Constructing a Single Tryptophan Arrestin-3 Variant A3V9W by site-direct mutagenesis

To study the potential binding-induced conformational changes on arrestin-3, a multifunctional adaptor protein, we intend to incorporate a fluorinated tryptophan residue as reporter in this protein, then obtain the fluorine NMR spectroscopy of this protein. A3V9W variant was designed to place a single tryptophan residue at the N-terminal of arrestin-3, which was reported as a primary binding site for some arrestin-3 binding partners such as JNK3 and ERK1/2. Here, we report our efforts to construct this arrestin-3 variant expression plasmid (pTrc HisB) by site-direct mutagenesis

Incorporation of Fluorinated-Tryptophan into c-Jun N-terminal Kinase 3

c-Jun N-terminal Kinase 3 (JNK3) is a member of Mitogen-Activated Protein kinases (MAPK), which regulates a diverse signal transduction events related to many essential cellular processes including differentiation, apoptosis and prefoliation. JNK3 has been recognized as a therapeutic target for neurodegenerative diseases, such as Parkinson's and Alzheimer's. This project seeks to elucidate the potential binding induced conformational changes of JNK3 protein by using 19F Nuclear Magnetic Resonance (NMR) spectroscopy. This study uses site specific incorporation of fluorine tags onto proteins through the use of chemically defined media, which seeks to deprive cells of the amino acid tryptophan. Through the addition of 5-fluoro-indole to the media, a fluorinated precursor to tryptophan, fluorinated tryptophan can be synthesized in cell by tryptophan synthase. For expression of JNK3 in this media, we will optimize the conditions by adding several nutrient additives (serine, and PLP) to increase the amount of fluorinated proteins. Our results demonstrate that the addition of serine and PLP significantly increases the production of functional JNK3. Preliminary NMR data indicates a successful incorporation of fluorine using this method of unnatural amino acid synthesis

The endocrine milieu and CD4 T-lymphocyte polarization during pregnancy

Acceptance of the fetal semi-allograft by the mother's immune system has become the focus of intensive research. CD4+ T cells are important actors in the establishment of pregnancy. Th1/Th2 paradigm has been expanded to include CD4+ regulatory T (Treg) and T helper 17 (Th17) cells. Pregnancy hormones exert very significant modulatory properties on the maternal immune system. In this review, we describe mechanisms by which the endocrine milieu modulates CD4 T cell polarization during pregnancy. We first focused on Treg and Th17 cells and on their importance for pregnancy. Secondly, we review the effects of pregnancy hormones [progesterone (P4) and estradiol (E2)] on immune cells previously described, with a particular attention to human chorionic gonadotropin (hCG). The importance of Treg cells for pregnancy is evidenced. They are recruited before implantation and are essential for pregnancy maintenance. Decreased number or less efficient Treg cells are implicated in fertility disorders. As for Th17 cells, the few available studies suggest that they have a negative impact on fertility. Th17 frequency is increased in infertile patients. With the combination of its pro-effects on Th2 and Treg cells and anti-effects on Th1 and Th17 cells, P4 contributes to establishment of a favorable environment for pregnancy. E2 effects are more dependent on the context but it seems that E2 promotes Treg and Th2 cells while it inhibits Th1 cells. hCG positively influences activities of Treg and uterine natural killer cells. This embryo signal is an essential actor for the success of pregnancy, both as the endocrine factor regulating P4 secretion by the ovarian corpus luteum, but also as a paracrine agent during implantation as well as an angiogenic and immunologic mediator during the course of gestation. Luteinizing hormone (LH) immune properties begin to be studied but its positive impact on Treg cells suggests that LH could be a considerable immunomodulator in the mouse

Short Arrestin-3-Derived Peptides Activate JNK3 in Cells

Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate several signaling branches, including mitogen-activated protein kinase cascades. Arrestin-3-dependent activation of the JNK family can be recapitulated with peptide fragments, which are monofunctional elements distilled from this multi-functional arrestin protein. Here, we use maltose-binding protein fusions of arrestin-3-derived peptides to identify arrestin elements that bind kinases of the ASK1-MKK4/7-JNK3 cascade and the shortest peptide facilitating JNK signaling. We identified a 16-residue arrestin-3-derived peptide expressed as a Venus fusion that leads to activation of JNK3α2 in cells. The strength of the binding to the kinases does not correlate with peptide activity. The ASK1-MKK4/7-JNK3 cascade has been implicated in neuronal apoptosis. While inhibitors of MAP kinases exist, short peptides are the first small molecule tools that can activate MAP kinases