Electronic Circular Dichroism Detects Conformational Changes Associated with Proteasome Gating Confirmed Using AFM Imaging.

Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and ß-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and ß-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators.

Modulation of the 20S Proteasome Activity by Porphyrin Derivatives Is Steered through Their Charge Distribution.

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a "new" 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure-activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg-Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a "contracted" version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis.

Contacts with Macrophages Promote an Aggressive Nanomechanical Phenotype of Circulating Tumor Cells in Prostate Cancer.

Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of "mechanical fitness" in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell-macrophage cocultures revealed that macrophages promoted epithelial-mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis. SIGNIFICANCE: The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

Cooperative Binding of the Cationic Porphyrin Tris-T4 Enhances Catalytic Activity of 20S Proteasome Unveiling a Complex Distribution of Functional States.

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.

Electrostatic Map Of Proteasome α-Rings Encodes The Design of Allosteric Porphyrin-Based Inhibitors Able To Affect 20S Conformation By Cooperative Binding.

The importance of allosteric proteasome inhibition in the treatment of cancer is becoming increasingly evident. Motivated by this urgent therapeutic need, we have recently identified cationic porphyrins as a highly versatile class of molecules able to regulate proteasome activity by interfering with gating mechanisms. In the present study, the mapping of electrostatic contacts bridging the regulatory particles with the α-rings of the human 20S proteasome led us to the identification of (meso-tetrakis(4-N-methylphenyl pyridyl)-porphyrin (pTMPyPP4) as a novel non-competitive inhibitor of human 20S proteasome. pTMPyPP4 inhibition mechanism implies a positive cooperative binding to proteasome, which disappears when a permanently open proteasome mutant (α-3ΔN) is used, supporting the hypothesis that the events associated with allosteric proteasome inhibition by pTMPyPP4 interfere with 20S gating and affect its "open-closed" equilibrium. Therefore, we propose that the spatial distribution of the negatively charged residues responsible for the interaction with regulatory particles at the α-ring surface of human 20S may be exploited as a blueprint for the design of allosteric proteasome regulators.

Copper(II) ions affect the gating dynamics of the 20S proteasome: a molecular and in cell study.

Due to their altered metabolism cancer cells are more sensitive to proteasome inhibition or changes of copper levels than normal cells. Thus, the development of copper complexes endowed with proteasome inhibition features has emerged as a promising anticancer strategy. However, limited information is available about the exact mechanism by which copper inhibits proteasome. Here we show that Cu(II) ions simultaneously inhibit the three peptidase activities of isolated 20S proteasomes with potencies (IC50) in the micromolar range. Cu(II) ions, in cell-free conditions, neither catalyze red-ox reactions nor disrupt the assembly of the 20S proteasome but, rather, promote conformational changes associated to impaired channel gating. Notably, HeLa cells grown in a Cu(II)-supplemented medium exhibit decreased proteasome activity. This effect, however, was attenuated in the presence of an antioxidant. Our results suggest that if, on one hand, Cu(II)-inhibited 20S activities may be associated to conformational changes that favor the closed state of the core particle, on the other hand the complex effect induced by Cu(II) ions in cancer cells is the result of several concurring events including ROS-mediated proteasome flooding, and disassembly of the 26S proteasome into its 20S and 19S components.

Cationic porphyrins are tunable gatekeepers of the 20S proteasome.

The 20S proteasome is a barrel-shaped enzymatic assembly playing a critical role in proteome maintenance. Access of proteasome substrates to the catalytic chamber is finely regulated through gating mechanisms which involve aromatic and negatively charged residues located at the N-terminal tails of α subunits. However, despite the importance of gates in regulating proteasome function, up to now very few molecules have been shown to interfere with the equilibrium by which the catalytic channel exchanges between the open and closed states. In this light, and inspired by previous results evidencing the antiproteasome potential of cationic porphyrins, here we combine experimental (enzyme kinetics, UV stopped flow and NMR) and computational (bioinformatic analysis and docking studies) approaches to inspect proteasome inhibition by meso-tetrakis(4-N-methylpyridyl)-porphyrin (H2T4) and its two ortho- and meta-isomers. We show that in a first, fast binding event H2T4 accommodates in a pocket made of negatively charged and aromatic residues present in α1 (Asp10, Phe9), α3 (Tyr5), α5 (Asp9, Tyr8), α6 (Asp7, Tyr6) and α7 (Asp9, Tyr8) subunits thereby stabilizing the closed conformation. A second, slower binding mode involves interaction with the grooves which separate the α- from the ß-rings. Of note, the proteasome inhibition by ortho- and meta-H2T4 decreases significantly if compared to the parent compound, thus underscoring the role played by spatial distribution of the four peripheral positive charges in regulating proteasome-ligand interactions. We think that our results may pave the way to further studies aimed at rationalizing the molecular basis of novel, and more sophisticated, proteasome regulatory mechanisms.