Current Projects in the Veglia Lab
Cyclic AMP (cAMP) mediated cell signaling
A primary cAMP receptor is the cAMP-dependent protein kinase A (PKA). This enzyme was the first kinase to be crystallized and has been used as the prototypical example for the AGC protein kinase family. In the inactive form, PKA adopts a homotetrameric assembly (holoenzyme) with two catalytic subunits (C subunits or PKA-C) and two regulatory subunits (R subunits). The holoenzyme is anchored to the membrane via A-kinase anchoring protein (AKAP). The classical activation mechanism involves cAMP binding to the R subunits and the release of PKA-C, which is free to phosphorylate a plethora of substrates. While several aberrant mutations have been discovered in the R subunit, in the past decades, mutations, deletions, and fusions have been found in the PRKACA gene encoding for PKA-C. These modifications are responsible for dysregulating cAMP signaling and the progression of diseases such as Cushing’s syndrome, myxomas, and fibrolamellar hepatocellular carcinomas. Our group utilizes spectroscopic and biophysical methods to understand how these mutations perturb the structural dynamics and allosteric signaling of PKA-C, resulting in dysfunctional cAMP signaling and leading to disease.
Selected publications
Calcium transport in skeletal and heart muscle
Calcium transport is central to cardiac and skeletal muscle contractility. Its homeostatic balance is modulated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA), which handles ~70% of intracellular Ca2+ regulation in humans. SERCA is an integral membrane protein whose function is regulated by an array of single-pass membrane proteins called regulins. Regulins keep this ATPase’s activity within a narrow physiological window. Dysregulation of SERCA activity degenerates into muscle disease. So far, seven regulins have been sequenced: phospholamban (PLN), sarcolipin (SLN), endoregulin (ELN), another regulin (ALN), myoregulin (MLN), dwarf open reading frame (DWORF), and sarcolamban (SCL, Drosophila m.). These regulins are single-pass membrane proteins that bind SERCA in the transmembrane domain and allosterically control SERCA’s apparent affinity for Ca2+ ions. Some regulins are post-translationally modified (e.g., phosphorylated, lipidation, acetylation, etc.). These events reverse or augment regulins’ regulatory function. In the past decade, it was found that SERCA’s regulatome has a new player, HAX-1, an intrinsically disordered protein that interacts with the other regulins to enhance their function. We aim to understand how regulins and HAX-1 interact with SERCA to augment or decrease Ca2+ transport and concomitant muscle contractility. Understanding the molecular determinant for Ca2+ transport by SERCA is critical to devise new and innovative therapy to counteract muscle disease, including heart failure.
Selected publications
NMR methods development
Many of our projects necessitate the development of novel techniques to investigate the structure and dynamic interactions of these large protein complexes. Therefore, we dedicate a significant effort to developing new methods to improve solution- and solid-state NMR spectroscopy. Specifically, we have been pioneering methods for multiple acquisitions of solid-state NMR spectra utilizing orphan spin operators. These polarization optimized experiments (POE) constitute the basis for speeding up multidimensional solid-state NMR experiments and can be applied to static and magic angle spinning experiments of soluble and membrane-bound proteins. Currently, we are applying evolutionary algorithms and artificial intelligence to redesign RF pulses and pulse sequences to improve sensitivity and performance and upgrade them for applications of NMR spectroscopy at high- and ultra-high magnetic fields. We developed a new software (GENETICS-AI) to design high-fidelity RF shapes that are highly compensated for inhomogeneity for possible MR spectroscopy and imaging applications.
Selected publications/patents