Biology subjects

Bertinetti, D.

Publications and source records attributed to Bertinetti, D..

5 recordsLinked to original sources

bioRxiv2025

Kinetic and thermodynamic analysis of PKA-R-cAMP interactions in crude media using focal molography

Quantifying biomolecular interactions under near-physiological conditions is essential for understanding biological processes. However, this remains a challenge in established techniques: for surface plasmon resonance, bulk refractive-index shifts and non-specific adsorption in hydrogel matrices can dominate the signal in serum, whereas for isothermal titration calorimetry, complex media primarily introduce large, composition-dependent background heats and run-to-run variability that obscure the binding enthalpy. Here, we demonstrate that focal molography, a label-free optical biosensing method, can reliably measure the kinetics and thermodynamics of the protein kinase A regulatory subunit binding to cyclic AMP derivatives in both buffer and 50% human serum. By performing the kinetic measurement over a range of temperatures, we were able to reveal that binding remains highly favorable in both environments, yet the results suggest that the balance of driving forces shifts toward enthalpic contributions in serum. This hints at the substantial difference of interaction mechanisms in a complex biological media compared to buffer systems. Our findings show that focal molography reliably quantifies protein-ligand interactions in complex media, providing consistent kinetic and thermodynamic data while overcoming limitations of existing methods. This highlights the potential of focal molography as a valuable tool for studying interactions under near-native conditions.

biophysics↗

bioRxiv2024

A Structural Mechanism for Noncanonical GPCRSignal Transduction in the Hedgehog Pathway

SUMMARY PARAGRAPHThe Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced via an unusual mechanism: upon agonist-induced phosphorylation, the noncanonical G protein-coupled receptor SMOOTHENED (SMO) binds the catalytic subunit of protein kinase A (PKA-C) and physically blocks its enzymatic activity. By combining computational structural approaches with biochemical and functional studies, we show that SMO mimics strategies prevalent in canonical GPCR and PKA signaling complexes, despite little sequence or secondary structural homology. An intrinsically disordered region of SMO binds the PKA-C active site, resembling the PKA regulatory subunit (PKA-R) / PKA-C holoenzyme, while the SMO transmembrane domain binds a conserved PKA-C interaction hub, similar to other GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key structural elements within the SMO cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural mechanism for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly.

biochemistry↗

bioRxiv2024

Allosteric modulation of protein kinase A in individuals affected by NLPD-PKA , a neurodegenerative disease in which the RIβ-L50R variant is expressed

Protein kinase A (PKA) is a crucial signaling enzyme in neurons, with its dysregulation being implicated in neurodegenerative diseases. Assembly of the PKA holoenzyme, comprising a dimer of heterodimers of regulatory (R) and catalytic (C) subunits, ensures allosteric regulation and functional specificity. Recently, we defined the RI{beta}-L50R variant as a causative mutation that triggers protein aggregation in a rare neurodegenerative disease. However, the mechanism underlying uncontrolled PKA allosteric regulation and its connection to the functional outcomes leading to clinical symptoms remain elusive. In this study, we established an in vitro model using patient-derived cells for a personalized approach and employed direct measurements of purified proteins to investigate disease mechanisms in a controlled environment. Structural analysis and circular dichroism spectroscopy revealed that cellular proteins aggregation resulted from misfolded RI{beta}-subunits, preventing holoenzyme assembly and anchoring through A Kinase Anchoring Proteins (AKAPs). While maintaining high affinity to the C subunit, the resulting RI{beta}-L50R:C heterodimer exhibits reduced cooperativity, requiring lower cAMP concentrations for dissociation. Consequently, there was an increased translocation of C-subunit into the nucleus, impacting gene expression. We successfully controlled C subunit translocation by introducing a mutation that decreased RI{beta}:C dissociation in response to elevated cAMP levels. This research thus sets the stage for developing therapeutic strategies that modulate PKA assembly and allostery, thus exerting control over the unique molecular signatures identified in the disease-associated transcriptome profile.

cell biology↗

bioRxiv2023

Tripartite interactions of PKA catalytic subunit and C-terminal domains of cardiac Ca2+ channel modulate its β-adrenergic regulation

The adrenergic nervous system augments cardiac contraction by increasing the activity of L-type voltage-gated CaV1.2 channels. Dysregulation of this process is linked to severe cardiac dysfunctions. The signaling cascade involves activation of {beta}-adrenergic receptors, elevation of cAMP levels, separation of protein kinase A (PKA) regulatory subunit (PKAR) from catalytic subunit (PKAC), and phosphorylation of the inhibitory protein Rad leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2 (1C) exists in two forms: full-length (FL) or proteolytically processed (truncated), lacking the distal C-terminus (dCT). Specificity and efficiency in the cascade are believed to emanate from unique protein-protein interactions, such as anchoring PKA (via PKAR) to 1C by A-kinase anchoring proteins (AKAPs). However, most AKAPs do not interact with the truncated 1C, and their role in {beta}AR regulation of cardiac CaV1.2 remains unclear. Here we show that PKAC, independently of PKAR or AKAPs, directly interacts with 1C at two domains in 1C-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Furthermore, we find that DCRD competes with PCRD and reduces its interaction with PKAC. The physiological consequences of these complex interactions are incompletely understood; our data suggest that they may fine-tune the {beta}AR regulation of CaV1.2. We propose that the newly discovered interactions take part in governing colocalization of regulatory proteins within the {beta}AR-CaV1.2 multimolecular signaling complexes in cardiomyocytes.

biochemistry↗

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↗