Neddylation of phosphoenolpyruvate carboxykinase 1 controls glucose metabolism.

Neddylation is a post-translational mechanism that adds a ubiquitin-like protein, namely neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Here, we show that neddylation in mouse liver is modulated by nutrient availability. Inhibition of neddylation in mouse liver reduces gluconeogenic capacity and the hyperglycemic actions of counter-regulatory hormones. Furthermore, people with type 2 diabetes display elevated hepatic neddylation levels. Mechanistically, fasting or caloric restriction of mice leads to neddylation of phosphoenolpyruvate carboxykinase 1 (PCK1) at three lysine residues-K278, K342, and K387. We find that mutating the three PCK1 lysines that are neddylated reduces their gluconeogenic activity rate. Molecular dynamics simulations show that neddylation of PCK1 could re-position two loops surrounding the catalytic center into an open configuration, rendering the catalytic center more accessible. Our study reveals that neddylation of PCK1 provides a finely tuned mechanism of controlling glucose metabolism by linking whole nutrient availability to metabolic homeostasis.

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.

GWAS of genetic factors affecting white blood cell morphological parameters in Sardinians uncovers influence of chromosome 11 innate immunity gene cluster on eosinophil morphology.

Few genome-wide association studies (GWAS) analyzing genetic regulation of morphological traits of white blood cells have been reported. We carried out a GWAS of 12 morphological traits in 869 individuals from the general population of Sardinia, Italy. These traits, included measures of cell volume, conductivity and light scatter in four white-cell populations (eosinophils, lymphocytes, monocytes, neutrophils). This analysis yielded seven statistically significant signals, four of which were novel (four novel, PRG2, P2RX3, two of CDK6). Five signals were replicated in the independent INTERVAL cohort of 11 822 individuals. The most interesting signal with large effect size on eosinophil scatter (P-value = 8.33 x 10-32, beta = -1.651, se = 0.1351) falls within the innate immunity cluster on chromosome 11, and is located in the PRG2 gene. Computational analyses revealed that a rare, Sardinian-specific PRG2:p.Ser148Pro mutation modifies PRG2 amino acid contacts and protein dynamics in a manner that could possibly explain the changes observed in eosinophil morphology. Our discoveries shed light on genetics of morphological traits. For the first time, we describe such large effect size on eosinophils morphology that is relatively frequent in Sardinian population.

Phosphorylation disrupts long-distance electron transport in cytochrome c.

It has been recently shown that electron transfer between mitochondrial cytochrome c and the cytochrome c1 subunit of the cytochrome bc1 can proceed at long-distance through the aqueous solution. Cytochrome c is thought to adjust its activity by changing the affinity for its partners via Tyr48 phosphorylation, but it is unknown how it impacts the nanoscopic environment, interaction forces, and long-range electron transfer. Here, we constrain the orientation and separation between cytochrome c1 and cytochrome c or the phosphomimetic Y48pCMF cytochrome c, and deploy an array of single-molecule, bulk, and computational methods to investigate the molecular mechanism of electron transfer regulation by cytochrome c phosphorylation. We demonstrate that phosphorylation impairs long-range electron transfer, shortens the long-distance charge conduit between the partners, strengthens their interaction, and departs it from equilibrium. These results unveil a nanoscopic view of the interaction between redox protein partners in electron transport chains and its mechanisms of regulation.

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.

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.

Mitochondrial cytochrome c shot towards histone chaperone condensates in the nucleus.

Despite mitochondria being key for the control of cell homeostasis and fate, their role in DNA damage response is usually just regarded as an apoptotic trigger. However, growing evidence points to mitochondrial factors modulating nuclear functions. Remarkably, after DNA damage, cytochrome c (Cc) interacts in the cell nucleus with a variety of well-known histone chaperones, whose activity is competitively inhibited by the haem protein. As nuclear Cc inhibits the nucleosome assembly/disassembly activity of histone chaperones, it might indeed affect chromatin dynamics and histone deposition on DNA. Several histone chaperones actually interact with Cc Lys residues through their acidic regions, which are also involved in heterotypic interactions leading to liquid-liquid phase transitions responsible for the assembly of nuclear condensates, including heterochromatin. This relies on dynamic histone-DNA interactions that can be modulated by acetylation of specific histone Lys residues. Thus, Cc may have a major regulatory role in DNA repair by fine-tuning nucleosome assembly activity and likely nuclear condensate formation.

Proposed mechanism for regulation of H2 O2 -induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins.

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant H2 O2 -induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon H2 O2 treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death.

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.

Exploring protein phosphorylation by combining computational approaches and biochemical methods.

Post-translational modifications of proteins expand their functional diversity, regulating the response of cells to a variety of stimuli. Among these modifications, phosphorylation is the most ubiquitous and plays a prominent role in cell signaling. The addition of a phosphate often affects the function of a protein by altering its structure and dynamics. However, these alterations are often difficult to study and the functional and structural implications remain unresolved. New approaches are emerging to overcome common obstacles related to the production and manipulation of these samples. Here, we summarize the available methods for phosphoprotein purification and phosphomimetic engineering, highlighting the advantages and disadvantages of each. We propose a general workflow for protein phosphorylation analysis combining computational and biochemical approaches, building on recent advances that enable user-friendly and easy-to-access Molecular Dynamics simulations. We hope this innovative workflow will inform the best experimental approach to explore such post-translational modifications. We have applied this workflow to two different human protein models: the hemeprotein cytochrome c and the RNA binding protein HuR. Our results illustrate the usefulness of Molecular Dynamics as a decision-making tool to design the most appropriate phosphomimetic variant.

Physical contact between cytochrome c1 and cytochrome c increases the driving force for electron transfer.

In oxidative phosphorylation, the transfer of electrons from reduced cofactors to molecular oxygen via the electron transport chain (ETC) sustains the electrochemical transmembrane potential needed for ATP synthesis. A key component of the ETC is complex III (CIII, cytochrome bc1), which transfers electrons from reduced ubiquinone to soluble cytochrome c (Cc) coupled to proton translocation into the mitochondrial intermembrane space. One electron from every two donated by hydroquinone at site P is transferred to Cc via the Rieske-cytochrome c1 (Cc1) pathway. According to recent structural analyses of CIII and its transitory complex with Cc, the interaction between the Rieske subunit and Cc1 switches intermittently during CIII activity. However, the electrochemical properties of Cc1 and their function as a wire between Rieske and Cc are rather unexplored. Here, temperature variable cyclic voltammetry provides novel data on the thermodynamics and kinetics of interfacial electron transfer of immobilized Cc1. Findings reveal that Cc1 displays two channels for electron exchange, with a remarkably fast heterogeneous electron transfer rate. Furthermore, the electrochemical properties are strongly modulated by the binding mode of the protein. Additionally, we show that electron transfer from Cc1 to Cc is thermodynamically favored in the immobilized Cc1-Cc complex. Nuclear Magnetic Resonance, HADDOCK, and Surface Plasmon Resonance experiments provide further structural and functional data of the Cc1-Cc complex. Our data supports the Rieske-Cc1-Cc pathway acting as a unilateral switch thyristor in which redox potential modulation through protein-protein contacts are complemented with the relay-like Rieske behavior.

Wheel and Deal in the Mitochondrial Inner Membranes: The Tale of Cytochrome c and Cardiolipin.

Cardiolipin oxidation and degradation by different factors under severe cell stress serve as a trigger for genetically encoded cell death programs. In this context, the interplay between cardiolipin and another mitochondrial factor-cytochrome c-is a key process in the early stages of apoptosis, and it is a matter of intense research. Cytochrome c interacts with lipid membranes by electrostatic interactions, hydrogen bonds, and hydrophobic effects. Experimental conditions (including pH, lipid composition, and post-translational modifications) determine which specific amino acid residues are involved in the interaction and influence the heme iron coordination state. In fact, up to four binding sites (A, C, N, and L), driven by different interactions, have been reported. Nevertheless, key aspects of the mechanism for cardiolipin oxidation by the hemeprotein are well established. First, cytochrome c acts as a pseudoperoxidase, a process orchestrated by tyrosine residues which are crucial for peroxygenase activity and sensitivity towards oxidation caused by protein self-degradation. Second, flexibility of two weakest folding units of the hemeprotein correlates with its peroxidase activity and the stability of the iron coordination sphere. Third, the diversity of the mode of interaction parallels a broad diversity in the specific reaction pathway. Thus, current knowledge has already enabled the design of novel drugs designed to successfully inhibit cardiolipin oxidation.

New moonlighting functions of mitochondrial cytochrome c in the cytoplasm and nucleus.

Cytochrome c (Cc) is a protein that functions as an electron carrier in the mitochondrial respiratory chain. However, Cc has moonlighting roles outside mitochondria driving the transition of apoptotic cells from life to death. When living cells are damaged, Cc escapes its natural mitochondrial environment and, once in the cytosol, it binds other proteins to form a complex named the apoptosome-a platform that triggers caspase activation and further leads to controlled cell dismantlement. Early released Cc also binds to inositol 1,4,5-triphosphate receptors on the ER membrane, which stimulates further massive Cc release from mitochondria. Besides the well-characterized binding proteins contributing to the proapoptotic functions of Cc, many novel protein targets have been recently described. Among them, histone chaperones were identified as key partners of Cc following DNA breaks, indicating that Cc might modulate chromatin dynamics through competitive binding to histone chaperones. In this article, we review the ample set of recently discovered antiapoptotic proteins-involved in DNA damage, transcription, and energetic metabolism-reported to interact with Cc in the cytoplasm and even the nucleus upon DNA breaks.

Cytochrome c: Surfing Off of the Mitochondrial Membrane on the Tops of Complexes III and IV.

The proper arrangement of protein components within the respiratory electron transport chain is nowadays a matter of intense debate, since altering it leads to cell aging and other related pathologies. Here, we discuss three current views-the so-called solid, fluid and plasticity models-which describe the organization of the main membrane-embedded mitochondrial protein complexes and the key elements that regulate and/or facilitate supercomplex assembly. The soluble electron carrier cytochrome c has recently emerged as an essential factor in the assembly and function of respiratory supercomplexes. In fact, a 'restricted diffusion pathway' mechanism for electron transfer between complexes III and IV has been proposed based on the secondary, distal binding sites for cytochrome c at its two membrane partners recently discovered. This channeling pathway facilitates the surfing of cytochrome c on both respiratory complexes, thereby tuning the efficiency of oxidative phosphorylation and diminishing the production of reactive oxygen species. The well-documented post-translational modifications of cytochrome c could further contribute to the rapid adjustment of electron flow in response to changing cellular conditions.

Oxidative stress is tightly regulated by cytochrome c phosphorylation and respirasome factors in mitochondria.

Respiratory cytochrome c has been found to be phosphorylated at tyrosine 97 in the postischemic brain upon neuroprotective insulin treatment, but how such posttranslational modification affects mitochondrial metabolism is unclear. Here, we report the structural features and functional behavior of a phosphomimetic cytochrome c mutant, which was generated by site-specific incorporation at position 97 of p-carboxymethyl-l-phenylalanine using the evolved tRNA synthetase method. We found that the point mutation does not alter the overall folding and heme environment of cytochrome c, but significantly affects the entire oxidative phosphorylation process. In fact, the electron donation rate of the mutant heme protein to cytochrome c oxidase, or complex IV, within respiratory supercomplexes was higher than that of the wild-type species, in agreement with the observed decrease in reactive oxygen species production. Direct contact of cytochrome c with the respiratory supercomplex factor HIGD1A (hypoxia-inducible domain family member 1A) is reported here, with the mutant heme protein exhibiting a lower affinity than the wild-type species. Interestingly, phosphomimetic cytochrome c also exhibited a lower caspase-3 activation activity. Altogether, these findings yield a better understanding of the molecular basis for mitochondrial metabolism in acute diseases, such as brain ischemia, and thus could allow the use of phosphomimetic cytochrome c as a neuroprotector with therapeutic applications.