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thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished).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.Programmed cell death (PCD)¹ is a fundamental event for the development of multicellular organisms and the homeostasis of their tissues. It is an evolutionarily conserved mechanism present in organisms ranging from yeast to mammals (1–3).In mammals, cytochrome c (Cc) and dATP bind to apoptosis protease-activating factor-1 (Apaf-1) in the cytoplasm, a process leading to the formation of the Apaf-1/caspase-9 complex known as apoptosome. This apoptosome subsequently activates caspases-3 and -7 (4, 5). In other organisms, such as Caenorhabditis elegans or Drosophila melanogaster, however, Cc is not essential for the assembly and activation of the apoptosome (6) despite the presence of proteins homologous to Apaf-1—cell death abnormality-4 (CED-4) in C. elegans and Drosophila Apaf-1-related killer (Dark) in D. melanogaster—which have been found to be essential for caspase cascade activation. Furthermore, other organisms such as Arabidopsis thaliana lack Apaf-1 (7). In fact, only highly distant caspase homologues (metacaspases) (8, 9), serine proteases (saspases) (10), phytaspases (11) and VEIDases (12–14) with caspase-like activity have been detected in plants; however, their targets remain veiled and whether they are activated by Cc remains unclear.Intriguingly, the release of Cc from mitochondria into the cytoplasm during the onset of PCD is an evolutionarily conserved event found in organisms ranging from yeast (15) and plants (16) to flies (17), and mammals (18). However, understanding of the roles of this phenomenon in different species can be said to be uneven at best. In fact, the release of Cc from mitochondria has thus far been considered a random event in all organisms, save mammals. Thus, the participation of Cc in the onset and progression of PCD needs to be further elucidated.Even in the case of mammals, the role(s) of Cc in the cytoplasm during PCD remain(s) controversial. Recently, new putative functions of Cc, going beyond the already-established apoptosome assembly process, have been proposed in the nucleus (19, 20) and the endoplasmic reticulum (21–23). Neither these newly proposed functions nor other arising functions, such as oxidative stress (24), are as yet fully understood. This current state of affairs demands deeper exploration of the additional roles played by Cc in nonmammalian species.In this study, putative novel Cc-partners involved in plant PCD were identified. For this identification, a proteomic approach was employed based on affinity chromatography and using Cc as bait. The Cc-interacting proteins were identified using nano-liquid chromatography tandem mass spectrometry (NanoLC-MS/MS). These Cc-partners were then further confirmed in vivo through bimolecular fluorescence complementation (BiFC) in A. thaliana protoplasts and human HEK293T cells, as a heterologous system. Finally, the Cc-GLY2, Cc-NRP1 and Cc-TCL interactions were corroborated in vitro using surface plasmon resonance (SPR).These results indicate that Cc is able to interact with targets in the plant cell cytoplasm during PCD. Moreover, they provide new ways of understanding why Cc release is an evolutionarily well-conserved event, and allow us to propose Cc as a signaling messenger, which somehow controls different essential events during PCD.     

Significant Modifications of the Salivary Proteome Potentially Associated with Complications of Down Syndrome Revealed by Top-down Proteomics

People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10–17 yr and 18–50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization–MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer''s disease, which is frequently associated with Down syndrome.Down syndrome (DS)¹ is a frequent genetic disorder in humans characterized by premature aging (1). A clinical feature of people with DS is the increased prevalence and severity of periodontal disease compared with age-matched subjects of similar levels of intellectual impairment and compared with the general population (2). Common conditions observed in DS are marginal gingivitis, acute and subacute necrotizing gingivitis, advanced periodontitis, gingival recession, and pocket formation (3, 4). It is known that saliva plays an important role in maintaining oral and dental health, because of the presence of a variety of antimicrobial peptides mainly derived from gland secretion, oral epithelial cells, and neutrophils (5). Several papers reported that neutrophils and T-lymphocyte function is impaired in people with DS (6–9). However, the salivary secretion of the antimicrobial LL-37 in young individuals with DS was found normal (10). A review of the literature (11, 12) reveals only sporadic and contradictory reports that attempt to explain the role of saliva in the oral health of subjects with DS, and on the whole, information on the biochemical composition of their saliva is scarce. On the basis of the above information, in the present study, we proposed to investigate the salivary proteome of DS subjects by an intact protein-based “top-down” approach. The spectrum of salivary peptides of DS subjects was compared with that of sex and age-matched healthy control groups to determine qualitative and quantitative differences. Interestingly, the results showed that several members of the S100A family, which possess different biological functions, and also described as potential markers of the Alzheimer Disease, were significantly increased in saliva of Down syndrome subjects with respect to controls.     

Development of gene-specific markers for acid soil/aluminium tolerance in barley (Hordeum vulgare L.)

Acid soil/aluminium toxicity is one of the major constraints on barley production around the world. Genetic improvement is the best solution and molecular-marker-assisted selection has proved to be an efficient tool for developing barley cultivars with acid soil/aluminium tolerance. In this study, barley variety Svanhals—introduced from CYMMIT (International Maize and Wheat Improvement Center)—was identified as acid soil/aluminium tolerant and the tolerance was mapped to chromosome 4H in 119 doubled haploid (DH) lines from a cross of Hamelin/Svanhals. The HvMATE gene, encoding an aluminium-activated citrate transporter, was selected as a candidate gene and gene-specific molecular markers were developed to detect acid soil/aluminium tolerance based on the polymerase chain reaction. Sequence analysis of the HvMATE gene identified a 21-bp indel (insertion–deletion) between the tolerant and sensitive cultivars. The new marker was further mapped to the QTL (quantitative trait loci) region on chromosome 4H for acid soil tolerance and accounted for 66.9 % of phenotypic variation in the DH population. Furthermore, the polymorphism was confirmed in other tolerant varieties which have been widely used as a source of acid soil tolerance in Australian barley breeding programs. The new gene-specific molecular marker provides an effective and simple molecular tool for selecting the acid soil tolerance gene from multiple tolerance sources.     

Progression,Symptoms and Psychosocial Concerns among Those Severely Affected by Multiple Sclerosis: A Mixed-Methods Cross-Sectional Study of Black Caribbean and White British People

Objective

Multiple sclerosis is now more common among minority ethnic groups in the UK but little is known about their experiences, especially in advanced stages. We examine disease progression, symptoms and psychosocial concerns among Black Caribbean (BC) and White British (WB) people severely affected by MS.

Design

Mixed methods study of 43 BC and 43 WB people with MS (PwMS) with an Expanded Disability Status Scale (EDSS) ≥6 involving data from in clinical records, face-to-face structured interviews and a nested-qualitative component. Progression Index (PI) and Multiple Sclerosis Severity Score (MSSS) were calculated. To control for selection bias, propensity scores were derived for each patient and adjusted for in the comparative statistical analysis; qualitative data were analysed using the framework approach.

Results

Median EDSS for both groups was (6.5; range: 6.0–9.0). Progression Index (PI) and Multiple Sclerosis Severity Score (MSSS) based on neurological assessment of current EDSS scores identified BC PwMS were more likely to have aggressive disease (PI F = 4.04, p = 0.048, MSSS F = 10.30, p<0.001). Patients’ reports of the time required to reach levels of functional decline equivalent to different EDSS levels varied by group; EDSS 4: BC 2.7 years v/s WB 10.2 years (U = 258.50, p = 0.013), EDSS 6∶6.1 years BC v/s WB 12.7 years (U = 535.500, p = 0.011), EDSS 8: BC 8.7 years v/s WB 10.2 years. Both groups reported high symptom burden. BC PwMS were more cognitively impaired than WB PwMS (F = 9.65, p = 0.003). Thematic analysis of qualitative interviews provides correspondence with quantitative findings; more BC than WB PwMS referred to feelings of extreme frustration and unresolved loss/confusion associated with their rapidly advancing disease. The interviews also reveal the centrality, meanings and impact of common MS-related symptoms.

Conclusions

Delays in diagnosis should be avoided and more frequent reviews may be justified by healthcare services. Culturally acceptable interventions to better support people who perceive MS as an assault on identity should be developed to help them achieve normalisation and enhance self-identity.     

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.     

C-Terminus-Mediated Voltage Gating of Arabidopsis Guard Cell Anion Channel QUAC1

Anion transporters in plants play a fundamental role in volume regulation and signaling. Currently, two plasma membrane-located anion channel familiesmSLAC/SLAH and ALMTmare known. Among the ALMT family, the root-expressed ALuminium-activated Malate Transporter 1 was identified by comparison of aluminum-tolerant and Al3＋-sensitive wheat cultivars and was subsequently shown to mediate voltage-independent malate currents. In con- trast, ALMT12/QUAC1 （QUickly activating Anion Channel1） is expressed in guard cells transporting malate in an Al3＋- insensitive and highly voltage-dependent manner. So far, no information is available about the structure and mechanism of voltage-dependent gating with the QUAC1 channel protein. Here, we analyzed gating of QUACl-type currents in the plasma membrane of guard cells and QUACl-expressing oocytes revealing similar voltage dependencies and activation- deactivation kinetics. In the heterologous expression system, QUAC1 was electrophysiologically characterized at increas- ing extra- and intracellular malate concentrations. Thereby, malate additively stimulated the voltage-dependent QUAC1 activity. In search of structural determinants of the gating process, we could not identify transmembrane domains com- mon for voltage-sensitive channels. However, site-directed mutations and deletions at the C-terminus of QUAC1 resulted in altered voltage-dependent channel activity. Interestingly, the replacement of a single glutamate residue, which is con- served in ALMT channels from different clades, by an alanine disrupted QUAC1 activity. Together with C- and N-terminal tagging, these results indicate that the cytosolic C-terminus is involved in the voltage-dependent gating mechanism of QUAC1.     

Molecular dynamics study of hell’s gate globin I (HGbI) from a methanotrophic extremophile: oxygen migration through a large cavity

Hell’s gate globin I (HGbI), a heme-containing protein from the extremophile Methylacidiphilum infernorum, has fast oxygen-binding/slow release characteristics due to its distal residues Gln and Tyr. The combination of Gln/Tyr distal iron coordination, adaptation to extreme environmental conditions, and lack of a D helix suggests that ligand migration in HGbI differs from other previously studied globins. Locally enhanced molecular dynamics trajectories of oxygen migration indicate a large internal cavity. This may increase the tendency of oxygen to exit from portals other than the most direct exit from the space near the heme. Oxygen may reside transiently in shallow surface depressions around the exits. Such surface trapping may enhance both oxygen uptake by increasing contact time between molecules, and decrease release by increasing the probability of oxygen reentry from the vicinity of the portal.

Barriers to interspecific hybridization between Juglans nigra L. and J. regia L species

Juglans nigra and Juglans regia are phylogenetically divergent species. Despite the economic interest in Juglans?×?intermedia (J. nigra?×? J. regia), walnut hybridization is rare under natural conditions and still difficult using controlled pollination. Here, we evaluated some reproductive mechanisms that may prevent successful natural hybridization. The study of flowering phenology of 11 J. nigra and 50 J. regia trees growing in a plantation provided information regarding the opportunity for interspecific crosses. Variation in flower size, pollen quality of putative donors, and variation in seed yield and rate of hybrid production among putative maternal trees were examined. DNA fingerprinting and parentage analyses based on nine microsatellites permitted the identification of hybrids and hybridogenic parent. Our data indicated that overlap occurred between the staminate flowering of protogynous J. regia and the beginning of pistillate flowering of protogynous J. nigra. Differences in floral size were computed between walnut species. Only three hybrids among 422 offspring of eleven J. nigra progenies were identified. Interspecific hybridization involving pollination of one early-flowering-protogynous J. nigra by three protogynous J. regia trees was detected. The correct development of J. regia male gametophytes, high pollen viability (86.5 %), and germination (57.6 %) ruled out the possibility that low pollen quality contributed to depressed hybrid production. Our findings indicated that these two species tended to remain reproductively isolated. The substantial disjunction in flowering time and additional prezygotic barriers such as differences in floral size and conspecific pollen advance may affect interspecific gene flow between J. regia and J. nigra.     

Identification of Annexin A1 interacting proteins in chronic myeloid leukemia KCL22 cells

In the present study, we used a functional proteomic approach to identify Annexin A1 (Anxa1) interacting proteins in the Philadelphia‐positive KCL22 cell line. We focused on Anxa1 because it is one of the major proteins upregulated in imatinib‐sensitive KCL22S cells versus imatinib‐resistant KCL22R. Our proteomic strategy revealed 21 interactors. Bioinformatic analysis showed that most of these proteins are involved in cell death processes. Among the proteins identified, we studied the interaction of Anxa1 with two phosphatases, Shp1 and Shp2, which were recently identified as biomarkers of imatinib sensitivity in patients affected by chronic myeloid leukemia. Our data open new perspectives in the search for annexin‐mediated signaling pathways and may shed light on mechanisms of resistance to imatinib that are unrelated to Bcr‐Abl activity. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000030.