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.

Mitochondrial Factors in the Cell Nucleus.

The origin of eukaryotic organisms involved the integration of mitochondria into the ancestor cell, with a massive gene transfer from the original proteobacterium to the host nucleus. Thus, mitochondrial performance relies on a mosaic of nuclear gene products from a variety of genomes. The concerted regulation of their synthesis is necessary for metabolic housekeeping and stress response. This governance involves crosstalk between mitochondrial, cytoplasmic, and nuclear factors. While anterograde and retrograde regulation preserve mitochondrial homeostasis, the mitochondria can modulate a wide set of nuclear genes in response to an extensive variety of conditions, whose response mechanisms often merge. In this review, we summarise how mitochondrial metabolites and proteins-encoded either in the nucleus or in the organelle-target the cell nucleus and exert different actions modulating gene expression and the chromatin state, or even causing DNA fragmentation in response to common stress conditions, such as hypoxia, oxidative stress, unfolded protein stress, and DNA damage.

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.

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.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48.

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-methyl-l-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.

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.

Histone chaperone activity of Arabidopsis thaliana NRP1 is blocked by cytochrome c.

Higher-order plants and mammals use similar mechanisms to repair and tolerate oxidative DNA damage. Most studies on the DNA repair process have focused on yeast and mammals, in which histone chaperone-mediated nucleosome disassembly/reassembly is essential for DNA to be accessible to repair machinery. However, little is known about the specific role and modulation of histone chaperones in the context of DNA damage in plants. Here, the histone chaperone NRP1, which is closely related to human SET/TAF-Iß, was found to exhibit nucleosome assembly activity in vitro and to accumulate in the chromatin of Arabidopsis thaliana after DNA breaks. In addition, this work establishes that NRP1 binds to cytochrome c, thereby preventing the former from binding to histones. Since NRP1 interacts with cytochrome c at its earmuff domain, that is, its histone-binding domain, cytochrome c thus competes with core histones and hampers the activity of NRP1 as a histone chaperone. Altogether, the results obtained indicate that the underlying molecular mechanisms in nucleosome disassembly/reassembly are highly conserved throughout evolution, as inferred from the similar inhibition of plant NRP1 and human SET/TAF-Iß by cytochrome c during DNA damage response.

Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4.

The four member family of "Cyclin and Cystathionine ß-synthase (CBS) domain divalent metal cation transport mediators", CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iß by cytochrome c.

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iß interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iß to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iß. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iß, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iß's histone chaperone activity.

Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47.

Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c - namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death - is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.

Structural and functional analysis of novel human cytochrome C targets in apoptosis.

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.

Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome.

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique.

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe-Met bond in the ferric form of cytochrome c, thereby lowering the pKa value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.

New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants.

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.

Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles.

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

Antimalarial activity of cupredoxins: the interaction of Plasmodium merozoite surface protein 119 (MSP119) and rusticyanin.

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.

Targeted delivery of pharmacological chaperones for Gaucher disease to macrophages by a mannosylated cyclodextrin carrier.

Gaucher disease (GD) is a rare monogenetic disorder leading to dysfunction of acid ß-glucosidase (ß-glucocerebrosidase; GCase) and accumulation of glucosylceramide in lysosomes, especially in macrophages (Gaucher cells). Many of the mutations at the origin of GD do not impair the catalytic activity of GCase, but cause misfolding and subsequent degradation by the quality control system at the endoplasmic reticulum. Pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the endogenous mutant enzyme represent promising alternatives to the currently available enzyme replacement and substrate reduction therapies (ERT and SRT, respectively), but unfavorable biodistribution and potential side-effects remain important issues. We have now designed a strategy to enhance the controlled delivery of PCs to macrophages that exploit the formation of ternary complexes between the PC, a trivalent mannosylated ß-cyclodextrin (ßCD) conjugate and the macrophage mannose receptor (MMR). First, PC candidates with appropriate relative avidities towards the ßCD cavity and the GCase active site were selected to ensure efficient transfer of the PC cargo from the host to the GCase active site. Control experiments confirmed that the ßCD carrier was selectively recognized by mannose-specific lectins and that the corresponding PC:mannosylated ßCD supramolecular complex retained both the chaperoning activity, as confirmed in human GD fibroblasts, and the MMR binding ability. Finally, fluorescence microscopy techniques proved targeting and cellular uptake of the PC-loaded system in macrophages. Altogether, the results support that combined cyclodextrin encapsulation and glycotargeting may improve the efficacy of PCs for GD.

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets.

Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5'-mer U-rich RNA stretches through the solvent exposed side of its ß-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain.

SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer.

The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.