A Framework for Studying Crucial Steps in Proteasome Core Particle Assembly

The ability of proteins to repeatedly and reliably self-assemble in the cell is a critical element of the maintenance of life. Despite this, the mechanisms that underlie these events are poorly understood. The proteasome core particle from Rhodococcus erythropolis is an excellent model system for understanding assembly processes. This system has a two step assembly pathway where individual subunits first assemble into half proteasomes. Then, two half proteasomes dimerize to produce a full proteasome core particle. The beta subunit of this complex is synthesized in an inactive with an N-terminal propeptide that is cleaved after assembly is complete, rendering the CP enzymatically active. Evidence suggests that the propeptides plays a crucial role in both steps of the assembly process. To date, however, it has been impossible to fully characterize the role of the propeptide in assembly because this protein is typically produced as a heterogeneous mixture with a variety of N-terminally truncations in the propeptides itself. Here, we used Ligation Independent Cloning to produce a beta variant, which we call D3, that is homogeneous for the full-length propeptide. We also used Native PAGE to begin to characterize the kinetics of half proteasome dimerization. We found that there is a temperature-dependent effect on the dimerization process and that the presence of the full propeptide dramatically slowed assembly when compared to the heterogeneous beta. Using these methods, we can now study the thermodynamics and kinetics of this system much more rigorously than has been possible to date

Proteins are Not Recruited: A Plea for Better Diction

Nearly all biological processes proceed or are controlled by protein-protein or protein-ligand binding reactions. Using anthropomorphic language to describe these interactions conveys an incorrect physical description of these processes while simultaneously minimizing the importance of the thermodynamics underpinning the associated interactions. Indeed, we should never say that proteins are recruited to binding partners or binding sites since this implies both a non- existent level of communication within biological systems and a non-existent process by which proteins or binding sites actively seek other proteins. Both of these fictions hinder our ability to determine quantitatively or qualitatively distinct biophysical descriptions of the associated systems. Here we present examples of how interactions typically described as protein recruitment can be more accurately and often more simply described as variations within binding equilibria. We argue that this approach is better for describing protein-protein and protein-ligand binding, even when the objective is only a qualitative description, especially for discussions with students in courses and research groups as it provides testable models for these interaction

Mimicking Elementary Reactions of Manganese Lipoxygenase Using Mn-hydroxo and Mn-alkylperoxo Complexes

Manganese lipoxygenase (MnLOX) is an enzyme that converts polyunsaturated fatty acids to alkyl hydroperoxides. In proposed mechanisms for this enzyme, the transfer of a hydrogen atom from a substrate C-H bond to an active-site MnIII-hydroxo center initiates substrate oxidation. In some proposed mechanisms, the active-site MnIII-hydroxo complex is regenerated by the reaction of a MnIII-alkylperoxo intermediate with water by a ligand substitution reaction. In a recent study, we described a pair of MnIII-hydroxo and MnIII-alkylperoxo complexes supported by the same amide-containing pentadentate ligand (6Medpaq). In this present work, we describe the reaction of the MnIII-hydroxo unit in C-H and O-H bond oxidation processes, thus mimicking one of the elementary reactions of the MnLOX enzyme. An analysis of kinetic data shows that the MnIII-hydroxo complex [MnIII(OH)(6Medpaq)]+ oxidizes TEMPOH (2,2′-6,6′-tetramethylpiperidine-1-ol) faster than the majority of previously reported MnIII-hydroxo complexes. Using a combination of cyclic voltammetry and electronic structure computations, we demonstrate that the weak MnIII-N(pyridine) bonds lead to a higher MnIII/II reduction potential, increasing the driving force for substrate oxidation reactions and accounting for the faster reaction rate. In addition, we demonstrate that the MnIII-alkylperoxo complex [MnIII(OOtBu)(6Medpaq)]+ reacts with water to obtain the corresponding MnIII-hydroxo species, thus mimicking the ligand substitution step proposed for MnLOX

Near-UV and Visible Light Degradation of Iron (III)-Containing Citrate Buffer: Formation of Carbon Dioxide Radical Anion via Fragmentation of a Sterically Hindered Alkoxyl Radical

Citrate is a commonly used buffer in pharmaceutical formulations which forms complexes with adventitious metals such as Fe3+. Fe3+-citrate complexes can act as potent photosensitizers under near-UV and visible light exposure, and recent studies reported evidence for the photo-production of a powerful reductant, carbon dioxide radical anion (•CO2–), from Fe3+-citrate complexes (Subelzu, N.; Schöneich, N., Mol. Pharm.2020, 17, 4163−4179). The mechanisms of •CO2– formation are currently unknown but must be established to devise strategies against •CO2– formation in pharmaceutical formulations which rely on the use of citrate buffer. In this study, we first established complementary evidence for the photolytic generation of •CO2– from Fe3+-citrate through spin trapping and electron paramagnetic resonance (EPR) spectroscopy, and subsequently used spin trapping in conjunction with tandem mass spectrometry (MS/MS) for mechanistic studies on the pathways of •CO2– formation. Experiments with stable isotope-labeled citrate suggest that the central carboxylate group of citrate is the major source of •CO2–. Competition studies with various inhibitors (alcohols and dimethyl sulfoxide) reveal two mechanisms of •CO2– formation, where one pathway involves β-cleavage of a sterically hindered alkoxyl radical generated from the hydroxyl group of citrate