Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

Structural determinants of the interactions of catechins with Aß oligomers and lipid membranes.

The aberrant self-assembly of intrinsically disordered proteins (IDPs) into soluble oligomers and their interactions with biological membranes underlie the pathogenesis of numerous neurodegenerative diseases, including Alzheimer's disease. Catechins have emerged as useful tools to reduce the toxicity of IDP oligomers by modulating their interactions with membranes. However, the structural determinants of catechin binding to IDP oligomers and membranes remain largely elusive. Here, we assemble a catechin library by combining several naturally occurring chemical modifications and, using a coupled NMR-statistical approach, we map at atomic resolution the interactions of such library with the Alzheimer's-associated amyloid-beta (Aß) oligomers and model membranes. Our results reveal multiple catechin affinity drivers and show that the combination of affinity-reducing covalent changes may lead to unexpected net gains in affinity. Interestingly, we find that the positive cooperativity is more prevalent for Aß oligomers than membrane binding, and that the determinants underlying catechin recognition by membranes are markedly different from those dissected for Aß oligomers. Notably, we find that the unanticipated positive cooperativity arises from the critical regulatory role of the gallate catechin moiety, which recruits previously disengaged substituents into the binding interface and leads to an overall greater compaction of the receptor-bound conformation. Overall, the previously elusive structural attributes mapped here provide an unprecedented foundation to establish structure-activity relationships of catechins.

Allosteric pluripotency: challenges and opportunities.

Allosteric pluripotency arises when the functional response of an allosteric receptor to an allosteric stimulus depends on additional allosteric modulators. Here, we discuss allosteric pluripotency as observed in the prototypical Protein Kinase A (PKA) as well as in other signaling systems, from typical multidomain signaling proteins to bacterial enzymes. We identify key drivers of pluripotent allostery and illustrate how hypothesizing allosteric pluripotency may solve apparent discrepancies currently present in the literature regarding the dual nature of known allosteric modulators. We also outline the implications of allosteric pluripotency for cellular signaling and allosteric drug design, and analyze the challenges and opportunities opened by the pluripotent nature of allostery.

Catechins as Tools to Understand the Molecular Basis of Neurodegeneration.

Protein misfolding as well as the subsequent self-association and deposition of amyloid aggregates is implicated in the progression of several neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Modulators of amyloidogenic aggregation serve as essential tools to dissect the underlying molecular mechanisms and may offer insight on potential therapeutic solutions. These modulators include green tea catechins, which are potent inhibitors of amyloid aggregation. Although catechins often exhibit poor pharmacokinetic properties and bioavailability, they are still essential tools for identifying the drivers of amyloid aggregation and for developing other aggregation modulators through structural mimicry. As an illustration of such strategies, here we review how catechins have been used to map the toxic surfaces of oligomeric amyloid-like species and develop catechin-based phenolic compounds with enhanced anti-amyloid activity.

Inhibition of toxic metal-alpha synuclein interactions by human serum albumin.

Human serum albumin (HSA), the most abundant protein in plasma and cerebrospinal fluid, not only serves as a crucial carrier of various exogenous and endogenous ligands but also modulates the aggregation of amyloidogenic proteins, including alpha synuclein (αSyn), which is associated with Parkinson's disease and other α-synucleinopathies. HSA decreases αSyn toxicity through the direct binding to monomeric and oligomeric αSyn species. However, it is possible that HSA also sequesters metal ions that otherwise promote aggregation. Cu(ii) ions, for example, enhance αSyn fibrillization in vitro, while also leading to neurotoxicity by generating reactive oxygen species (ROS). However, it is currently unclear if and how HSA affects Cu(ii)-binding to αSyn. Using an integrated set of NMR experiments, we show that HSA is able to chelate Cu(ii) ions from αSyn more efficiently than standard chelators such as EDTA, revealing an unexpected cooperativity between the HSA metal-binding sites. Notably, fatty acid binding to HSA perturbs this cooperativity, thus interfering with the sequestration of Cu(ii) ions from αSyn. We also observed that glycation of HSA diminished Cu(ii)-binding affinity, while largely preserving the degree of cooperativity between the HSA metal-binding sites. Additionally, our results show that Cu(ii)-binding to HSA stabilizes the interactions of HSA with αSyn primarily at two different regions, i.e. the N-terminus, Tyr 39 and the majority of the C-terminus. Our study not only unveils the effect of fatty acid binding and age-related posttranslational modifications, such as glycation, on the neuroprotective mechanisms of HSA, but also highlights the potential of αSyn as a viable NMR-based sensor to investigate HSA-metal interactions.

Early-Onset Parkinson Mutation Remodels Monomer-Fibril Interactions to Allosterically Amplify Synuclein's Amyloid Cascade.

Alpha synuclein (αS) aggregates are the main component of Lewy bodies (LBs) associated with Parkinson's disease (PD). A longstanding question about αS and PD pertains to the autosomal dominant E46K αS mutant, which leads to the early onset of PD and LB dementias. The E46K mutation not only promotes αS aggregation but also stabilizes αS monomers in "closed" conformers, which are compact and aggregation-incompetent. Hence, the mechanism of action of the E46K mutation is currently unclear. Here, we show that αS monomers harboring the E46K mutation exhibit more extensive interactions with fibrils compared to those of WT. Such monomer-fibril interactions are sufficient to allosterically drive transitions of αS monomers from closed to open conformations, enabling αS aggregation. We also show that E46K promotes head-to-tail monomer-monomer interactions in early self-association events. This multipronged mechanism provides a new framework to explain how the E46K mutation and possibly other αS variants trigger early-onset PD.

Interactions of intrinsically disordered proteins with the unconventional chaperone human serum albumin: From mechanisms of amyloid inhibition to therapeutic opportunities.

Human Serum Albumin (HSA), the most abundant protein in plasma, serves a diverse repertoire of biological functions including regulation of oncotic pressure and redox potential, transport of serum solutes, but also chaperoning of misfolded proteins. Here we review how HSA interacts with a wide spectrum of client proteins including intrinsically disordered proteins (IDPs) such as Aß, the islet amyloid peptide (IAPP), alpha synuclein and stressed globular proteins such as insulin. The comparative analysis of the HSA chaperone - client interactions reveals that the amyloid-inhibitory function of HSA arises from at least four emerging mechanisms. Two mechanisms (the monomer stabilizer model and the monomer competitor model) involve the direct binding of HSA to either IDP monomers or oligomers, while other mechanisms (metal chelation and membrane protection) rely on the indirect modulation by HSA of other factors that drive IDP aggregation. While HSA is not the only extracellular chaperone, given its abundance, HSA is likely to account for a significant fraction of the chaperoning effects in plasma, thus opening new therapeutic opportunities in the context of the peripheral sink hypothesis.

Non-Canonical Allostery in Cyclic Nucleotide Dependent Kinases.

The cAMP- and cGMP-dependent protein kinases (PKA and PKG) are canonically activated by the corresponding cyclic nucleotides. However, both systems are also sensitive to a wide range of non-canonical allosteric effectors, such as reactive oxygen species, which induce the formation of regulatory inter- and intra-molecular disulfide bridges, and disease-related mutations (DRMs). Here, we present a combined analysis of representative non-canonical allosteric effectors for PKA and PKG, and we identify common molecular mechanisms underlying non-canonical allostery in these kinases, from shifts in dynamical regulatory equilibria to modulation of inter-protomer interactions. In addition, mutations may also drive oligomerization beyond dimerization, and possibly phase transitions, causing loss of kinase inhibitory function and amplifying the allosteric effects of DRMs. Hence non-canonical allosteric stimuli often result in constitutive kinase activation underlying either physiological control of downstream signaling pathways or pathological outcomes, from aortic aneurisms to cancer predisposition. Overall, PKA and PKG emerge as "pan-sensors" going well beyond canonical cyclic nucleotide activation, revealing their versatile roles as central signaling hubs.