How Mitochondrial Metabolism Contributes to Macrophage Phenotype and Functions

Metabolic reprogramming of cells from the innate immune system is one of the most noteworthy topics in immunological research nowadays. Upon infection or tissue damage, innate immune cells, such as macrophages, mobilize various immune and metabolic signals to mount a response best suited to eradicate the threat. Current data indicate that both the immune and metabolic responses are closely interconnected. On account of its peculiar position in regulating both of these processes, the mitochondrion has emerged as a critical organelle that orchestrates the coordinated metabolic and immune adaptations in macrophages. Significant effort is now underway to understand how metabolic features of differentiated macrophages regulate their immune specificities with the eventual goal to manipulate cellular metabolism to control immunity. In this review, we highlight some of the recent work that place cellular and mitochondrial metabolism in a central position in the macrophage differentiation program.     

Implications of oxidative stress in the cytotoxicity of Pseudomonas aeruginosa ExoU

ExoU PLA2-like activity has been shown to account for membrane lysis and acute death of infected cells. Translocation of effector proteins by the type III secretion systems depends on close contact between microbial and host cells. Our finding that both the ExoU-producing PA103 Pseudomonas aeruginosa and its mutant obtained by deletion of exoU adhered poorly to endothelial cells (EC) led to the hypothesis that, in some cells, the amount of injected toxin may not be enough to induce cell lysis but cells would suffer from a long-term effect of ExoU intoxication. To address this question, cells were exposed to both bacteria for 1 h and then treated with gentamicin-containing medium, to eliminate infecting microorganisms. After 24 h, the percentage of viable EC in PA103-infected cultures was significantly lower than in cultures exposed to the mutant, as determined by the MTT assay. Cell death was not likely to depend on the ExoU lytic activity since cell labeling with propidium iodide was similar in cultures infected with both bacterial strains. Bacterial cytotoxicity was significantly reduced by MAFP, a specific inhibitor of cPLA2 and iPLA2. Since the PLA2 activity on membrane phospholipids generates free fatty acid, including arachidonic acid (AA), we next compared the bacterial ability to release AA from infected EC. PA103 was shown to induce a potent AA release that was inhibited by MAFP. AA oxidation by oxygenases generates eicosanoids, known to induce both cell death and proliferation. However neither inhibitors of cyclooxygenases (ibuprofen) nor lipoxygenases (NDGA) reduced the ExoU toxicity. Since non-enzymatic oxidation of AA generates reactive radicals, we next investigated the PA103 ability to induce oxidative stress in infected cells. FACS analysis of cell labeling with the C-11 fluor probe and with anti-4-hydroxynonel antibody revealed a significant peroxidation of cell membrane lipids. These results, together with our finding that PA103-infected EC death was significantly attenuated by alpha-tocopherol, led to the conclusion that AA-induced oxidative stress may be another mechanism of cell damage in the course of infection by ExoU-producing P. aeruginosa.     

Molecular determinants of self-association and rearrangement of a trimeric intermediate during the assembly of a parvovirus capsid

The minute virus of mice (MVM) provides a simple model for the dissection of the molecular determinants of the self-assembly, stability, and dynamics of a biological supramolecular complex. MVM assembly involves the trimerization of capsid subunits in the cytoplasm; trimers are transported to the nucleus, where they suffer a conformational change and are made competent for capsid formation. Our previous study revealed that capsid assembly from trimers is dependent on stronger intertrimer interactions that are equally spaced in an equatorial belt surrounding each trimer. We have now targeted the interfaces between monomers within each trimer to identify the molecular determinants of trimerization and the rearrangement needed for capsid assembly. Twenty-eight amino acid residues per monomer were individually mutated to alanine to remove most of the stronger intersubunit interactions. The effects on trimer and capsid assembly and virus infectivity in cells were analyzed. No side chain was individually required for trimer assembly in the cytoplasm; in contrast, half of them were required to make the trimers competent for nuclear capsid assembly, even though none was close to intertrimer interfaces. These critical side chains are conserved and participate in extensive hydrophobic contacts, buried hydrogen bonds, or salt bridges between subunits. This study on MVM capsid assembly reveals that: (i) trimerization is a robust process, insensitive to removal of individual intersubunit interactions; and (ii) the rearrangement of the trimer intermediate required for capsid assembly is a global process that depends on the establishment of many interactions along the protein-protein interfaces within each trimer.     

ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites.     

Iron deficiency causes changes in chlorophyll fluorescence due to the reduction in the dark of the Photosystem II acceptor side

Iron deficiency was found to affect the redox state of the Photosystem II acceptor side in dark-adapted, attached leaves of sugar beet (Beta vulgaris L.). Dark-adapted iron-deficient leaves exhibited relatively high F_o and F_pl levels in the Kautsky chlorophyll fluorescence induction curve when compared to the iron-sufficient controls. However, far-red illumination led to marked decreases in the apparent F_o and F_pl levels. Modulated fluorescence showed that far-red light decreased the fluorescence yield to the true F_o levels by increasing photochemical quenching, without inducing changes in the level of non-photochemical quenching. In dark-adapted, iron-deficient leaves, far-red illumination induced a faster fluorescence decay in the µs-ms time domain, indicating an improvement in the electron transport after the primary quinone acceptor in the reducing side of Photosystem II. All these data indicate that in iron-deficient leaves the plastoquinone pool was reduced in the dark. The extent of the plastoquinone reduction in sugar beet depended on the chlorophyll concentration of the leaf, on the time of preillumination and on the duration of dark adaptation. The dark reduction of plastoquinone was observed not only in sugar beet but also in other plant species affected by iron deficiency both in controlled conditions and in the field.     

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

Background

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats.

Results

ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development.

Conclusions

We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.     

The Fat from Frozen Mammals Reveals Sources of Essential Fatty Acids Suitable for Palaeolithic and Neolithic Humans

The elucidation of the sources of n-3 fatty acids available for the humans in the Upper Palaeolithic and Neolithic is highly relevant in order to ascertain the availability of such nutrients in that time frame as well as to draw useful conclusions about healthy dietary habits for present-day humans. To this end, we have analysed fat from several frozen mammals found in the permafrost of Siberia (Russia). A total of 6 specimens were included in this study: 2 mammoths, i.e. baby female calf called “Lyuba” and a juvenile female called “Yuka”, both specimens approximately from the same time, i.e. Karginian Interstadial (41,000 and 34,000 years BP); two adult horses from the middle Holocene (4,600 and 4,400 years BP); and two bison very close to the Early Holocene (8,200 and 9,300 years BP). All samples were analysed by gas-liquid chromatography-mass spectrometry (GLC-MS) and GLC-flame ionization detector (GLC-FID). As demonstrated in this work, the fat of single-stomached mammals often consumed by Palaeolithic/Neolithic hunters contained suitable amounts of n-3 and n-6 fatty acids, possibly in quantities sufficient to meet the today''s recommended daily intake for good health. Moreover, the results also suggest that mammoths and horses at that time were hibernators.     

Iron deficiency interrupts energy transfer from a disconnected part of the antenna to the rest of Photosystem II

Iron deficiency changed markedly the shape of the leaf chlorophyll fluorescence induction kinetics during a dark-light transition, the so-called Kautsky effect. Changes in chlorophyll fluorescence lifetime and yield were observed, increasing largely the minimal and the intermediate chlorophyll fluorescence levels, with a marked dip between the intermediate and the maximum levels and loss of the secondary peak after the maximum. During the slow changes, the lifetime-yield relationship was found to be linear and curvilinear (towards positive lifetime values) in control and Fe-deficient leaves, respectively. These results suggested that part of the Photosystem II antenna in Fe-deficient leaves emits fluorescence with a long lifetime. In dark-adapted Fe-deficient leaves, measurements in the picosecond-nanosecond time domain confirmed the presence of a 3.3-ns component, contributing to 15% of the total fluorescence. Computer simulations revealed that upon illumination such contribution is also present and remains constant, indicating that energy transfer is partially interrupted in Fe-deficient leaves. Photosystem II-enriched membrane fractions containing different pigment-protein complexes were isolated from control and Fe-deficient leaves and characterized spectrophotometrically. The photosynthetic pigment composition of the fractions was also determined. Data revealed the presence of a novel pigment-protein complex induced by Fe deficiency and an enrichment of internal relative to peripheral antenna complexes. The data suggest a partial disconnection between internal Photosystem II antenna complexes and the reaction center, which could lead to an underestimation of the Photosystem II efficiency in dark-adapted, low chlorophyll Fe-deficient leaves, using chlorophyll fluorescence. This revised version was published online in August 2006 with corrections to the Cover Date.     

Stature estimation from complete long bones in the Middle Pleistocene humans from the Sima de los Huesos, Sierra de Atapuerca (Spain)

Systematic excavations at the site of the Sima de los Huesos (SH) in the Sierra de Atapuerca (Burgos, Spain) have allowed us to reconstruct 27 complete long bones of the human species Homo heidelbergensis. The SH sample is used here, together with a sample of 39 complete Homo neanderthalensis long bones and 17 complete early Homo sapiens (Skhul/Qafzeh) long bones, to compare the stature of these three different human species. Stature is estimated for each bone using race- and sex-independent regression formulae, yielding an average stature for each bone within each taxon. The mean length of each long bone from SH is significantly greater (p < 0.05) than the corresponding mean values in the Neandertal sample. The stature has been calculated for male and female specimens separately, averaging both means to calculate a general mean. This general mean stature for the entire sample of long bones is 163.6 cm for the SH hominins, 160.6 cm for Neandertals and 177.4 cm for early modern humans. Despite some overlap in the ranges of variation, all mean values in the SH sample (whether considering isolated bones, the upper or lower limb, males or females or more complete individuals) are larger than those of Neandertals. Given the strong relationship between long bone length and stature, we conclude that SH hominins represent a slightly taller population or species than the Neandertals. However, compared with living European Mediterranean populations, neither the Sima de los Huesos hominins nor the Neandertals should be considered ‘short’ people. In fact, the average stature within the genus Homo seems to have changed little over the course of the last two million years, since the appearance of Homo ergaster in East Africa. It is only with the emergence of H. sapiens, whose earliest representatives were ‘very tall’, that a significant increase in stature can be documented.