Regulation of tyrosine hydroxylase is preserved across different homo- and heterodimeric 14-3-3 proteins

Tyrosine hydroxylase (TH) is regulated by members of the 14-3-3 protein family. However, knowledge about the variation between 14-3-3 proteins in their regulation of TH is still limited. We examined the binding, effects on activation and dephosphorylation kinetics of tyrosine hydroxylase (TH) by abundant midbrain 14-3-3 proteins (β, η, ζ, γ and ε) of different dimer composition. All 14-3-3 homodimers and their respective 14-3-3ε-heterodimers bound with similar high affinity (Kd values of 1.4–3.8 nM) to serine19 phosphorylated human TH (TH-pS19). We similarly observed a consistent activation of bovine (3.3- to 4.4-fold) and human TH-pS19 (1.3–1.6 fold) across all the different 14-3-3 dimer species, with homodimeric 14-3-3γ being the strongest activator. Both hetero- and homodimers of 14-3-3 strongly inhibited dephosphorylation of TH-pS19, and we speculate if this is an important homeostatic mechanism of 14-3-3 target-protein regulation in vivo. We conclude that TH is a robust interaction partner of different 14-3-3 dimer types with moderate variability between the 14-3-3 dimers on their regulation of TH.publishedVersio

Arc is a flexible modular protein capable of reversible self-oligomerization

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes.publishedVersio

Structural and thermodynamic insight into phenylalanine hydroxylase from the human pathogen Legionella pneumophila

Phenylalanine hydroxylase from Legionella pneumophila (lpPAH) has a major functional role in the synthesis of the pigment pyomelanin, which is a potential virulence factor. We present here the crystal structure of lpPAH, which is a dimeric enzyme that shows high thermostability, with a midpoint denaturation temperature of 79 °C, and low substrate affinity. The structure revealed a dimerization motif that includes ionic interactions and a hydrophobic core, composed of both β-structure and a C-terminal region, with the specific residues (P255, P256, Y257 and F258) interacting with the same residues from the adjacent subunit within the dimer. This unique dimerization interface, together with a number of aromatic clusters, appears to contribute to the high thermal stability of lpPAH. The crystal structure also explains the increased aggregation of the enzyme in the presence of salt. Moreover, the low affinity for substrate l-Phe could be explained from three consecutive glycine residues (G181, 182, 183) located at the substrate-binding site. This is the first structure of a dimeric bacterial PAH and provides a framework for interpreting the molecular and kinetic properties of lpPAH and for further investigating the regulation of the enzyme.publishedVersio

Inhibition of the Human Hsc70 System by Small Ligands as a Potential Anticancer Approach

Heat shock protein (Hsp) synthesis is upregulated in a wide range of cancers to provide the appropriate environment for tumor progression. The Hsp110 and Hsp70 families have been associated to cancer cell survival and resistance to chemotherapy. In this study, we explore the strategy of drug repurposing to find new Hsp70 and Hsp110 inhibitors that display toxicity against melanoma cancer cells. We found that the hits discovered using Apg2, a human representative of the Hsp110 family, as the initial target bind also to structural regions present in members of the Hsp70 family, and therefore inhibit the remodeling activity of the Hsp70 system. One of these compounds, the spasmolytic agent pinaverium bromide used for functional gastrointestinal disorders, inhibits the intracellular chaperone activity of the Hsp70 system and elicits its cytotoxic activity specifically in two melanoma cell lines by activating apoptosis. Docking and molecular dynamics simulations indicate that this compound interacts with regions located in the nucleotide-binding domain and the linker of the chaperones, modulating their ATPase activity. Thus, repurposing of pinaverium bromide for cancer treatment appears as a promising novel therapeutic approach

Arc is a flexible modular protein capable of reversible self-oligomerization

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes