Structural Contour Map of the Iota Carbonic Anhydrase from the Diatom Thalassiosira pseudonana Using a Multiprong Approach.

Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO2 and HCO3-. The "iota" class (ι-CA) was first found in the marine diatom Thalassiosira pseudonana (tpι-CA) and is widespread among photosynthetic microalgae and prokaryotes. The ι-CA has a domain COG4875 (or COG4337) that can be repeated from one to several times and resembles a calcium-calmodulin protein kinase II association domain (CaMKII-AD). The crystal structure of this domain in the ι-CA from a cyanobacterium and a chlorarachniophyte has been recently determined. However, the three-dimensional organization of the four domain-containing tpι-CA is unknown. Using biophysical techniques and 3-D modeling, we show that the homotetrameric tpι-CA in solution has a flat "drone-like" shape with a core formed by the association of the first two domains of each monomer, and four protruding arms formed by domains 3 and 4. We also observe that the short linker between domains 3 and 4 in each monomer confers high flexibility, allowing for different conformations to be adopted. We propose the possible 3-D structure of a truncated tpι-CA containing fewer domain repeats using experimental data and discuss the implications of this atypical shape on the activity and metal coordination of the ι-CA.

Insights on the Functions and Ecophysiological Relevance of the Diverse Carbonic Anhydrases in Microalgae.

Carbonic anhydrases (CAs) exist in all kingdoms of life. They are metalloenzymes, often containing zinc, that catalyze the interconversion of bicarbonate and carbon dioxide-a ubiquitous reaction involved in a variety of cellular processes. So far, eight classes of apparently evolutionary unrelated CAs that are present in a large diversity of living organisms have been described. In this review, we focus on the diversity of CAs and their roles in photosynthetic microalgae. We describe their essential role in carbon dioxide-concentrating mechanisms and photosynthesis, their regulation, as well as their less studied roles in non-photosynthetic processes. We also discuss the presence in some microalgae, especially diatoms, of cambialistic CAs (i.e., CAs that can replace Zn by Co, Cd, or Fe) and, more recently, a CA that uses Mn as a metal cofactor, with potential ecological relevance in aquatic environments where trace metal concentrations are low. There has been a recent explosion of knowledge about this well-known enzyme with exciting future opportunities to answer outstanding questions using a range of different approaches.

Storage Compound Accumulation in Diatoms as Response to Elevated CO2 Concentration.

Accumulation of reserve compounds (i.e., lipids and chrysolaminarin) in diatoms depends on the environmental conditions, and is often triggered by stress conditions, such as nutrient limitation. Manipulation of CO2 supply can also be used to improve both lipids and carbohydrates accumulation. Given the high diversity among diatoms, we studied the two marine model diatoms-Thalassiosira pseudonana and Phaeodactylum tricornutum, a freshwater diatom, Asterionella formosa, and Navicula pelliculosa-found in fresh- and sea-water environments. We measured the accumulation of reserve compounds and the activity of enzymes involved in carbon metabolism in these diatoms grown at high and atmospheric CO2. We observed that biomass and lipid accumulation in cells grown at high CO2 differ among the diatoms. Lipid accumulation increased only in P. tricornutum and N. pelliculosa grown in seawater in response to elevated CO2. Moreover, accumulation of lipids was also accompanied by an increased activity of the enzymes tested. However, lipid accumulation and enzyme activity decreased in N. pelliculosa cultured in fresh water. Chrysolaminarin accumulation was also affected by CO2 concentration; however, there was no clear relation with lipids accumulation. Our results are relevant to understand better the ecological role of the environment in the diatom adaptation to CO2 and the mechanisms underpinning the production of storage compounds considering diatom diversity.

A new widespread subclass of carbonic anhydrase in marine phytoplankton.

Most aquatic photoautotrophs depend on CO2-concentrating mechanisms (CCMs) to maintain productivity at ambient concentrations of CO2, and carbonic anhydrase (CA) plays a key role in these processes. Here we present different lines of evidence showing that the protein LCIP63, identified in the marine diatom Thalassiosira pseudonana, is a CA. However, sequence analysis showed that it has a low identity with any known CA and therefore belongs to a new subclass that we designate as iota-CA. Moreover, LCIP63 unusually prefers Mn2+ to Zn2+ as a cofactor, which is potentially of ecological relevance since Mn2+ is more abundant than Zn2+ in the ocean. LCIP63 is located in the chloroplast and only expressed at low concentrations of CO2. When overexpressed using biolistic transformation, the rate of photosynthesis at limiting concentrations of dissolved inorganic carbon increased, confirming its role in the CCM. LCIP63 homologs are present in the five other sequenced diatoms and in other algae, bacteria, and archaea. Thus LCIP63 is phylogenetically widespread but overlooked. Analysis of the Tara Oceans database confirmed this and showed that LCIP63 is widely distributed in marine environments and is therefore likely to play an important role in global biogeochemical carbon cycling.

Copper deficiency-induced anemia is caused by a mitochondrial metabolic reprograming in erythropoietic cells.

The lack of copper has been associated with anemia, myelodysplastic syndromes and leukemia as well as with a loss in complex IV activity and an enlarged mitochondrial morphology. Mitochondria play a key role during the differentiation of hematopoietic stem cells by regulating the passage from a glycolytic to oxidative metabolism. The former is associated with cell proliferation and the latter with cell differentiation. Oxidative metabolism, which occurs inside mitochondria, is sustained by the respiratory chain, where complex IV is copper-dependent. We have hypothesized that a copper deficiency induces a mitochondrial metabolic reprogramming, favoring cell expansion over cell differentiation in erythropoiesis. Erythroid progression analysis of the bone marrow of mice fed with a copper deficient diet and of the in vitro erythropoiesis of human CD34+ cells treated with a bathocuproine - a copper chelator - showed a major expansion of progenitor cells and a decreased differentiation. Under copper deficiency, mitochondria switched to a higher membrane potential, lower oxygen consumption rate and lower ROS levels as compared with control cells. In addition, mitochondrial biomass was increased and an up-regulation of the mitochondrial fusion protein mitofusin 2 was observed. Most copper-deficient phenotypes were mimicked by the pharmacological inhibition of complex IV with azide. We concluded that copper deficiency induced a mitochondrial metabolic reprogramming, making hematopoietic stem cells favor progenitor cell expansion over cell differentiation.

Hyperglycemia Induces Bioenergetic Changes in Adipose-Derived Stromal Cells While Their Pericytic Function Is Retained.

Diabetic retinopathy (DR) is a hyperglycemia (HG)-mediated microvascular complication. In DR, the loss of pericytes and subsequently endothelial cells leads to pathologic angiogenesis in retina. Adipose-derived stromal cells (ASC) are a promising source of therapeutic cells to replace lost pericytes in DR. To date, knowledge of the influence of HG on the bioenergetics and pericytic function of ASC is negligible. Human ASC were cultured in normoglycemia medium (5 mM d-glucose) or under HG (30 mM d-glucose) and assessed. Our data showed that HG increased the level of apoptosis and reactive oxygen species production in ASC, yet their proliferation rate was not affected. HG induced alterations in mitochondrial function and morphology in ASC. HG also strongly affected the bioenergetic status of ASC in which both the maximum oxygen consumption rate and extracellular acidification rate were decreased. This was corroborated by a reduced uptake of glucose under HG. In spite of these observations, in vitro, ASC promoted the formation of vascular-like networks of human umbilical vein endothelial cells on monolayers of ASC under HG with minimally affected.

Non-cytotoxic copper overload boosts mitochondrial energy metabolism to modulate cell proliferation and differentiation in the human erythroleukemic cell line K562.

Copper is integral to the mitochondrial respiratory complex IV and contributes to proliferation and differentiation, metabolic reprogramming and mitochondrial function. The K562 cell line was exposed to a non-cytotoxic copper overload to evaluate mitochondrial dynamics, function and cell fate. This induced higher rates of mitochondrial turnover given by an increase in mitochondrial fusion and fission events and in the autophagic flux. The appearance of smaller and condensed mitochondria was also observed. Bioenergetics activity included more respiratory complexes, higher oxygen consumption rate, superoxide production and ATP synthesis, with no decrease in membrane potential. Increased cell proliferation and inhibited differentiation also occurred. Non-cytotoxic copper levels can modify mitochondrial metabolism and cell fate, which could be used in cancer biology and regenerative medicine.

Adaptive responses of mitochondria to mild copper deprivation involve changes in morphology, OXPHOS remodeling and bioenergetics.

Copper is an essential cofactor of complex IV of the electron transfer chain, and it is directly involved in the generation of mitochondrial membrane potential. Its deficiency induces the formation of ROS, large mitochondria and anemia. Thus, there is a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis. Copper depletion might end in cellular apoptosis or necrosis. However, before entering into those irreversible processes, mitochondria may execute a series of adaptive responses. Mitochondrial adaptive responses (MAR) may involve multiple and diverse mechanisms for preserving cell life, such as mitochondrial dynamics, OXPHOS remodeling and bioenergetics output. In this study, a mild copper deficiency was produced in an animal model through intraperitoneal injections of bathocuproine disulfonate in order to study the MAR. Under these conditions, a new type of mitochondrial morphology was discovered in the liver. Termed the "butternut squash" mitochondria, it coexisted with normal and swollen mitochondria. Western blot analyses of mitochondrial dynamics proteins showed an up-regulation of MFN-2 and OPA1 fusion proteins. Furthermore, isolated liver mitochondria displayed OXPHOS remodeling through a decrease in supercomplex activity with a concomitant increase at an individual level of complexes I and IV, higher respiratory rates at complex I and II levels, higher oligomycin-insensitive respiration, and lower respiratory control ratio values when compared to the control group. As expected, total ATP and ATP/ADP values were not significantly different, since animal's health was not compromised. As a whole, these results describe a compensatory and adaptive response of metabolism and bioenergetics under copper deprivation.

Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells.

Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive.