The Metabolic Status Drives Acclimation of Iron Deficiency Responses in Chlamydomonas reinhardtii as Revealed by Proteomics Based Hierarchical Clustering and Reverse Genetics

Iron is a crucial cofactor in numerous redox-active proteins operating in bioenergetic pathways including respiration and photosynthesis. Cellular iron management is essential to sustain sufficient energy production and minimize oxidative stress. To produce energy for cell growth, the green alga Chlamydomonas reinhardtii possesses the metabolic flexibility to use light and/or carbon sources such as acetate. To investigate the interplay between the iron-deficiency response and growth requirements under distinct trophic conditions, we took a quantitative proteomics approach coupled to innovative hierarchical clustering using different “distance-linkage combinations” and random noise injection. Protein co-expression analyses of the combined data sets revealed insights into cellular responses governing acclimation to iron deprivation and regulation associated with photosynthesis dependent growth. Photoautotrophic growth requirements as well as the iron deficiency induced specific metabolic enzymes and stress related proteins, and yet differences in the set of induced enzymes, proteases, and redox-related polypeptides were evident, implying the establishment of distinct response networks under the different conditions. Moreover, our data clearly support the notion that the iron deficiency response includes a hierarchy for iron allocation within organelles in C. reinhardtii. Importantly, deletion of a bifunctional alcohol and acetaldehyde dehydrogenase (ADH1), which is induced under low iron based on the proteomic data, attenuates the remodeling of the photosynthetic machinery in response to iron deficiency, and at the same time stimulates expression of stress-related proteins such as NDA2, LHCSR3, and PGRL1. This finding provides evidence that the coordinated regulation of bioenergetics pathways and iron deficiency response is sensitive to the cellular and chloroplast metabolic and/or redox status, consistent with systems approach data.The green alga Chlamydomonas reinhardtii has an enormous metabolic versatility (1) and possesses the flexibility to grow in the presence of different carbon sources. It may use carbon dioxide (CO₂) for photoautotrophic, acetate for heterotrophic, and both carbon sources for mixotrophic growth. In this alga CO₂ is fixed via the Calvin Benson Bassham cycle (2), while acetate can be taken up, converted to acetyl-CoA, and enter the glyoxylate cycle where it may be incorporated into C4 acids (3). In addition to the use of acetate as a source of energy and carbon backbone for biosynthetic processes, acetate can control respiration and photosynthesis in conjunction with the light intensity and CO₂ availability (4–6). Moreover, acclimation responses to iron- and copper-deficiencies significantly vary in photoautotrophic versus heterotrophic conditions (7–10), indicating that the metabolic status of the cells influence overall cellular acclimation responses.Transition metals like copper, manganese, and iron possess the ability to donate and accept electrons, making these metals suitable cofactors in enzymes that catalyze redox reactions. In particular, iron is used as a cofactor in numerous biochemical pathways and is therefore an essential nutrient. Cells require relatively high levels of iron because it is present in heme-, iron-sulfur and other proteins that function in respiratory and photosynthetic energy transducing. Correspondingly, in eukaryotic cells, the mitochondrion is a major iron-utilizing compartment. It is well established that iron is transported into mitochondria for heme synthesis and iron-sulfur cluster assembly. This is required for the formation of a functional respiratory electron transport machinery (11). Therefore, mitochondrial metabolism in mammals, fungi and plants is significantly affected under iron deficiency, as demonstrated by a number of studies (12–14). In plants, the chloroplasts are a primary target of iron deficiency. Changes in chloroplast structure, photosynthetic capacity and the composition of thylakoid membranes have been described for plants deprived of iron (15–21).Plants have devised various strategies for acquiring iron (22). Generally, iron deficiency leads to the activation of the iron uptake systems in photosynthetic organisms. For example, the accumulation of the ferroxidase, a component of the high affinity iron uptake system in C. reinhardtii, is very rapidly enhanced when iron becomes limiting (23). Inactivation of IRT1, the most prevalent Fe²⁺ transporter in Arabidopsis thaliana leads to a dramatic iron deficiency that is reflected by chlorosis (24–26). Despite the evolution of elaborate iron-uptake mechanisms in plants, iron deficiency-induced chlorosis remains a major agricultural problem (27, 28).The global impact of iron deficiency on photosynthetic productivity has been also shown in vast ocean regions, which are severely limited for iron (29, 30). Generally, one can conclude that photosynthesis in the oceans and on land can occur in environment where iron availability is restricted.Photosystem I (PSI) is a prime target of iron deficiency as it contains 12 atoms of iron per core complex. In algae, the degradation of PSI is also linked to remodeling of PSI-associated light-harvesting antenna (LHCI) (31–33). Cyanobacteria respond to iron deficiency by degradation of light harvesting phycobilisomes (34) and induction of the “iron-stress-induced” gene isiA. The ISIA protein, which has significant sequence similarity with CP43, a chlorophyll a-binding protein of photosystem II (PSII; (35, 36), forms a ring of 18 molecules around a PSI trimeric reaction center, as shown by electron microscopy (37, 38). The overall reorganization of the PSI complex from 900 kDa into 1.7 MDa complex highlights the large adaptive nature of the cellular response to iron deficiency, which helps to optimize the architecture of the photosynthetic apparatus to conditions in which iron is a limiting factor.The marine diatom Thalassiosira oceanica shows a remarkable retrenchment of cellular metabolism and remodeling of bioenergetic pathways in response to iron availability (39). Low iron triggers a reduction in the level of iron-rich photosynthetic proteins while iron-rich mitochondrial proteins are preserved. Furthermore, iron deprivation causes a remodeling of the photosynthetic machinery resulting in the adjustment of light energy use to an overall decline in the level of photosynthetic electron transport complexes (39). These responses, reported for green algae such as C. reinhardtii (31, 40, 41), are important for minimizing photo-oxidative stress and optimizing photosynthetic function. As observed for T. oceanica, under conditions of low iron availability (in the presence of organic carbon) a hierarchy of iron allocation responses in C. reinhardtii result in the down-regulation of iron-rich photosynthetic complexes while iron-rich mitochondrial complexes remain stable (41). Notably, under photoautotrophic and mixotrophic conditions C. reinhardtii displays distinct iron deprivation responses, suggesting that the cell''s response to iron deficiency is also dependent on trophic conditions (7–9). Thus bioenergetics pathways are remodeled in response to iron availability as well as to the type of carbon source available. Moreover, recent data has indicated that the regulation of iron-induced remodeling of the photosynthetic apparatus is linked to energy metabolism. Depletion of Proton Gradient Regulation Like1 protein (PGRL1) in C. reinhardtii has revealed a decreased efficiency of cyclic electron transfer under low iron conditions resulting in higher vulnerability toward iron deprivation (42).It was our aim to generate a more comprehensive picture of how the proteome of C. reinhardtii varies in response to low iron under distinct trophic conditions and how these changes compare with differences observed for cells grown under photoautotrophic and photoheterotrophic iron replete conditions. Quantitative proteomics in conjunction with a novel hierarchical clustering approach revealed information about the responses of C. reinhardtii to low iron conditions and the iron requirements of photoautotrophic growth. These analyses provide novel insights into the relationships between protein networks required for photosynthesis and iron deprivation-elicited stress responses; these studies are providing the knowledge required for modulating the level of available iron to improve the photosynthetic performance of plants (43, 44).     

Characterization of UPF peptides,members of the glutathione analogues library,on the basis of their effects on oxidative stress-related enzymes

Previously the authors have designed and synthesized a library of antioxidative glutathione analogues called UPF peptides which are superior to glutathione in hydroxyl radical elimination. This paper is a follow-up study which investigated the effects of the most promising members of the library (UPF1 and UPF17) on oxidative stress-related enzymes. At concentrations used in vivo experiments neither UPF peptide influenced the activity of glutathione peroxidase (GPx) when purified enzyme or erythrocyte lysate was used. At higher concentrations they inhibited GPx activity. UPF peptides had no effect on glutathione reductase (GR) activity. Also they, as well as glutathione itself, slightly increased MnSOD activity in human brain mitochondria and inhibited oxidative burst caused by neutrophil NAD(P)H oxidase. RT-PCR measurements showed that UPF1 and UPF17 have no effect on GPx and MnSOD expression level in human blood mononuclear cells. The results of this study confirm that investigated UPF peptides do not interfere with the enzymatic mechanisms of antioxidative defence and can be used as themselves or as a lead for the protector molecule design against excessive oxidative stress.     

ADH1 expression inversely correlates with CDR1 and CDR2 in Candida albicans from chronic oral candidosis in APECED (APS-I) patients

Expression of the alcohol dehydrogenase gene ADH1, which converts ethanol into carcinogenic acetaldehyde, significantly inversely correlated with the expression of CDR1 and CDR2, genes linked to azole resistance in Candida albicans isolated from chronic oral candidosis in autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy (APECED, APS-I) patients. This is a novel link between candidal two-carbon metabolism genes and azole resistance.     

Highly Divergent Type 2 and 3 Vaccine-Derived Polioviruses Isolated from Sewage in Tallinn,Estonia

Highly divergent vaccine-derived polioviruses (VDPVs) have been isolated from sewage in Tallinn, Estonia, since 2002. Sequence analysis of VDPVs of serotypes 2 and 3 showed that they shared common noncapsid region recombination sites, indicating origination from a single trivalent oral polio vaccine dose, estimated to have been given between 1986 and 1998. The sewage isolates closely resemble VDPVs chronically excreted by persons with common variable immunodeficiency, but no chronic excretors have yet been identified in Estonia.     

Crh, the paralogue of the phosphocarrier protein HPr, controls the methylglyoxal bypass of glycolysis in Bacillus subtilis

The histidine protein HPr has a key role in regulation of carbohydrate utilization in low-GC Gram-positive bacteria. Bacilli possess the paralogue Crh. Like HPr, Crh becomes phosphorylated by kinase HPrK/P in response to high fructose-1,6-bisphosphate concentrations. However, Crh can only partially substitute for the regulatory functions of HPr leaving its role mysterious. Using protein co-purification, we identified enzyme methylglyoxal synthase MgsA as interaction partner of Crh in Bacillus subtilis. MgsA converts dihydroxyacetone-phosphate to methylglyoxal and thereby initiates a glycolytic bypass that prevents the deleterious accumulation of phospho-sugars under carbon overflow conditions. However, methylgyloxal is toxic and its production requires control. We show here that exclusively the non-phosphorylated form of Crh interacts with MgsA in vivo and inhibits MgsA activity in vitro. Accordingly, Crh inhibits methylglyoxal formation in vivo under nutritional famine conditions that favour a low HPr kinase activity. Thus, Crh senses the metabolic state of the cell, as reflected by its phosphorylation state, and accordingly controls flux through the harmful methylglyoxal pathway. Interestingly, HPr is unable to bind and regulate MgsA, making this a bona fide function of Crh. Four residues that differ in the interaction surfaces of HPr and Crh may account for this difference.     

Substance P is a key mediator of stress-induced protection from allergic sensitization via modified antigen presentation

Interaction between the nervous and immune systems greatly contributes to inflammatory disease. In organs at the interface between our body and the environment, the sensory neuropeptide substance P (SP) is one key mediator of an acute local stress response through neurogenic inflammation but may also alter cytokine balance and dendritic cell (DC) function. Using a combined murine allergic inflammation/noise stress model with C57BL/6 mice, we show in this paper that SP--released during repeated stress exposure--has the capacity to markedly attenuate inflammation. In particular, repeated stress exposure prior to allergen sensitization increases DC-nerve fiber contacts, enhances DC migration and maturation, alters cytokine balance, and increases levels of IL-2 and T regulatory cell numbers in local lymph nodes and inflamed tissue in a neurokinin 1-SP-receptor (neurokinin-1 receptor)-dependent manner. Concordantly, allergic inflammation is significantly reduced after repeated stress exposure. We conclude that SP/repeated stress prior to immune activation acts protolerogenically and thereby beneficially in inflammation.     

Comparison of the stability of glutathione and related synthetic tetrapeptides by HPLC and capillary electrophoresis.

Glutathione and related peptides are interesting targets as protectors of biological systems against an oxidative injury. Two novel glutathione analogues, UPF1 and UPF15, have been designed and synthesised. As a result of different reactions taking place, the thiol-containing compounds oxidise to disulfides. In this study, the stability of UPF1, UPF15 and glutathione in various solutions was investigated by using HPLC and CE. The results showed that UPF1 and UPF15 are powerful hydroxyl radical scavengers and their dimerisation process velocity is higher than that of glutathione.     

A new species of “sangre de drago” (Croton section Cyclostigma, Euphorbiaceae) from coastal Ecuador

Croton churutensis is described as a new species ofCroton sectionCyclostigma endemic to lowland deciduous forests in coastal Ecuador. Its red latex is used locally in Guayas Province to treat wounds, stomach ulcers, and some skin conditions caused by fungal infections. The new species differs from its closest apparent relative,Croton hibiscifolius, in its arching-pendent inflorescences, short-pedicellate female flowers with quadrifid stigmas, more numerous stamens, laciniate stipules, and lower elevation habitat.     

Bone morphogenetic proteins 4 and 2/7 induce osteogenic differentiation of mouse skin derived fibroblast and dermal papilla cells

Heterotopic ossification is a pathological condition in which bone forms outside the skeletal system. It can also occur in skin, which is the case in some genetic disorders. In addition to precursor cells and the appropriate tissue environment, heterotopic ossification requires inductive signals such as bone morphogenetic proteins (BMP). BMPs are growth and differentiation factors that have the ability to induce cartilage and bone formation in ectopic sites. The objective of this study is to explore the effect of the BMP-4 homodimer and BMP-2/7 heterodimer on the osteogenic differentiation of primary mouse skin fibroblasts and hair follicle dermal papilla (DP) cells. Osteogenic differentiation was induced by osteogenic induction medium (OS) containing 10 nM dexamethasone. The effect of BMP-4 and BMP-2/7 was studied using alkaline phosphatase (ALP) and calcium assays after 1.5, 3 and 5 weeks of differentiation. Fibroblasts and DP cells were able to differentiate into osteoblast-like matrix mineralizing cells. The first visible sign of differentiation was the change of morphology from rounded to more spindle-shaped cells. BMP-4 and BMP-2/7 exposure elevated ALP activity and calcium production significantly more than OS alone. The osteogenic response to BMP-4 and BMP-2/7 was similar in fibroblasts, whereas, in DP cells, BMP-2/7 was more potent than BMP-4. OS alone could not induce osteogenic differentiation in DP cells. Clear and consistent results show that dermal fibroblasts and stem cells from the dermal papilla were capable of osteogenic differentiation. The BMP-2/7 heterodimer was significantly more effective on hair follicular dermal stem cell differentiation.