Biophysical analysis of protein-protein and protein-small molecule interactions

The validation of small molecule inhibitors identified by high throughput screening requires a set of robust assays for interaction analysis. Here I describe the implementation of three methods: bioluminescence resonance energy transfer (BRET) for the analysis of protein/protein interaction in cells, surface plasmon resonance (SPR) for the measurement of binding kinetics in vitro and capture compound mass spectrometry (CCMS) for the determination of binding specificity in proteome. All the methods described were tested on cytohesins, a family of guanine nucleotide exchange factors. The very recent discovery of their additional role in the regulation of receptor tyrosine kinases (RTKs) signalling and the availability of specific small molecule inhibitors (the Secins) made them an interesting target. BRET was applied to the analysis of a possible binding of the cytohesin ARNO to the RTK EGF receptor (EGFR). Two strategies were devised: a direct, where the interaction of ARNO and EGFR was monitored, and an indirect, which followed the changes in the EGFR/EGFR interaction upon overexpression of ARNO. Two SPR approaches were developed to analyse the interaction between ARNO and the EGFR on the one hand, and to determine the kinetic parameters of binding of ARNO to its inhibitor Secin16 on the other hand. For CCMS, a photoreactive affinity based SecinH3 probe (SecinH3-TPD) was synthesised and its ability to specifically label ARNO was shown, although the labelling yield was limited by low solubility. A protocol for the enrichment, digestion and MS-analysis of the labelled proteins was established

Resveratrol inhibits the intracellular calcium increase and angiotensin/endothelin system activation induced by soluble uric acid in mesangial cells

Resveratrol (Resv) is natural polyphenol found in grapes. This study evaluated the protective effect of Resv against the effects of uric acid (UA) in immortalized human mesangial cells (ihMCs). ihMCs were preincubated with Resv (12.5 µM) for 1 h and treated with UA (10 mg/dL) for 6 or 12 h. The intracellular calcium concentration [Ca2+]i was quantified by fluorescence using flow cytometry. Angiotensinogen (AGT) and pre-pro endothelin-1 (ppET-1) mRNA were assayed by quantitative real-time RT-PCR. Angiotensin II (AII) and endothelin-1 (ET-1) were assayed by ELISA. UA significantly increased [Ca2+]i. Pre-incubation with Resv significantly reduced the change in [Ca2+]i induced by UA. Incubation with UA for 6 or 12 h also increased AGT mRNA expression and AII protein synthesis. Resv blunted these increases in AGT mRNA expression and AII protein. Incubation with UA in the ihMCs increased ppET-1 expression and ET-1 protein synthesis at 6 and 12 h. When ihMCs were pre-incubated with Resv, UA had a significantly diminished effect on ppET-1 mRNA expression and ET-1 protein synthesis at 6 and 12 h, respectively. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. The renin-angiotensin system may contribute to the pathogenesis of renal function and chronic kidney disease. Resv can minimize the impact of UA on AII, ET-1 and the increase of [Ca2+]i in mesangial cells, suggesting that, at least in part, Resv can prevent the effects of soluble UA in mesangial cells.Universidade Federal de São Paulo (UNIFESP) Departamento de Medicina Divisão de NefrologiaUNIFESP, Depto. de Medicina Divisão de NefrologiaSciEL

Inertial vs. mindful repetition of previous entry mode choices: do firms always learn from experience?

Experience, meant as the repetition of the same action, is considered a predictor of the entry mode choice in foreign markets because it allows reducing uncertainty. However, repetition does not necessarily increase the expected performance, depending on the learning stemming from previous experiences. Focusing on offshoring decisions, namely the choice between captive and outsourcing entry mode, we distinguish between the inertial repetition of routines vs. the mindful repetition of previous entry modes (where the company distinguishes and internalizes the outcomes of the past offshoring initiatives associated to the entry choices). We claim that: (i) the latter leads to higher growth perspectives for the focal offshoring initiative, and; (ii) learning is higher when repetition concerns captive entry modes. Our empirical analysis, run on 410 companies’ offshoring decisions undertaken from 2006 to 2011, confirms our expectation