Identifying conformational changes with site-directed spin labeling reveals that the GTPase domain of HydF is a molecular switch

[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase. The X-ray structure of HydF indicates that the protein is a homodimer with both monomers carrying two functional domains: a C-terminal FeS cluster-binding domain, where the precursor is assembled, and a N-terminal GTPase domain, whose exact contribution to cluster biogenesis and hydrogenase activation is still elusive. We previously obtained several hints suggesting that the binding of GTP to HydF could be involved in the interactions of this scaffold protein with the other maturases and with the hydrogenase itself. In this work, by means of site directed spin labeling coupled to EPR/PELDOR spectroscopy, we explored the conformational changes induced in a recombinant HydF protein by GTP binding, and provide the first clue that the HydF GTPase domain could be involved in the H-cluster assembly working as a molecular switch similarly to other known small GTPases

Relationship between activity and stability: Design and characterization of stable variants of human frataxin

The relationships between conformational dynamics, stability and protein function are not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex dedicated to the iron-sulfur (Fe–S) cluster assembly within the mitochondrial matrix. In humans, the loss of FXN expression or a decrease in its functionality results in Friedreich's Ataxia, a cardio-neurodegenerative disease. Recently, the way in which FXN interacts with the rest of the subunits of the supercomplex was uncovered. This opens a window to explore relationships between structural dynamics and function. In this study, we prepared a set of FXN variants spanning a broad range of conformational stabilities. Variants S160I, S160M and A204R were more stable than the wild-type and showed similar biological activity. Additionally, we prepared SILCAR, a variant that combines S160I, L203C and A204R mutations. SILCAR was 2.4 kcal mol−1 more stable and equally active. Some of the variants were significantly more resistant to proteolysis than the wild-type FXN. SILCAR showed the highest resistance, suggesting a more rigid structure. It was corroborated by means of molecular dynamics simulations. Relaxation dispersion NMR experiments comparing SILCAR and wild-type variants suggested similar internal motions in the microsecond to millisecond timescale. Instead, variant S157I showed higher denaturation resistance but a significant lower function, similarly to that observed for the FRDA variant N146K. We concluded that the contribution of particular side chains to the conformational stability of FXN might be highly subordinated to their impact on both the protein function and the stability of the functional supercomplex.Fil: Castro, Ignacio Hugo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bringas, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Doni, Davide. Universita Di Padova. Dipartimento Di Biología; ItaliaFil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Capece, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Aran, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Blaustein Kappelmacher, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; ArgentinaFil: Costantini, Paola. Universita Di Padova. Dipartimento Di Biología; ItaliaFil: Santos, Javier. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Exploring iron-binding to human frataxin and to selected Friedreich ataxia mutants by means of NMR and EPR spectroscopies

The neurodegenerative disease Friedreich ataxia results from a deficiency of frataxin, a mitochondrial protein. Most patients have a GAA expansion in the first intron of both alleles of frataxin gene, whereas a minority of them are heterozygous for the expansion and contain a mutation in the other allele. Frataxin has been claimed to participate in iron homeostasis and biosynthesis of FeS clusters, however its role in both pathways is not unequivocally defined. In this work we combined different advanced spectroscopic analyses to explore the iron-binding properties of human frataxin, as isolated and at the FeS clusters assembly machinery. For the first time we used EPR spectroscopy to address this key issue providing clear evidence of the formation of a complex with a low symmetry coordination of the metal ion. By 2D NMR, we confirmed that iron can be bound in both oxidation states, a controversial issue, and, in addition, we were able to point out a transient interaction of frataxin with a N-terminal 6his-tagged variant of ISCU, the scaffold protein of the FeS clusters assembly machinery. To obtain insights on structure/function relationships relevant to understand the disease molecular mechanism(s), we extended our studies to four clinical frataxin mutants. All variants showed a moderate to strong impairment in their ability to activate the FeS cluster assembly machinery in vitro, while keeping the same iron-binding features of the wild type protein. This supports the multifunctional nature of frataxin and the complex biochemical consequences of its mutations.Fil: Bellanda, Massimo. Università di Padova; ItaliaFil: Maso, Lorenzo. Università di Padova; ItaliaFil: Doni, Davide. Università di Padova; ItaliaFil: Bortolus, M.. Università di Padova; ItaliaFil: De Rosa, E.. Università di Padova; ItaliaFil: Lunardi, Federica. Università di Padova; ItaliaFil: Alfonsi, Arianna. Università di Padova; ItaliaFil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Carbonera, Donatella. Università di Padova; ItaliaFil: Costantini, Paola. Università di Padova; Itali

Effect of systemic therapies or best supportive care after disease progression to both nivolumab and cabozantinib in metastatic renal cell carcinoma: The Meet‐Uro 19BEYOND study

Background Nivolumab and cabozantinib are currently approved agents in metastatic renal cell carcinoma (mRCC) but there are no data available for patients progressing to both treatments. The aim of this study was to compare active therapeutic options and best supportive care (BSC) after progression to nivolumab and cabozantinib in mRCC. Methods In this retrospective study, we selected 50 patients from eight Italian centers. The primary endpoint of the study was the overall survival (OS) of patients on active treatment versus BSC. Secondary endpoints were the progression-free survival (PFS) and objective response rate (ORR). The efficacy of active therapy was also investigated. Results After progression to both nivolumab and cabozantinib, 57.1% of patients were given active treatment (mainly everolimus and sorafenib) while 42.9% received BSC. The median OS was 13 months (95% CI: 4-NR) in actively treated patients and 3 months (95% CI: 2–4) in BSC patients (p = 0.001). Patients treated with sorafenib had better disease control than those treated with everolimus (stable disease: 71.4% vs. 16.7%, progression disease: 14.3% vs. 58.3%; p = 0.03), with no significant differences in PFS (5 and 3 months, 95% CI: 1–6 vs. 2–5; p = 0.6) and OS (12 and 4 months, 95% CI: 3-NR vs. 2-NR; p = 0.2). Conclusion After treatment with both nivolumab and cabozantinib, the choice of a safe active systemic therapy offered better outcomes than BSC

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Studio della Patogenesi dell'Atassia di Friedreich, una Malattia Neurodegenerativa: un Approccio Molecolare, Biochimico e Cellulare Integrato

L'atassia di Friedreich (FRDA) è una malattia cardio- e neurodegenerativa, dovuta a una riduzione dei livelli di fratassina (FXN), una proteina mitocondriale. Diversi ruoli sono stati proposti per FXN, dalla partecipazione alla biogenesi dei centri ferro-zolfo (ISCs, Iron Sulfur Clusters) e dell'eme (cofattori redox della catena respiratoria mitocondriale), all’attività di legame/stoccaggio o chaperone per il ferro. Tali funzioni sono in accordo con le caratteristiche biochimiche osservate nelle cellule dei pazienti affetti da FRDA, cioè un deficit di proteine contenenti ISCs come cofattori, un’alterata omeostasi del ferro e una maggiore suscettibilità allo stress ossidativo. Tuttavia, una chiara relazione tra i livelli di FXN, la deregolazione dell'assemblaggio degli ISCs e difetti bioenergetici non è stata ancora univocamente chiarita. Nella prima parte del mio progetto, è stato analizzato il fenotipo mitocondriale di linee cellulari linfoblastoidi (LCL) sia di pazienti affetti da FRDA che di un controllo sano. Sono stati osservati difetti nell'efficienza bioenergetica cellulare, un’ultrastruttura mitocondriale alterata e una ridotta capacità di associazione dei complessi respiratori in supercomplessi (SCs), strutture supramolecolari che, in condizioni fisiologiche, garantiscono di migliorare l’efficienza respiratoria mitocondriale. Si è scoperto che, nelle cellule sane, FXN e alcune proteine chiave del macchinario di assemblaggio degli ISCs sono arricchite nelle criste mitocondriali, il sub compartimento funzionale che ospita i (super)complessi respiratori; al contrario, nelle cellule FRDA, parte della FXN residua tende a traslocare in matrice, diversamente dalle proteine appartenenti al macchinario di assemblaggio degli ISCs. Questa evidenza suggerisce che i cambiamenti ultrastrutturali mitocondriali osservati nelle cellule FRDA potrebbero essere correlati alla parziale traslocazione di FXN dalle criste e che ciò potrebbe influenzare l’interazione funzionale con il macchinario di assemblaggio degli ISCs, così come con la catena respiratoria. L'ipotesi di un'interazione funzionale tra FXN e la catena di trasporto elettronico è stata indagata ulteriormente e i risultati ottenuti suggeriscono un coinvolgimento di FXN nell'assemblaggio e/o nella stabilità dei supercomplessi, specialmente quelli contenenti il complesso I. La seconda parte del mio progetto è stata dedicata allo studio delle proprietà di legame del ferro di FXN umana attraverso l’utilizzo di diverse tecniche spettroscopiche, tra cui spettroscopia di fluorescenza e di dicroismo circolare. Per ottenere informazioni su potenziali cambiamenti conformazionali di FXN promossi dal legame con il ferro, si è inoltre ricorsi ad un approccio mai utilizzato prima nello studio di FXN, cioè la tecnica SDSL accoppiata alla spettroscopia EPR. Questa tecnica, basata sul labeling di cisteine con sonde paramagnetiche in specifiche posizioni, permette di rilevare, a livello locale, possibili cambiamenti conformazionali della proteina. I risultati ottenuti dalla combinazione di SDSL-EPR, fluorescenza e dicroismo circolare hanno permesso di far luce sulle proprietà di legame col ferro di FXN, in relazione ai suoi possibili ruoli fisiologici. Inoltre, sulla base di un recente studio in vitro che ha dimostrato un'interazione diretta tra FXN di lievito e le superossido dismutasi (SODs), enzimi coinvolti nella difesa contro lo stress ossidativo, tramite la medesima tecnica di SDSL-EPR, abbiamo valutato se anche FXN umana fosse in grado di interagire con SOD2, l'isoforma mitocondriale. Gli esperimenti EPR, combinati con analisi di fluorescenza e con un approccio in silico, hanno permesso di dimostrare che SOD2 interagisce con FXN umana in vitro, supportando quindi l'ipotesi di un coinvolgimento di FXN nella protezione dal danno ossidativo a livello cellulare.Friedreich’s ataxia (FRDA) is a cardio-neurodegenerative disease, resulting from a severe decrease of the mitochondrial protein frataxin (FXN). Several roles have been proposed for FXN, ranging from biogenesis of iron-sulfur clusters (ISCs) and heme (key redox cofactors of the mitochondrial respiratory chain), to iron-binding/storage and iron chaperone. All these functions are in agreement with the biochemical features that characterize FRDA patients’ cells, i.e. a deficit of proteins containing ISCs as cofactors, imbalance in iron homeostasis and increased susceptibility to oxidative stress. However, a definite relationship between FXN levels, ISCs assembly dysregulation and bioenergetics failure has not been established yet. In the first part of my research, lymphoblastoid cell lines (LCLs) from different FRDA patients, along with a LCL from a healthy control, were thoroughly analyzed in terms of mitochondrial phenotype. Defects in bioenergetics efficiency and in mitochondrial ultrastructure were disclosed, along with a reduced capability to assemble the respiratory complexes in supercomplexes (SCs), supramolecular structures which in healthy cells improve the overall mitochondrial respiration. It was found that, in healthy cells, FXN and key proteins of the ISCs assembly machinery are enriched in the mitochondrial cristae, the functional subcompartment housing the respiratory (super)complexes. On the contrary, in FRDA cells, while proteins belonging to the ISCs assembly machinery retain their association with the mitochondrial cristae as in healthy cells, part of the residual FXN moves towards the matrix. This evidence suggests that the mitochondrial ultrastructural changes observed in FRDA cells could be connected to the partial loss of FXN from the cristae to the matrix and that this displacement could in turn affect its functional interaction with the ISCs assembly machinery, as well as with the mitochondrial respiratory chain. The hypothesis of a functional interaction between FXN and the respiratory chain was further investigated and, taken together, the obtained results suggest an involvement of FXN in the assembly and/or stability of supercomplexes, especially those containing complex I. The second part of my research was dedicated to study the iron-binding properties of human FXN through different spectroscopic techniques. By means of fluorescence spectroscopy and circular dichroism, the behavior of FXN in the presence of ferrous and ferric iron has been investigated. Moreover, to gain more information about the potential structural changes of FXN promoted by iron-binding, it was taken advantage of an approach never used before in the study of FXN, i.e. SDSL-EPR spectroscopy. This technique is based on labeling cysteines in selected positions of the protein with paramagnetic probes allowing to detect, at local level, possible conformational changes of the backbone dynamics. The results obtained by the combination of SDSL-EPR, fluorescence and circular dichroism spectroscopies have allowed to shed light on the iron-binding properties of FXN, in relation to its possible physiological roles. Moreover, based on a recent in vitro study showing a direct interaction between yeast FXN and superoxide dismutases (SODs), enzymes involved in the cellular antioxidant defense, it was addressed by SDSL-EPR if also human FXN is potentially able to interact with human mitochondrial SOD2. EPR experiments, combined with fluorescence analyses and with an in silico approach, demonstrated that human SOD2 interacts with human FXN in vitro, supporting the hypothesis that FXN could also be involved in the protection against oxidative damage, which commonly characterizes FRDA disease

Overview of the maturation machinery of the H-cluster of [FeFe]-hydrogenases with a focus on HydF

Hydrogen production in nature is performed by hydrogenases. Among them, [FeFe]-hydrogenases have a peculiar active site, named H-cluster, that is made of two parts, synthesized in different pathways. The cubane sub-cluster requires the normal iron-sulfur cluster maturation machinery. The [2Fe] sub-cluster instead requires a dedicated set of maturase proteins, HydE, HydF, and HydG that work to assemble the cluster and deliver it to the apo-hydrogenase. In particular, the delivery is performed by HydF. In this review, we will perform an overview of the latest knowledge on the maturation machinery of the H-cluster, focusing in particular on HydF

Sustainable Soil Management: Effects of Clinoptilolite and Organic Compost Soil Application on Eco-Physiology, Quercitin, and Hydroxylated, Methoxylated Anthocyanins on <i>Vitis vinifera</i>

Climate change and compostinS1g methods have an important junction on the phenological and ripening grapevine phases. Moreover, the optimization of these composting methods in closed-loop corporate chains can skillfully address the waste problem (pomace, stalks, and pruning residues) in viticultural areas. Owing to the ongoing global warming, in many wine-growing regions, there has been unbalanced ripening, with tricky harvests. Excessive temperatures in fact impoverish the anthocyanin amount of the must while the serious water deficits do not allow a correct development of the berry, stopping its growth processes. This experiment was created to improve the soil management and the quality of the grapes, through the application of a new land conditioner (Zeowine) to the soil, derived from the compost processes of industrial wine, waste, and zeolite. Three treatments on a Sangiovese vineyard were conducted: Zeowine (ZW) (30 tons per ha), Zeolite (Z) (10 tons per ha), and Compost (C) (20 tons per ha). During the two seasons (2021–2022), measurements were made of single-leaf gas exchange and leaf midday water potential, as well as chlorophyll fluorescence. In addition, the parameters of plant yield, yeast assimilable nitrogen, technological maturity, fractionation of anthocyanins (Cyanidin-3-glucoside, Delphinidin-3-glucoside, Malvidin-3-acetylglucoside, Malvidin-3-cumarylglucoside, Malvidin-3-glucoside, Peonidin-3-acetylglucoside, Peonidin-3-cumarylglucoside, Peonidin-3-glucoside, and Petunidin-3-glucoside), Caffeic Acid, Coumaric Acid, Gallic Acid, Ferulic Acid, Kaempferol-3-O-glucoside, Quercetin-3-O-rutinoside, Quercetin-3-O-glucoside, Quercetin-3-O-galactoside, and Quercetin-3-O-glucuronide were analyzed. The Zeowine and zeolite showed less negative water potential, higher photosynthesis, and lower leaf temperature. Furthermore, they showed higher levels of anthocyanin accumulation and a lower level of quercetin. Finally, the interaction of the beneficial results of Zeowine (soil and grapevines) was evidenced by the embellishment of the nutritional and water efficiency, the minimizing of the need for fertilizers, the closure of the production cycle of waste material from the supply chain, and the improvement of the quality of the wines

A Combined Spectroscopic and In Silico Approach to Evaluate the Interaction of Human Frataxin with Mitochondrial Superoxide Dismutase

Frataxin (FXN) is a highly conserved mitochondrial protein whose deficiency causes Friedreich&rsquo;s ataxia, a neurodegenerative disease. The precise physiological function of FXN is still unclear; however, there is experimental evidence that the protein is involved in biosynthetic iron&ndash;sulfur cluster machinery, redox imbalance, and iron homeostasis. FXN is synthesized in the cytosol and imported into the mitochondria, where it is proteolytically cleaved to the mature form. Its involvement in the redox imbalance suggests that FXN could interact with mitochondrial superoxide dismutase (SOD2), a key enzyme in antioxidant cellular defense. In this work, we use site-directed spin labelling coupled to electron paramagnetic resonance spectroscopy (SDSL-EPR) and fluorescence quenching experiments to investigate the interaction between human FXN and SOD2 in vitro. Spectroscopic data are combined with rigid body protein&ndash;protein docking to assess the potential structure of the FXN-SOD2 complex, which leaves the metal binding region of FXN accessible to the solvent. We provide evidence that human FXN interacts with human SOD2 in vitro and that the complex is in fast exchange. This interaction could be relevant during the assembly of iron-sulfur (FeS) clusters and/or their incorporation in proteins when FeS clusters are potentially susceptible to attacks by reactive oxygen species