Creative Biolabs provides a versatile genetic tool MAPPIT that can be used to analyze and detect new protein-protein interactions in mammalian cells. Discerning the interacting partners of a protein is a straightforward method to gain insight into the protein's functionality and to position it in an interaction network within cells. Varieties of biotechnologies have been described to serve this purpose, and some of them are specially used to study posttranslational modifications in mammalian proteins so as to clarify their intrinsic physiological context. Whereas, several inherent constraints restrict the use of these techniques and most are not suitable for discovering new interacting partners. It’s worth noting that a novel protocol termed Mammalian Protein-Protein Interaction Trap (MAPPIT) can work as a versatile genetic tool to detect and analyze unknown protein-protein interactions in mammalian cells.
Fig.1 Overview of the different MAPPIT approaches.
(a) MAPPIT (b) Heteromeric MAPPIT (c) Reverse MAPPIT (d) MASPIT (Mammalian small molecule-protein interaction trap)
Physical interactions between proteins play an important role in cellular processes. Focus on drawing the protein interaction maps is ongoing in a number of model organisms and will provide a scaffold for further detailed functional research by a diversity of approaches. MAPPIT is a mammalian two-hybrid system that allows the detection and analysis of protein-protein interactions (PPI), particularly in their native environment. This method is on the basis of type I cytokine receptor signal transduction. A bait protein of interest is fused to the signaling-deficient cytokine receptor chimera, the signaling competence of which is restored upon recruitment of a prey protein coupled to a functional cytokine receptor domain. Ultimately, the result leads to the transcription of a reporter or marker gene under the control of the promoter.
Mechanisms of MAPPIT
MAPPIT stands out as a pioneering PPI technology which is found on a true mammalian signal transduction pathway, the Janus kinase-signal transducer and activator of transcription (JAK-STAT) cascade, other than derived from yeast or other lower animal models. MAPPIT has arisen from the knowledge of the classic JAK-STAT pathway. In mammals, there are four JAK kinases (JAK1, JAK2, JAK3, and Tyk2) and seven STAT transcription factors (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6) having been identified. The canonical pattern of JAK-STAT signaling is activated by the binding of specific peptide ligands (e.g. cytokines as interleukins and colony-stimulating factors, several hormones as leptin and growth hormone) to transmembrane receptors.
The MAPPIT assay typically utilizes a signaling-deficient chimeric type I cytokine receptor whose extracellular domain is derived from the homodimeric erythropoietin receptor (EpoR) and is fused to the transmembrane domain and cytoplasmic tail of the leptin receptor (LepR). This chimeric receptor causes signaling-deficient by mutating the three tyrosines (Tyr) presented in the cytoplasmic tail to phenylalanine (Phe). Through ligand administration, the associated JAKs of this chimeric receptor is activated. However, since there is no tyrosine left in the receptor tail to be phosphorylated, no STAT recruitment sites will be initiated and no signal is transmitted to the nucleus as well. The protein of interest that will serve as bait is C-terminally fused to this signaling-deficient receptor. The prey protein referred to test for interaction with the bait one is coupled to a portion of another cytokine receptor, the glycoprotein 130 receptor (gp130). The usage of gp130 domain contains Tyr motifs after phosphorylation via the JAKs that work as STAT3 recruitment sites. If bait and prey, two proteins of interest interact, the JAKs will be able to phosphorylate Tyrs with cytokine ligand activation. That result generates functional STAT3 binding sites and recruited STATs upon activation by JAKs will migrate to the nucleus to activate the transcription of a luciferase reporter gene.
Fig.2 Schematic representation of the MAPPIT principle.
The interaction between bait and prey results in the recruitment of a gp130 fragment containing STAT3 recruitment sites and thereby facilitating the complementation of the signaling-deficient chimeric receptor bait. In a nutshell, MAPPIT depends on a dysfunctional JAK-STAT pathway, of which the activity is only restored when PPI of specific bait and prey occurs.
Strengths and Development of MAPPIT
Creative Biolabs has seasoned technicians to help worldwide scientists to detect designated protein-protein interactions for their research. MAPPIT system has been proven to be valuable to study interactions between proteins in mammalian cells where offer a normal physiological context for mammalian proteins to be tested. This highlighted asset ensures proper folding and provides the necessary cofactors and regulatory proteins participating in post-translational modifications or assisting any conformational alterations to achieve protein’s interaction.
Other strengths of MAPPIT technique include being sensitivity, robustness, and scalability. It exhibits a significant signal-to-noise ratio, identifies various protein interactions including transient and indirect ones, and has been shown to be highly complementary to other two-hybrid assays. Since the conception of the original MAPPIT, the platform has been expanded assay variations and permitted large-scale PPI screening, mapping of PPI interfaces, PPI inhibitor screening, and drug profiling which open up new fields of application. Meanwhile, there are additional tools of MAPPIT aiming at increasing the versatility of that platform.
Fig.3 The MAPPIT toolbox and its applications.