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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.

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.

Neurodegeneration with brain iron accumulation (NBIA) is a group of inherited neurologic disorders in which iron accumulates in the basal ganglia resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric abnormalities, and optic atrophy or retinal degeneration. The most prevalent form of NBIA is pantothenate kinase-associated neurodegeneration (PKAN) associated with mutations in the gene of pantothenate kinase 2 (PANK2), which is essential for coenzyme A (CoA) synthesis. There is no cure for NBIA nor is there a standard course of treatment. In the current work, we describe that fibroblasts derived from patients harbouring PANK2 mutations can reproduce many of the cellular pathological alterations found in the disease, such as intracellular iron and lipofuscin accumulation, increased oxidative stress, and mitochondrial dysfunction. Furthermore, mutant fibroblasts showed a characteristic senescent morphology. Treatment with pantothenate, the PANK2 enzyme substrate, was able to correct all pathological alterations in responder mutant fibroblasts with residual PANK2 enzyme expression. However, pantothenate had no effect on mutant fibroblasts with truncated/incomplete protein expression. The positive effect of pantothenate in particular mutations was also confirmed in induced neurons obtained by direct reprograming of mutant fibroblasts. Our results suggest that pantothenate treatment can stabilize the expression levels of PANK2 in selected mutations. These results encourage us to propose our screening model as a quick and easy way to detect pantothenate-responder patients with PANK2 mutations. The existence of residual enzyme expression in some affected individuals raises the possibility of treatment using high dose of pantothenate. Instituto de Salud Carlos III FIS PI16/00786 Junta de Andalucía CTS-5725, BIO-122 Dirección General de Investigación Científica y Técnica BFU2015-64536-R

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methylL-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methylL-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects. España, MINECO BFU2015-71017-P/BMC and BFU2015- 19451/BMC Unión Europea, Bio-NMR-00130 and CALIPSO-312284 España, Ministerio de Educación AP2009-4092

The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed, and the 19-kDa C-terminal fragment (MSP119) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth, MSP119 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based on BiGGER is employed on MSP119-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP119 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria treatment and provide valuable information for drug design.

1 página., The role of cytochrome c in apoptosis is well-established, but its participation in signaling pathways in vivo remains still poorly understood due to its essential role in mitochondrial respiration.

El presente trabajo forma parte del realizado por el grupo al que el doctorando se encuentra adscrito, en el que se busca una mejor compresión de la relación estructura-función en proteínas redox. En este sentido, se están realizando análisis comparados del citocromo c6 y la plastocianina como donadores de electrones del fotosistema I (Hervás et al., 1992a; 1992c; 1994; Medina et al., 1993; Díaz et al., 1994), así como de la ferredoxina y la flavodoxina como aceptores de electrones de dicho fotosistema (Hervás et al., 1992b; 1992c; Medina at al., 1992). El estudio comparado de dichas proteínas tiene interés desde el punto de vista evolutivo, no sólo por la selección de la plastocianina y la ferredocina como donador y aceptor de electrones -respectivamente- del PSI en plantas superiores, en lugar de sus respectivos análogos, sino por el esclarecimiento de una posible evolución en los mecanismos de reacción de dichas proteínas (Hervás et al., 1994). Por otro lado, se pretende determinar qué residuos intervienen en la interacción de las metaloproteínas con el PSI mediante el estudio de proteínas modificadas por mutagénesis dirigida, para lo que se ha clonado el gen petE de Synechocystis sp. PCC 6803, que codifica la plastociannina (Hervás et al., 1993). En el presente trabajo se muestra un estudio sobre la naturaleza de la interacción del PSI de Monoraphidium con sus donadores de electrones y se compara con resultados obtenidos con PSI y metaloproteínas obtenidas de Anabaena, donde las fuerzas electrostáticas de largo alcance son atractivas, en lugar de repulsivas. Además, se ha clonado en E. coli el gen petJ de Synechocystis sp. PCC 6803, que codifica el citocromo c6, y se ha demostrado que la proteína se sintetiza y madura correctamente en la enterobacteria. Estos resultados abren la posibilidad a posteriores estudios sobre los aminoácidos específicos que intervienen en la interacción con el PSI y en la transferencia electrónica, empleando proteínas modificadas en determinados residuos, mediante mutagénesis dirigida.

Trabajo presentado por la Licenciada Dª. Estrella Frutos Beltrán para optar al título de Doctor en Bioquímica por la Universidad de Sevilla

32 pags., 17 figs., 1 tab., The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins., We thank the staf at the NMR facility at CITIUS (University of Seville), the microscopy facility at CABIMER (Seville) and the ALBA Synchrotron (Barcelona). We are grateful to the Spanish Government (grant nos. PID2021-126663NB-I00, PGC2018-096049-B-I00/FEDER, BIO2015-70092-R, BFU2015-71017/BMC, BFU2016-75984/BMC, PID2019-105872GB and BFU2017-90030-P, FEDER/Ministerio de Ciencia e Innovación–Agencia Estatal de Investigación), European Regional Development Fund (FEDER), the Regional Government of Andalusia (grant nos. BIO-198; US-1254317, US-1257019, P18-FR-3487 and P18-HO-4091, US/JUNTA/FEDER, UE), the European Commission: European Regional Development Fund and European Research Council (CONCERT, contract number 648201) and the Ramón Areces Foundation. This work has been supported by Infrastructure for NMR, EM and X-rays for Translational Research (iNEXT, grant no. PID 3407) funded by the Horizon 2020 program of the European Commission, Cámara Foundation (C.A.E.R.´s fellowship), the Spanish Ministry of Education, Culture and Sports (grant nos. FPU18/06577, FPU16/01513 and FPU013/04373; M.A.C.-C., A.V.-C. and F.R.-R.’s fellowships, respectively), La Caixa Foundation (E.S.’s fellowship), Severo Ochoa Award of Excellence from MINECO (Government of Spain, IRB Barcelona and CNB Madrid) and the Spanish Association Against Cancer Scientific Foundation (FC AECC, M.P.G.’s postdoctoral grant)

Post-translational modifications often modulate protein function. Actually, phosphorylation of cytochrome c occurs in vivo at threonine 28, serine 47 and tyrosines 48 and 97. Phosphorylation of the two latters, in particular, is related to a wide range of human diseases as cytochrome c plays a pleiotropic role serving as an electron carrier in the respiratory electron transfer and acting as a cell death signal at the onset of apoptosis. The effect that phosphorylation of threonine 28 and serine 47 bears on the physiological functions of this protein remains concealed. The low yield of phosphorylated cytochrome c purification from cell extracts makes its analysis challenging. Also the specific kinases acting on the protein remain unknown. Hence, it has resorted to mutations to mimic targeted phosphorylation. Here, we have replaced threonine 28 and serine 47 by aspartate. And the analysis of tyrosine 48 and 97 phosphorylation has been performed by using the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF), which is a close phosphorylation mimic of tyrosine. Noteworthy, the Y48pCMF mutation significantly lowers the value for the alkaline transition pKa of oxidized cytochrome c. The negative charges at positions 28 and 48 cause a decrease in the midpoint redox potential value of 30 mV and 60 mV, respectively, and lower the affinity towards the distal site of cytochrome c1 in complex III. However, the phosphomimic variants at position 28, 47 and 48 are more efficient as electron donors to cytochrome c oxidase than the wild-type species. Concerning the role of cytochrome c in programmed cell death, negative charges at positions 48 and 97 hinder its ability to triger caspase-3 activation. In addition, any modification of residue 47 affects the pro-apoptotic function of cytochrome c. In summary, phosphorylation of cytochrome c modulates its distinct functions depending on the targeted residue, and can thus be the basis to understand an ample set of molecular diseases. Premio Extraordinario de Doctorado US

La vida se encuentra en casi cualquier lugar de la Tierra, tanto en las grietas hidrotermales de los suelos marinos o en las aguas en ebullición de los géiseres, como en los picos de la cordillera del Himalaya o en las frías extensiones de la Antártida. Los seres vivos se pueden clasificar en función del ambiente en el que habitan y de su adaptación al mismo. Así encontramos organismos psicrófilos o termófilos, adaptados a bajas o altas temperaturas respectivamente; halófilos, en ambientes con una alta concentración salina; acidófilos o alcalífilos propios de ambientes de bajos o altos pH respectivamente; los barófilos en condiciones de alta presión, etcétera. En general, estos organismos se denominan extremófilos y han llamado la atención de numerosos científicos desde los primeros estudios llevados a cabo por Perutz y colaboradores en los años 70 (Perutz y Raidt, 1975; Perutz, 1978). Según la temperatura óptima de crecimiento (TOC), los organismos se clasifican en cuatro grupos: psicrófilos, con una TOC en el rango de -5 a 15 ºC, mesófilos con una TOC entre 15 y 45 ºC, termófilos con una TOC entre 45 y 80 ºC, y los hipertermófilos (o termófilos extremos) con una TOC superior a 80 ºC (Vieille y Zeikis, 2001; Li et al., 2005). Las proteínas originarias de organismos mesófilos se denominan proteínas mesofílicas, y termofílicas las de los organismos termófilos o hipertermófilos (Pantazaki et al., 2008). Desde el punto de vista de la biología celular, los componentes de la membrana y pequeñas moléculas protectoras juegan un papel importante en el caso de las adaptaciones a salinidad, pH o presión extremas, (Jaenicke, 1991; Yancey et al., 1982; van de Vossenberg et al., 1998). En las adaptaciones a temperaturas extremas, los componentes celulares como por ejemplo, las proteínas, deben ser termoestables (Jaenicke y Zavodszky, 1990).

Las interacciones trasitorias entre biomoléculas se han convertido en el centro de la investigación de muchas áreas de la Bioquímica Moderna. La caracterización estructural continuada, utilizando cualquiera de las técnicas estructurales de alta resolución, es vital para comprender la función de este tipo de estos complejos. El trabajo de esta Tesis Doctoral se ha planteado con un doble objetivo. Por un lado, profundizar en el conocimiento acerca de las interacciones moleculares y la caracterización estructural de los complejos transitorios entre proteínas, teniendo como objetivo final la compresión de los factores que controlan la relación entre la estructura y la función de las proteínas, así como el modo de interacción y formación de este tipo de complejos inestables o débiles. Se ha hecho especial énfasis en aquellos rasgos que confieren a una proteína su capacidad para interaccionar especifica y transitoria con otras. El segundo objetivo supone un avance en el desarrollo y conocimiento de dos espectroscopias actualmente imprescindibles, para los estudios de interacción entre biomoléculas en disolución: la Resonancia Magnética Nuclear RMN y la absorción de Rayos X XAS. Este desarrollo tiene a su vez una doble vertiente: a nivel experimental, optimizando la calidad de los espectros de RMN y XAS en concentraciones límite.a nivel metodológico, aplicando, de manera pionera, la XAS al estudio de complejos entre biomoléculas y abordando, por primera vez, la caracterización estructural entre proteínas solubles y de membrana por NMR en disolución. Premio Extraordinario de Doctorado US