PD-1 and CTLA-4 exert additive control of effector regulatory T cells at homeostasis.

At homeostasis, a substantial proportion of Foxp3 T regulatory cells (T) have an activated phenotype associated with enhanced TCR signals and these effector T cells (eT) co-express elevated levels of PD-1 and CTLA-4. Short term blockade of the PD-1 or CTLA-4 pathways results in increased eT populations, while combination blockade of both pathways had an additive effect. Mechanistically, combination blockade resulted in a reduction of suppressive phospho-SHP2 Y580 in eT cells which was associated with increased proliferation, enhanced production of IL-10, and reduced dendritic cell and macrophage expression of CD80 and MHC-II.

PD-L1-PD-1 interactions limit effector regulatory T cell populations at homeostasis and during infection.

Phenotypic and transcriptional profiling of regulatory T (T) cells at homeostasis reveals that T cell receptor activation promotes T cells with an effector phenotype (eT) characterized by the production of interleukin-10 and expression of the inhibitory receptor PD-1. At homeostasis, blockade of the PD-1 pathway results in enhanced eT cell activity, whereas during infection with Toxoplasma gondii, early interferon-γ upregulates myeloid cell expression of PD-L1 associated with reduced T cell populations. In infected mice, blockade of PD-L1, complete deletion of PD-1 or lineage-specific deletion of PD-1 in T cells prevents loss of eT cells.

Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics.

Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B.