Spatiotemporal control of kinases and the biomolecular tools to trace activity.

The delicate balance of cell physiology is implicitly tied to the expression and activation of proteins. Post-translational modifications offer a tool to dynamically switch protein activity on and off to orchestrate a wide range of protein-protein interactions to tune signal transduction during cellular homeostasis and pathological responses. There is a growing acknowledgment that subcellular locations of kinases define the spatial network of potential scaffolds, adaptors, and substrates.

Bile Acid-Targeted Hyaluronic Acid Nanoparticles for Enhanced Oral Absorption of Deferoxamine.

Patients with β-thalassemia and sickle cell disease often rely on blood transfusions which can lead to hemochromatosis and chronic oxidative stress in cells and tissues. Deferoxamine (DFO) is clinically approved to treat hemochromatosis but is suboptimal to patients due to its poor pharmacokinetics which requires long-term infusion regimens. Although the oral route is preferable, DFO has limited oral bioavailability.

Fluorescence resonance energy transfer (FRET) spatiotemporal mapping of atypical P38 reveals an endosomal and cytosolic spatial bias.

Mitogen-activated protein kinase (MAPK) p38 is a central regulator of intracellular signaling, driving physiological and pathological pathways. With over 150 downstream targets, it is predicted that spatial positioning and the availability of cofactors and substrates determines kinase signaling specificity. The subcellular localization of p38 is highly dynamic to facilitate the selective activation of spatially restricted substrates.

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells.

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein-coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase-dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined.

The Structure and Immune Regulatory Implications of the Ubiquitin-Like Tandem Domain Within an Avian 2'-5' Oligoadenylate Synthetase-Like Protein.

Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host's ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered.

aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete, β-arrestin-2-mediated SphK1-S1PR1-Akt signaling axis.

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses.

ATF6 is essential for human cone photoreceptor development.

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying disease variants and from gene-edited null hESCs.

Heat shock protein 27 activity is linked to endothelial barrier recovery after proinflammatory GPCR-induced disruption.

Vascular inflammation causes endothelial barrier disruption and tissue edema. Several inflammatory mediators act through G protein–coupled receptors (GPCRs), including protease-activated receptor-1 (PAR1), to elicit inflammatory responses. The activation of PAR1 by its ligand thrombin stimulates proinflammatory, p38 mitogen-activated protein kinase (MAPK) signaling that promotes endothelial barrier disruption.

Atypical p38 Signaling, Activation, and Implications for Disease.

The mitogen-activated protein kinase (MAPK) p38 is an essential family of kinases, regulating responses to environmental stress and inflammation. There is an ever-increasing plethora of physiological and pathophysiological conditions attributed to p38 activity, ranging from cell division and embryonic development to the control of a multitude of diseases including retinal, cardiovascular, and neurodegenerative diseases, diabetes, and cancer. Despite the decades of intense investigation, a viable therapeutic approach to disrupt p38 signaling remains elusive.

Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function.

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling.

Ubiquitination as a Key Regulator of Endosomal Signaling by GPCRs.

G protein-coupled receptors (GPCRs) represent the largest family of therapeutic targets for FDA approved drugs. Therefore, understanding the molecular regulation of their signaling pathways is of paramount importance. Similarly, the mitogen activated protein kinase (MAPK) p38 is a critical mediator of proinflammatory disease.

G protein-coupled receptors activate p38 MAPK via a non-canonical TAB1-TAB2- and TAB1-TAB3-dependent pathway in endothelial cells.

Endothelial dysfunction is induced by inflammatory mediators including multiple G protein-coupled receptor (GPCR) agonists. However, the GPCR signaling pathways that promote endothelial dysfunction are incompletely understood. We previously showed that thrombin promotes endothelial barrier disruption through autophosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via a non-canonical transforming growth factor-β-activated protein kinase-1-binding protein-1 (TAB1) and TAB2-dependent pathway rather than the canonical three-tiered kinase cascade.

A Tyrosine Switch on NEDD4-2 E3 Ligase Transmits GPCR Inflammatory Signaling.

Ubiquitination is essential for protein degradation and signaling and pivotal to many physiological processes. Ubiquitination of a subset of G-protein-coupled receptors (GPCRs) by the E3 ligase NEDD4-2 is required for p38 activation, but how GPCRs activate NEDD4-2 to promote ubiquitin-mediated signaling is not known. Here, we report that the GPCR protease-activated receptor-1 (PAR1) stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4-2 to promote p38 signaling and endothelial barrier disruption.

ALIX Regulates the Ubiquitin-Independent Lysosomal Sorting of the P2Y1 Purinergic Receptor via a YPX3L Motif.

Endocytic sorting and lysosomal degradation are integral to the regulation of G protein-coupled receptor (GPCR) function. Upon ligand binding, classical GPCRs are activated, internalized and recycled or sorted to lysosomes for degradation, a process that requires receptor ubiquitination. However, recent studies have demonstrated that numerous GPCRs are sorted to lysosomes independent of receptor ubiquitination.

Integration of endothelial protease-activated receptor-1 inflammatory signaling by ubiquitin.

Purpose Of Review: The maintenance and integrity of the endothelial barrier is essential for vascular homeostasis. Endothelial barrier dysfunction is mediated by various inflammatory factors, many of which act through G protein-coupled receptors including protease-activated receptors (PARs). PARs are expressed in multiple cell types in the vasculature and mediate cellular responses to thrombin, the key effector protease of the coagulation cascade.

Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins.

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization.

The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein-coupled receptor lysosomal sorting.

The sorting of G protein-coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known.

Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes.

Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability.

Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection.

Azithromycin is a potent macrolide antibiotic with poorly understood antiinflammatory properties. Long-term use of azithromycin in patients with chronic inflammatory lung diseases, such as cystic fibrosis (CF), results in improved outcomes. Paradoxically, a recent study reported that azithromycin use in patients with CF is associated with increased infection with nontuberculous mycobacteria (NTM).