) RT-PCR of β-Gal, GFP and β-Actin mRNA in pcDNA3-β-Gal and pcDNA3-GFP transfected cells. (<b>C</b>) Radioimmunoprecipitation of PDGF-B protein expressed in pCMV-PDGF-B transfected cells using an N-terminal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014408#pone.0014408-Rao2" target="_blank">[69]</a>. (<b>D</b>) Levels of β-Gal, GFP and β-Tubulin proteins in pcDNA3- β-Gal and -GFP transfected cells. 50 µg of transfected cell lysate was analyzed for GFP and β-Gal levels by SDS-PAGE. (<b>E</b>) β-galactosidase assay using the β-Gal ELISA Kit from Roche Diagnostics. (<b>F</b>) Fluorescence microscopy and bright field (BF) images of 293T, PC3BM, HeLa and PC3 cells transfected with pcDNA-GFP reporter gene construct. (<b>G</b>) Analysis of the fold differences in expression of the reporter mRNA and protein levels between PC3 and HeLa, and PC3 and PC3BM.</p

Western blot analysis of key target proteins of mTOR pathway.

<p>Analysis of total protein and/or phosphorylated forms of (<b>A</b>) mTOR and TSC2, (<b>B</b>) eIF4E, (<b>C</b>) 4EBP1, (<b>D</b>) S6, (<b>E</b>) eEF2 and eEF2K (<b>F</b>) p70S6K, (<b>G</b>) PI3K and PDK1, (<b>H</b>) Akt and (<b>I</b>) ERK1/2, p38 MAPK and PKC. 50 µg of cell lysate was used for analysis of mTOR, TSC2, PI3K, PDK1, Akt, eEF2, 4EBP1 and S6 and 100 µg was used for detection of phosphorylated forms and other proteins.</p

Comparative analysis of the translational regulatory proteins and different signaling pathway proteins in PC3 and 293 T cells.

<p>(<b>A</b>) Analysis of mTOR target proteins 4EBP1, S6, eEF2K and p70S6K. (<b>B</b>) mTOR and TSC2. (<b>C</b>) PI3K/Akt and ERK1/2. (<b>D</b>) PKC. 200 µg of cell lysate was used for PKC analysis and 50 µg was used for analysis of other proteins.</p

Virtual Screening of Curcumin and Its Analogs Against the Spike Surface Glycoprotein of SARS-CoV-2 and SARS-CoV

COVID-19, a new pandemic caused by SARS-CoV-2, was first identified in 2019 in Wuhan, China. The novel corona virus SARS-CoV-2 and the 2002 SARS-CoV have 74 % identity and use similar mechanisms to gain entry into the cell. Both the viruses enter the host cell by binding of the viral spike glycoprotein to the host receptor, angiotensin converting enzyme 2 (ACE2). Targeting entry of the virus has a better advantage than inhibiting the later stages of the viral life cycle. The crystal structure of the SARS-CoV (6CRV: full length S protein) and SARS-CoV-2 Spike proteins (6M0J: Receptor binding domain, RBD) was used to determine potential small molecule inhibitors. Curcumin, a naturally occurring phytochemical in Curcuma longa, is known to have broad pharmacological properties. In the present study, curcumin and its derivatives were docked, using Autodock 4.2, onto the 6CRV and 6M0J to study their capability to act as inhibitors of the spike protein and thereby, viral entry. The curcumin and its derivatives displayed binding energies, ΔG, ranging from -10.98 to -5.12 kcal/mol (6CRV) and -10.01 to -5.33 kcal/mol (6M0J). The least binding energy was seen in bis-demethoxycurcumin with: ΔG = -10.98 kcal/mol (6CRV) and -10.01 kcal/mol (6M0J). A good binding energy, drug likeness and efficient pharmacokinetic parameters suggest the potential of curcumin and few of its derivatives as SARS-CoV-2 spike protein inhibitors. However, further research is necessary to investigate the ability of these compounds as viral entry inhibitors.<br /

DenHunt - A Comprehensive Database of the Intricate Network of Dengue-Human Interactions

<div><p>Dengue virus (DENV) is a human pathogen and its etiology has been widely established. There are many interactions between DENV and human proteins that have been reported in literature. However, no publicly accessible resource for efficiently retrieving the information is yet available. In this study, we mined all publicly available dengue–human interactions that have been reported in the literature into a database called DenHunt. We retrieved 682 direct interactions of human proteins with dengue viral components, 382 indirect interactions and 4120 differentially expressed human genes in dengue infected cell lines and patients. We have illustrated the importance of DenHunt by mapping the dengue–human interactions on to the host interactome and observed that the virus targets multiple host functional complexes of important cellular processes such as metabolism, immune system and signaling pathways suggesting a potential role of these interactions in viral pathogenesis. We also observed that 7 percent of the dengue virus interacting human proteins are also associated with other infectious and non-infectious diseases. Finally, the understanding that comes from such analyses could be used to design better strategies to counteract the diseases caused by dengue virus. The whole dataset has been catalogued in a searchable database, called DenHunt (<a href="http://proline.biochem.iisc.ernet.in/DenHunt/" target="_blank">http://proline.biochem.iisc.ernet.in/DenHunt/</a>).</p></div