Biology subjects

Veglia, G.

Publications and source records attributed to Veglia, G..

3 recordsLinked to original sources

bioRxiv2021

A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction

The Hedgehog (Hh) cascade is central to development, tissue homeostasis, and cancer. A pivotal step in Hh signal transduction is the activation of GLI transcription factors by the atypical G protein-coupled receptor (GPCR) Smoothened (SMO). How SMO activates GLI has remained unclear for decades. Here we show that SMO employs a decoy substrate sequence to physically block the active site of the PKA catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular, and organismal models, we demonstrate that interfering with SMO / PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism we uncovered echoes one utilized by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a new mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

biochemistry↗

bioRxiv2021

A Kink in DWORF Helical Structure Controls the Activation of the Sarco-plasmic Reticulum Ca2+-ATPase

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase embedded in the sarcoplasmic reticulum. For each enzymatic cycle, SERCA transports 2 Ca2+ ions per ATP hydrolyzed in exchange for 2 to 3 H+ ions. SERCA is responsible for approximately 70% of Ca2+ transport and plays a central role in muscle relaxation. SERCAs function is regulated by endogenous regulins, single-pass membrane proteins that bind the ATPase within the membrane. While most of the regulins, such as phospholamban and sarcolipin, inhibit SERCAs activity, a newly discovered protein DWarf Open Reading Frame (DWORF) has a unique activating effect. DWORF is a 3.8 kDa bitopic membrane protein expressed in cardiac muscle. In this work, we determine the structure, topology, and per-residue lipid interactions of DWORF in lipid bilayers using a combination of high-resolution oriented sample solid-state NMR (OS-ssNMR) spectroscopy and refinement by replica-averaged orientationally-restrained molecular dynamics (RAOR-MD). We found that DWORFs structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64{degrees} and a transmembrane (TM) C-terminal helix with an angle of 32{degrees}. A kink induced by Pro15, unique to DWORF, separated the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORFs activating effect on SERCA. Overall, our findings directly link DWORFs structural topology to its unique activating effect on SERCA.

biophysics↗

bioRxiv2020

A Bitopic Miniprotein Regulates a Membrane-Embedded Enzyme via Topological Allostery

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to {beta}-adrenergic stimulation, thus modulating cardiac output during the fight- or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzymes function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLNs cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzymes active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally-modified bitopic membrane proteins.

biophysics↗