Biology subjects

Meloni, M.

Publications and source records attributed to Meloni, M..

10 recordsLinked to original sources

bioRxiv2025

Heterologous expression of the cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in Chlamydomonas reinhardtii causes increased cell size and biomass productivity in mixotrophic conditions

The Calvin-Benson-Bassham (CBB) cycle is the metabolic pathway responsible for CO assimilation in oxygenic photosynthetic organisms. Two key rate-limiting steps in this cycle are catalyzed by the enzymes fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase), making them promising targets for genetic enhancement to improve carbon fixation. A potential strategy involves overexpressing a cyanobacterial dual-function FBP/SBPase, which catalyzes both reactionsOverexpression of this enzyme in tobacco plants or in other plants led to an increase in growth rate and biomass accumulation. Here, the overexpression of the same enzyme was achieved in Chlamydomonas reinhardtii. The recombinant cyanobacterial FBP/SBPase isolated from C. reinhardtii exhibited the expected catalytic activity, being Mg2+ dependent and strongly activated in the presence of a reducing agent. The FBP/SBPase expressing lines exhibited an increased photosynthetic activity at the cell level and decreased production of singlet oxygen upon exposure to high irradiances, suggesting improved capacity to manage high excitation pressure of the photosynthetic apparatus. Increased cell volume was measured in FBP/SBPase-expressing lines under different growth conditions. However, increased growth and biomass productivity were observed only in mixotrophy when light and CO2 were limiting, leading to increased starch, protein, and lipid content on a cellular basis. This phenotype caused an increased sedimentation rate in the transformant lines: the expression of FBP/SBPase enzyme could thus be considered as a strategy to improve the cell harvesting process. These findings provide new insights into carbon metabolism in microalgae, and could, in the future, support improved biomass accumulation, paving the way for effective domestication and industrial use.

plant biology↗

bioRxiv2023

Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii

Improving photosynthetic efficiency in plants and microalgae is of utmost importance to support the growing world population and to enable the bioproduction of energy and chemicals. Limitations in photosynthetic light conversion efficiency can be directly attributed to kinetic bottlenecks within the Calvin-Benson-Bassham cycle (CBBC) responsible for carbon fixation. A better understanding of these bottlenecks in vivo is crucial to overcome these limiting factors through bio-engineering. The present study is focused on the analysis of phosphoribulokinase (PRK) in the unicellular green alga Chlamydomonas reinhardtii. We have characterized a PRK knock-out mutant strain and showed that in the absence of PRK, Chlamydomonas cannot grow photoautotrophically while functional complementation with a synthetic construct allowed restoration of photoautotrophy. Nevertheless, using standard genetic elements, the expression of PRK was limited to 40% of the reference level in complemented strains and could not restore normal growth in photoautotrophic conditions suggesting that the CBBC is limited. We were subsequently able to overcome this initial limitation by improving the design of the transcriptional unit expressing PRK using diverse combinations of DNA parts including PRK endogenous promoter and introns. This enabled us to obtain strains with PRK levels comparable to the reference strain and even overexpressing strains. A collection of strains with PRK levels between 16% and 250% of WT PRK levels was generated and characterized. Immunoblot and growth assays revealed that a PRK content of {approx}86% is sufficient to fully restore photoautotrophic growth. This result suggests that PRK is present in moderate excess in Chlamydomonas. Consistently, the overexpression of PRK did not increase photosynthetic growth indicating that that the endogenous level of PRK in Chlamydomonas is not limiting the Calvin-Benson-Bassham cycle under optimal conditions.

synthetic biology↗

bioRxiv2022

Structural and functional characterization of chloroplast ribulose-5-phosphate-3-epimerase from the model green microalga Chlamydomonas reinhardtii

Photosynthetic carbon fixation relies on Rubisco and ten additional enzymes in the conserved Calvin-Benson-Bassham (CBB) cycle. Epimerization of xylulose-5-phosphate (X5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco activity. Ribulose-5-phosphate-3-epimerase (RPE) catalyzes the formation of Ru5P but it can also operate in the pentose phosphate pathway (PPP) by catalyzing the reverse reaction. Here, we describe the catalytic and structural properties of the recombinant form of photosynthetic RPE isoform 1 from Chlamydomonas reinhardtii (CrRPE1). The enzyme shows catalytic parameters that are variably comparable to those of the paralogues involved in the PPP and CBB cycle but with some notable exceptions. CrRPE1 is a homo-hexamer that exposes a catalytic pocket on the top of an 8{beta}8 triose isomerase-type (TIM-) barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. Despite being identified as a putative target of thiol-based redox modifications, CrRPE1 activity is not altered by redox treatments, indicating that the enzyme does not bear redox sensitive thiol groups and is not regulated by thiol-switching mechanisms. We mapped phosphorylation sites on the crystal structure and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. Overall, this work provides a detailed description of the catalytic and regulatory properties of CrRPE along with structural data, which allow for a deeper understanding of the functioning of this enzyme of the CBB cycle and in setting the basis for possible strategies to improve the photosynthetic metabolism.

biochemistry↗

bioRxiv2022

AMPK deficiency in smooth muscles causes persistent pulmonary hypertension after birth and premature death

We address a paradox, that AMPK may facilitate hypoxic pulmonary vasoconstriction and its deficiency precipitate pulmonary hypertension. Here we show that AMPK-1/2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the newborn. Accordingly, dual AMPK-1/2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularization and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no edema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. KV1.5 potassium currents of pulmonary arterial myocytes are markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation is also evident. Importantly, there is no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction is attenuated by AMPK-1 or AMPK-2 deletion without triggering pulmonary hypertension.

pathology↗

bioRxiv2022

Structural snapshots of nitrosoglutathione binding and reactivity underlying S-nitrosylation of photosynthetic GAPDH

S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols. GSNO can also induce protein S-glutathionylation but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes enzyme inhibition by specifically inducing S-nitrosylation. Treatment with reducing agents restores CrGAPA activity completely. While the cofactor NADP+ only partially protects from GSNO-mediated S-nitrosylation, the resultant inactivation is completely blocked by the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible of CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. Quantum mechanical/molecular mechanical calculations were performed to investigate the reaction mechanism of GSNO with CrGAPA Cys149 and to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.

plant biology↗

bioRxiv2020

Contractile reprogramming of cardiac pericytes by MEK inhibition promotes arteriologenesis of the ischemic heart

BackgroundThe development of collateral arteries after a myocardial infarction (MI) was intensively studied, while the mechanism by which pericytes (PCs) contribute to arteriologenesis remains unexplored. This study aimed to 1) investigate if cardiac PCs gain functional features of contractile vascular smooth muscle cells (VSMCs) in vitro, and 2) determine if this potential can be evoked pharmacologically to encourage heart arteriologenesis in vivo. MethodsPCs were immunosorted as CD31neg/CD34pos cells from human and mouse hearts. Contractile reprogramming was induced by either depletion of growth factors or addition of PD0325901, a clinically available MEK inhibitor. Next generation RNA-Sequencing was performed in naive and differentiated human PCs to assess the whole-transcriptome profile. Three in vivo studies were conducted in C57BL6/J mice to determine: 1) the ability of human PCs to promote arteriole formation when implanted subcutaneously within PD0325901-containing Matrigel plugs, 2) the effect of orally administered PD0325901 on the arteriole density of normoperfused hearts, and 3) the possibility of promoting capillary formation and muscularization of the infarcted heart through the same pharmacological approach. ResultsRemoval of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) from the culture medium induced the differentiation of PCs into contractile VSMC-like cells. Because both growth factors induce the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling, we attempted to induce PC differentiation in vitro and in vivo using PD0325901. RNA-sequencing revealed that differentiated PCs were enriched in transcripts associated with smooth muscle contraction and biological function. PD0325901-treated PCs rapidly acquired antigenic and functional features of contractile VSMCs in vitro. Moreover, human PCs formed new arterioles when implanted subcutaneously within PD0325901-containing Matrigel plugs in mice. Oral administration of PD0325901 for two weeks increased the density and expression of contractile proteins in small-calibre arterioles of the murine heart, thereby increasing myocardial perfusion. Similarly, PD0325901 induced reparative arteriologenesis and capillarization, reduced the scar, and improved left ventricular performance in a murine model of MI. ConclusionWe propose a novel method to promote the heart vascularization through the pharmacological modulation of resident mural cells. This novel approach could have an immediate impact on the treatment of coronary artery disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIHuman myocardial pericytes have intrinsic vascular plasticity that can be pharmacologically evoked using PD0325901, a clinically available MEK inhibitor. C_LIO_LIIn mice, the pharmacological inhibition of ERK1/2 signalling, by the oral administration of PD0325901 for 2 weeks, encouraged the heart arteriologenesis through pericyte differentiation. C_LIO_LIIn a preclinical mouse model of myocardial infarction, the oral administration of PD0325901 for 2 weeks induced reparative arteriologenesis and capillarization, reduced the scar, and improved left ventricular performance. C_LI What are the clinical implications?O_LIThis novel drug-based therapeutic approach is readily available to all patients. C_LIO_LITherefore, it could have an immediate clinical impact for the treatment of coronary artery disease and other heart conditions associated with deficient coronary vascularization. C_LI

cell biology↗

bioRxiv2020

Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. The kinetic properties of CrGSNOR1 were determined and the high-resolution three-dimensional structures of the apo and NAD+-bound forms of the enzyme were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved 3D-folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed. One-sentence summaryGSNOR1 from Chlamydomonas reinhardtii displays an unusual variability of the catalytic zinc coordination environment and an unexpected resistance to thiol-based redox modifications

plant biology↗

bioRxiv2019

The pericardium promotes cardiac repair and remodelling post-myocardial infarction

The pericardium is widely recognised for its lubricating and bio-mechanical properties. It also contains fat-associated lymphoid clusters (FALCs) and its immune functions have been widely overlooked. Here we aimed to assess the inflammatory activity of the pericardium in patients who suffered a recent myocardial infarction (MI) and to determine its importance for repair and remodelling in a murine MI model induced by coronary artery ligation (CAL). By comparing 18F-fluorodeoxyglucose (FDG) activity in the pericardium of patients with stable coronary artery disease and patients who had a recent MI, we demonstrate that MI is associated with increased pericardial inflammation. We confirm in mice, that pericardial FALCs undergo a major expansion following CAL. We show that despite similar initial injury, removal of the pericardium prior to MI disrupted subsequent repair, resulting in 50% mortality due to cardiac rupture, while all mice with intact pericardia survived. Removal of the pericardium also led to decreased staining for Ym1, a marker of reparative macrophages and adverse cardiac fibrosis within the infarct area. Together, this work indicates a crucial role for the pericardium in regulating inflammation, macrophage polarisation and tissue remodelling in the heart following MI.

physiology↗

bioRxiv2019

Eosinophil deficiency promotes aberrant repair and adverse remodelling following acute myocardial infarction

BackgroundEosinophil count predicts outcome following myocardial infarction (MI) and eosinophils regulate tissue repair and regeneration in extra-cardiac settings.\n\nObjectivesTo investigate the role of eosinophils in regulating inflammation, repair and remodelling following MI.\n\nMethodsBlood eosinophil count was assessed in 732 patients undergoing primary percutaneous coronary intervention for ST-segment elevation MI (STEMI). Experimental MI was induced in wild-type (WT) and eosinophil-depleted mice ({Delta}dblGATA or anti-Siglec F antibody treated). Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of infarct digests.\n\nResultsBlood eosinophil count declined in the hours following STEMI in patients and following MI induction in mice. Eosinophils were subsequently identified in the myocardium of patients and mice. Genetic eosinophil depletion in mice increased LV dilatation (end-systolic area: 29.0{+/-}2.2cm2 v 21.6{+/-}1.6cm2; p=0.02) and reduced ejection fraction (22.0{+/-}3.6% v 34.3{+/-}4.0% in p=0.04,) in {Delta}dblGATA v WT following MI (n=8-9/group), an outcome reproduced by pharmacological depletion. {Delta}dblGATA mice had increased scar size with disrupted collagen deposition and altered expression of the collagen cross-linking genes plod2 and lox. CD206+pro-repair macrophages were less prevalent in the infarct zone of {Delta}dblGATA mice (27.0{+/-}2.3% v WT: 39.2.4{+/-}3.5%; p=0.01, n=9-11/group) but were restored by replenishment with bone marrow-derived eosinophils. Anti-inflammatory cytokine concentrations were reduced in {Delta}dblGATA mice and IL-4 complex administration 24h after MI rescued adverse remodelling.\n\nConclusionsEosinophils are recruited to the heart following MI and are required for effective repair and to prevent adverse remodelling. IL-4 therapy has potential to limit detrimental outcomes when eosinophil availability is low.\n\nHIGHLIGHTSA drop in eosinophil blood count is associated with recruitment of eosinophils to the heart during repair following clinical and experimental myocardial infarction.\n\nGenetic & pharmacological eosinophil depletion leads to increased adverse remodelling in experimental MI.\n\nEosinophils are required for aquisition of an anti-inflammatory macrophage phenotype and a shift to resolution of inflammation during infarct repair.\n\nInterleukin-4 therapy is able to rescue the adverse remodelling phenotype in conditions of eosinophil deficiency.\n\nCondensed abstractIn ST-segment elevation myocardial infarction (STEMI) of both patients and mice, there was a decline in blood eosinophil count, with activated eosinophils recruited to the infarct zone. Eosinophil deficiency resulted in attenuated anti-inflammatory pro-repair macrophage polarization, enhanced myocardial inflammation, increased scar size and deterioration of myocardial structure and function. Adverse cardiac remodelling in the setting of eosinophil deficiency was prevented by IL-4 therapy.

immunology↗

bioRxiv2018

High genetic diversity and presence of genetic structure characterise the Corso-Sardinian endemics Ruta corsica and Ruta lamarmorae (Rutaceae)

Corsica and Sardinia form one of the ten areas with highest biodiversity in the Mediterranean and are considered one of the priority regions for conservation in Europe. In order to preserve the high levels of endemism and biological diversity at different hierarchical levels, knowledge of the evolutionary history and current genetic structure of Corso-Sardinian endemics is instrumental. Microsatellite markers were newly developed and used to study the genetic structure and taxonomic status of Ruta corsica and Ruta lamarmorae, rare endemics of Corsica and Sardinia, respectively, and previously considered a single species. Our analyses identified high levels of genetic variation within each species (P=0.883, He=0.543 for R. corsica; P=0.972, He=0.627 for R. lamarmorae). Intrinsic traits of the species (hermaphroditism, proterandry and polyploidy) and island-dependent factors (i.e. age, origin and history of the islands) might explain the detected high levels of genetic variation. We discovered differentiation between R. corsica and R. lamarmorae, and genetic structure within each species, which are consistent with the observation of low dispersal ability for both species. Our genetic results support the recent taxonomic classification of R. corsica and R. lamarmorae as separate species and suggest that they diverge at only few loci. One R. corsica population (SA) strongly differed from all other studied populations and appeared to be the product of hybridization between the two species in STRUCTURE analyses. Our results provide important insights for the conservation of the two rare endemics. Further genetic analyses are recommended for R. lamarmorae and for population SA (R. corsica).

genetics↗