Mapping PPI networks is challenging as it

Mapping PPI networks is challenging, as it is difficult to express and purify large variaties of Pleuromutilin synthesis in sufficient quantities to allow the study of interactions between them. Membrane-bound proteins are particularly difficult to purify in functional form due to their amphiphilic nature. Furthermore, many molecular interactions are transient and weak, reducing the yield of bound material, and these interactions therefore require highly sensitive detection methods. Commonly used methods to study PPIs are the yeast-two hybrid (Y2H) system [[32], [33]] and affinity purification followed by mass spectrometry (AP-MS). Some proteins may not fold, function or interact properly in the environment of the yeast nucleus, for example membrane-bound proteins. This limitation has led in recent years to the development of protein complementation assays (PCAs), which are compatible with a broader spectrum of proteins [[34], [35]]. Although PCA is considered more reliable than Y2H, both methods are relatively low in sensitivity and in specificity. Another widely used method for characterizing PPIs is AP-MS [36], a biochemical approach adjusted for protein complexes rather than mapping direct binary interactions. Therefore, these technologies are insufficient for detecting short-lived and weak interactions with viral proteins. Microfluidic microarray technologies have several important advantages over commonly used microarray platforms, including low reagent consumption and therefore low reagent cost, as well as high throughput capabilities, which enable simultaneous screening for 100 s to 1000 s of PPIs [37]. In PPI assays, the microfluidic device is programmed with a ‘synthetic genes’ array, which is translated to a protein array. This ‘protein interaction network generator’ (PING) platform can be used to form both soluble protein arrays [38] and membrane protein arrays (MPAs) [39]. The microfluidic devices are made of soft, transparent, bio-compatible elastomers, most commonly PDMS, which can be easily molded into desired 3-D structures. Integrated microfluidic devices comprise micromechanical valves that regulate the flow of fluids in the device, as well as “button” valves that trap interacting molecules and provide a snapshot of interactions at equilibrium, allowing detection of weak and transient interactions [[40], [41]]. These numerous advantages make the microfluidic protein array a promising tool for detecting PPIs between any sets of proteins.
Materials and methods
Results
Discussion Following the screening for PPIs, we selected newly found interactions for validation by co-IP and for cleavage analysis. Five out of 10 tested proteins were verified by co-IP as new interactors of the NS3/4A protein; however, out of 20 interactions examined, only one protein, NRG1, was cleaved. The lack of proteolytic activity of the protease shows both its specificity towards very few proteins, as well as its multifunctionality in its interaction with a variety of other proteins. IL-28A and JAK2, which were newly found partners of NS3/4A, are known to be anti-viral, anti-tumor and immunomodulatory proteins. They play a critical role in anti-viral host defense via the activation of the JAK/STAT signaling pathway, resulting in the expression of IFN-stimulated genes [54,55]. JAK is involved in various processes such as cell growth, development, differentiation and histone modifications and also mediates essential signaling events in both innate and adaptive immunity [54]. In addition, CDK2, another new interactor of NS3, is a serine/threonine-protein kinase that controls the timing of entry into mitosis/meiosis via the subsequent phosphorylation of cyclin B/CDK1 in the centrosome and in the nucleus. We speculated that NS3/4A inhibition of JAK2, IL-28A and CDK1 involves their cleavage, but found no evidence of such cleavage. Nevertheless, NS3/4A inhibits protein activity through interaction and complex formation and not necessarily by cleavage, as, for example, in the case of P53 or TBK1 proteins.