Cytochrome c lysine acetylation regulates cellular respiration and cell death in ischemic skeletal muscle.

Skeletal muscle is more resilient to ischemia-reperfusion injury than other organs. Tissue specific post-translational modifications of cytochrome c (Cytc) are involved in ischemia-reperfusion injury by regulating mitochondrial respiration and apoptosis. Here, we describe an acetylation site of Cytc, lysine 39 (K39), which was mapped in ischemic porcine skeletal muscle and removed by sirtuin5 in vitro. Using purified protein and cellular double knockout models, we show that K39 acetylation and acetylmimetic K39Q replacement increases cytochrome c oxidase (COX) activity and ROS scavenging while inhibiting apoptosis via decreased binding to Apaf-1, caspase cleavage and activity, and cardiolipin peroxidase activity. These results are discussed with X-ray crystallography structures of K39 acetylated (1.50 Å) and acetylmimetic K39Q Cytc (1.36 Å) and NMR dynamics. We propose that K39 acetylation is an adaptive response that controls electron transport chain flux, allowing skeletal muscle to meet heightened energy demand while simultaneously providing the tissue with robust resilience to ischemia-reperfusion injury.

Electric field-induced functional changes in electrode-immobilized mutant species of human cytochrome c.

Post-translational modifications and naturally occurring mutations of cytochrome c have been recognized as a regulatory mechanism to control its biology. In this work, we investigate the effect of such in vivo chemical modifications of human cytochrome c on its redox properties in the adsorbed state onto an electrode. In particular, tyrosines 48 and 97 have been replaced by the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF), thus mimicking tyrosine phosphorylation. Additionally, tyrosine 48 has been replaced by a histidine producing the natural Y48H pathogenic mutant. Thermodynamics and kinetics of the interfacial electron transfer of wild-type cytochrome c and herein produced variants, adsorbed electrostatically under different local interfacial electric fields, were determined by means of variable temperature cyclic film voltammetry. It is shown that non-native cytochrome c variants immobilized under a low interfacial electric field display redox thermodynamics and kinetics similar to those of wild-type cytochrome c. However, upon increasing the strength of the electric field, the redox thermodynamics and kinetics of the modified proteins markedly differ from those of the wild-type species. The mutations promote stabilization of the oxidized form and a significant increase in the activation enthalpy values that can be ascribed to a subtle distortion of the heme cofactor and/or difference of the amino acid rearrangements rather than to a coarse protein structural change. Overall, these results point to a combined effect of the single point mutations at positions 48 and 97 and the strength of electrostatic binding on the regulatory mechanism of mitochondrial membrane activity, when acting as a redox shuttle protein.

Surprise-induced enhancements in the associability of Pavlovian cues facilitate learning across behavior systems.

Surprising violations of outcome expectancies have long been known to enhance the associability of Pavlovian cues; that is, the rate at which the cue enters into further associations. The adaptive value of such enhancements resides in promoting new learning in the face of uncertainty. However, it is unclear whether associability enhancements reflect increased associative plasticity within a particular behavior system, or whether they can facilitate learning between a cue and any arbitrary outcome, as suggested by attentional models of conditioning. Here, we show evidence consistent with the latter hypothesis. Violating the outcome expectancies generated by a cue in an appetitive setting (feeding behavior system) facilitated subsequent learning about the cue in an aversive setting (defense behavior system). In addition to shedding light on the nature of associability enhancements, our findings offer the neuroscientist a behavioral tool to dissociate their neural substrates from those of other, behavior system- or valence-specific changes. Moreover, our results present an opportunity to utilize associability enhancements to the advantage of counterconditioning procedures in therapeutic contexts. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Agency rescues competition for credit assignment among predictive cues from adverse learning conditions.

A fundamental assumption of learning theories is that the credit assigned to predictive cues is not simply determined by their probability of reinforcement, but by their ability to compete with other cues present during learning. This assumption has guided behavioral and neural science research for decades, and tremendous empirical and theoretical advances have been made identifying the mechanisms of cue competition. However, when learning conditions are not optimal (e.g., when training is massed), cue competition is attenuated. This failure of the learning system exposes the individual's vulnerability to form spurious associations in the real world. Here, we uncover that cue competition in rats can be rescued when conditions are suboptimal provided that the individual has agency over the learning experience. Our findings reveal a new effect of agency over learning on credit assignment among predictive cues, and open new avenues of investigation into the underlying mechanisms.

Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants.

Post-translational modifications frequently modulate protein functions. Lysine acetylation in particular plays a key role in interactions between respiratory cytochrome c and its metabolic partners. To date, in vivo acetylation of lysines at positions 8 and 53 has specifically been identified in mammalian cytochrome c, but little is known about the structural basis of acetylation-induced functional changes. Here, we independently replaced these two residues in recombinant human cytochrome c with glutamine to mimic lysine acetylation and then characterized the structure and function of the resulting K8Q and K53Q mutants. We found that the physicochemical features were mostly unchanged in the two acetyl-mimetic mutants, but their thermal stability was significantly altered. NMR chemical shift perturbations of the backbone amide resonances revealed local structural changes, and the thermodynamics and kinetics of electron transfer in mutants immobilized on gold electrodes showed an increase in both protein dynamics and solvent involvement in the redox process. We also observed that the K8Q (but not the K53Q) mutation slightly increased the binding affinity of cytochrome c to its physiological electron donor, cytochrome c1 -which is a component of mitochondrial complex III, or cytochrome bc1 -thus suggesting that Lys8 (but not Lys53) is located in the interaction area. Finally, the K8Q and K53Q mutants exhibited reduced efficiency as electron donors to complex IV, or cytochrome c oxidase.

Autotolerant ceruloplasmin based biocathodes for implanted biological power sources.

High-performance autotolerant bioelectrodes should be ideally suited to design implantable bioelectronic devices. Because of its high redox potential and ability to reduce oxygen directly to water, human ceruloplasmin, HCp, the only blue multicopper oxidase present in human plasma, appears to be the ultimate biocatalyst for oxygen biosensors and also biocathodes in biological power sources. In comparison to fungal and plant blue multicopper oxidases, e.g. Myrothecium verrucaria bilirubin oxidase and Rhus vernicifera laccase, respectively, the inflammatory response to HCp in human blood is significantly reduced. Partial purification of HCp allowed to preserve the native conformation of the enzyme and its biocatalytic activity. Therefore, electrochemical studies were carried out with the partially purified enzyme immobilised on nanostructured graphite electrodes at physiological pH and temperature. Amperometric investigations revealed low reductive current densities, i.e. about 1.65 µA cm-2 in oxygenated electrolyte and in the absence of any mediator, demonstrating nevertheless direct electron transfer based O2 bioelectroreduction by HCp for the first time. The reductive current density obtained in the mediated system was about 12 µA cm-2. Even though the inflammatory response of HCp is diminished in human blood, inadequate bioelectrocatalytic performance hinders its use as a cathodic bioelement in a biofuel cell.

Active Role of the Buffer in the Proton-Coupled Electron Transfer of Immobilized Iron Porphyrins.

Evaluation of the proton-coupled electron transfer thermodynamics of immobilized hemin is challenging due to the disparity of its electrochemical titration curves reported in the literature. Deviations from the one-electron, one-proton transfer at circumneutral pHs have been commonly ascribed to either the formation of dimeric species or the ionization of a second iron-bound water molecule. Herein, however, we report on non-idealities in the more acidic region, whose onset and extent vary with the nature and concentration of the commonly used phosphate and acetate buffers. It is shown that these deviations originate in the ligand-exchange binding between the oxidized aquo-hemin complex and the anionic components of the buffer, so that they are restricted to the pH interval where these forms coexist. A stepwise approach was developed to quantify unambiguously the apparent and intrinsic binding equilibrium constants. The apparent binding equilibrium constant exhibits a peak-shaped pH dependence, whose maximum is located at approximately the midpoint between the pKa of the iron-bound water and the first pKa of the buffer, and its magnitude is greater for the phosphate than for the acetate buffer. But strikingly, the opposite trend was found for the magnitude of the intrinsic binding equilibrium constants determined from the apparent ones, due to the different relative locations of the phosphoric and acetic pKa values with respect to that of the oxidized aquo-hemin. To probe the role of the heme propionic residues, a similar study was carried out with a propionic-free iron porphyrin containing eight ethyl residues. These substituents decrease the acidity of the iron-bound water, strengthen the iron(III)-acetate binding, weaken the iron(III)-dihydrogen phosphate binding, and enable the binding between iron(III) and monohydrogen phosphate, which was hampered in hemin by the presence of the negatively charged propionate residues. Overall, this work provides a more complete speciation of immobilized iron porphyrins under acidic conditions than previously considered, showing the substitutional lability of the aqua ligand in the oxidized state of the iron center and the reluctance of its hydroxyl counterpart to anion exchange. Knowledge of these redox- and pH-dependent bindings with the buffer components is crucial for a rigorous quantification of the proton-coupled electron transfer and the electrocatalytic activity of iron porphyrins.

Post-Translational Modifications of Cytochrome c in Cell Life and Disease.

Mitochondria are the powerhouses of the cell, whilst their malfunction is related to several human pathologies, including neurodegenerative diseases, cardiovascular diseases, and various types of cancer. In mitochondrial metabolism, cytochrome c is a small soluble heme protein that acts as an essential redox carrier in the respiratory electron transport chain. However, cytochrome c is likewise an essential protein in the cytoplasm acting as an activator of programmed cell death. Such a dual role of cytochrome c in cell life and death is indeed fine-regulated by a wide variety of protein post-translational modifications. In this work, we show how these modifications can alter cytochrome c structure and functionality, thus emerging as a control mechanism of cell metabolism but also as a key element in development and prevention of pathologies.

Effects on goal directed behavior and habit in two animal models of Parkinson's disease.

Instrumental conditioning involves two different processes: Goal-directed behavior, characterized by its dependence on the causal relationship between action and outcome and the sensitivity of actions to changes in the value of the outcome; and habits, characterized for its persistence and insensitivity to changes after conditioning. It is known that the dopaminergic system is involved in both kind of learning. The present experiments analyzed two animal models of Parkinson's disease. The 6-OHDA model causes selective damage of the catecholaminergic neurons, specifically affecting the dopaminergic neurons in nigro-striatal system. This model simulates degenerative process symptomatology of Parkinson's disease. On the other hand, the LPS model generates an inflammation process in the infusion area. This model simulates the early symptoms of this disorder, including neuroinflammation and microglia activation. In order to validate both parkinsonian models, we studied if 6-OHDA and LPS models cause the same behavioral effects. The results showed that the 6-OHDA model interfered with the process involved in habit formation. In contrast, animals treated with LPS showed a goal-directed learning deficit. Differences between these models could be due to the different effects on Substantia Nigra neurons. 6-OHDA model might disrupt the nigrostriatal pathway, while LPS could interfere on efferences and afferences to Substantia Nigra.

Protein crosslinking improves the thermal resistance of plastocyanin immobilized on a modified gold electrode.

Increasing the thermal stability of immobilized proteins is a motivating goal for improving the performance of electrochemical biodevices. In this work, we propose the immobilization of crosslinked plastocyanin from the thermophilic cyanobacterium Phormidium laminosum by simultaneous incubation of a mixture of plastocyanin and the coupling reagents. The thermal stability of the so built covalently immobilized protein films has been assessed by cyclic voltammetry in the 0-90 °C temperature range and has been compared to that of physisorbed films. It is shown that the protein loss along a thermal cycle is significantly reduced in the case of the crosslinked films, whose redox properties remain unaltered along a cyclic heating-cooling thermal scan, and can withstand the contact with 70 °C solutions for four hours. Comparison of thermal unfolding curves obtained by circular dichroism spectroscopy of both free and crosslinked protein confirms the improved thermic resistance of the crosslinked plastocyanin. Notably, the electron transfer thermodynamics of physisorbed and crosslinked plastocyanin films are quite similar, suggesting that the formation of intra- and inter-protein amide bonds do not affect the integrity and functionality of the copper redox centers. UV-Vis absorption and circular dichroism measurements corroborate that protein crosslinking does not alter the coordination geometry of the metal center.

Key Role of the Local Hydrophobicity in the East Patch of Plastocyanins on Their Thermal Stability and Redox Properties.

Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein.