Dissecting protein tyrosine phosphatase signaling by engineered chemogenetic control of its activity.

Protein tyrosine phosphatases (PTPases) are critical mediators of dynamic cell signaling. A tool capable of identifying transient signaling events downstream of PTPases is essential to understand phosphatase function on a physiological time scale. We report a broadly applicable protein engineering method for allosteric regulation of PTPases. This method enables dissection of transient events and reconstruction of individual signaling pathways. Implementation of this approach for Shp2 phosphatase revealed parallel MAPK and ROCK II dependent pathways downstream of Shp2, mediating transient cell spreading and migration. Furthermore, we show that the N-SH2 domain of Shp2 regulates MAPK-independent, ROCK II-dependent cell migration. Engineered targeting of Shp2 activity to different protein complexes revealed that Shp2-FAK signaling induces cell spreading whereas Shp2-Gab1 or Shp2-Gab2 mediates cell migration. We identified specific transient morphodynamic processes induced by Shp2 and determined the role of individual signaling pathways downstream of Shp2 in regulating these events. Broad application of this approach is demonstrated by regulating PTP1B and PTP-PEST phosphatases.

Engineered Allosteric Regulation of Protein Function.

Allosteric regulation of proteins has been utilized to study various aspects of cell signaling, from unicellular events to organism-wide phenotypes. However, traditional methods of allosteric regulation, such as constitutively active mutants and inhibitors, lack tight spatiotemporal control. This often leads to unintended signaling consequences that interfere with data interpretation. To overcome these obstacles, researchers employed protein engineering approaches that enable tight control of protein function through allosteric mechanisms. These methods provide high specificity as well as spatial and temporal precision in regulation of protein activity in vitro and in vivo. In this review, we focus on the recent advancements in engineered allosteric regulation and discuss the various bioengineered allosteric techniques available now, from chimeric GPCRs to chemogenetic and optogenetic switches. We highlight the benefits and pitfalls of each of these techniques as well as areas in which future improvements can be made. Additionally, we provide a brief discussion on implementation of engineered allosteric regulation approaches, demonstrating that these tools can shed light on elusive biological events and have the potential to be utilized in precision medicine.

Gestational Insulin Resistance Is Mediated by the Gut Microbiome-Indoleamine 2,3-Dioxygenase Axis.

BACKGROUND & AIMS: Normal gestation involves a reprogramming of the maternal gut microbiome (GM) that contributes to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of the GM-maternal metabolism interaction. METHODS: The GM and plasma metabolome of CD1, NIH-Swiss, and C57 mice were analyzed with the use of 16S rRNA sequencing and untargeted liquid chromatography-mass spectrometry throughout gestation. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM was studied with the use of fecal microbial transplants (FMTs). RESULTS: Significant variation in GM alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time point specific, with the species enriched at gestation day 15/19 (G15/19), a point of heightened IR, being distinct from those enriched before or after pregnancy. Metabolomics revealed elevated plasma kynurenine at G15/19 in all 3 mouse strains. IDO1, the rate-limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine level effects on IR derive from the GM in both mouse and human pregnancy. CONCLUSIONS: GM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation, and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1. (Gestational Gut Microbiome-IDO1 Axis Mediates Pregnancy Insulin Resistance; EMBL-ENA ID: PRJEB45047. MetaboLights ID: MTBLS3598).