Structural and biochemical insights of CypA and AIF interaction

The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370-394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward distinct CypA structural determinants governing the inhibitor selectivity and the differential biological effects of AIF and CsA, and provide new structural insights for designing CypA/AIF selective inhibitors with therapeutic relevance in neurodegenerative diseases

Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid-virus pSSVx: a novel peculiar member of the winged helix–turn–helix transcription factor family

The hybrid plasmid virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 aminoacid protein, namely Stf76, that does not show significant sequence homology with any protein with known three-dimensional structure. The recombinant protein recognises specifically two DNA binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. CD, spectrofluorimetric, light scattering and ITC experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix–turn–helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76-DNA complex has been built using as template a structurally related homolog

Design, Optimization, and Structural Characterization of an Apoptosis-Inducing Factor Peptide Targeting Human Cyclophilin A to Inhibit Apoptosis Inducing Factor-Mediated Cell Death

Blocking the interaction between the apoptosis-inducing factor (AIF) and cyclophilin A (CypA) by the AIF fragment AIF(370-394) is protective against glutamate-induced neuronal cell death and brain injury in mice. Starting from AIF(370-394), we report the generation of the disulfide-bridged and shorter variant AIF(381-389) and its structural characterization by nuclear magnetic resonance (NMR) in the free and CypA-bound state. AIF(381-389) in both the free and bound states assumes a β-hairpin conformation similar to that of the fragment in the AIF protein and shows a highly reduced conformational flexibility. This peptide displays a similar in vitro affinity for CypA, an improved antiapoptotic activity in cells and an enhanced proteolytic stability compared to the parent peptide. The NMR-based 3D model of the AIF(381-389)/CypA complex provides a better understanding of the binding hot spots on both the peptide and the protein and can be exploited to design AIF/CypA inhibitors with improved pharmacokinetic and pharmacodynamics features

High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations

The crucial role of integrin in pathological processes such as tumor progression and metastasis formation has inspired intense efforts to design novel pharmaceutical agents modulating integrin functions in order to provide new tools for potential therapies. In the past decade, we have investigated the biological proprieties of the chimeric peptide RGDechi, containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to specifically recognize αvβ3 without cross reacting with αvβ5 and αIIbβ3 integrin. Additionally, we have demonstrated using two RGDechi-derived peptides, called RGDechi1-14 and ψRGDechi, that chemical modifications introduced in the C-terminal part of the peptide alter or abolish the binding to the αvβ3 integrin. Here, to shed light on the structural and dynamical determinants involved in the integrin recognition mechanism, we investigate the effects of the chemical modifications by exploring the conformational space sampled by RGDechi1-14 and ψRGDechi using an integrated natural-abundance NMR/MD approach. Our data demonstrate that the flexibility of the RGD-containing cycle is driven by the echistatin C-terminal region of the RGDechi peptide through a coupling mechanism between the N- and C-terminal regions

A seismic-properties and wave-propagation analysis for the long-term monitoring of supercritical geothermal systems

This study is focused on the modeling of the seismic response in a supercritical geothermal system subjected to a long-term cold and isothermal re-injection during 20 years of exploitation. The modeling aims to assess the usability of seismic-methods for monitoring supercritical geothermal reservoir. We use a synthetic data set for the analysis of the seismic properties of the reservoir: pressure and temperature evolution is derived from a previous numerical study of a two-dimensional supercritical reservoir with pure water as the pore fluid. We calculate the related variations in time of the seismic properties of the reservoir saturated-rock formation using the Burgers–Gassmann mechanical model to evaluate the effects of high temperature on the rock frame, and to account for fluid-properties variations. We observe that, at the analyzed pressure and temperature conditions, the seismic properties of the reservoir formation are mainly determined by the thermophysical characteristics of the pore fluid. During the long-term cold re-injection we observe a decrease in the compressional seismic velocity in the cooled area near the injection well, with a radius that increases in time. We use a 2D full-waveform propagation algorithm based on the Burgers–Gassmann model to calculate synthetic signals in a single-well acquisition layout, with a time step of half year. We observe that in the cool injection scenario the changes in the anomalous area can be seismically monitored, while in the case of isothermal injection, changes due to the re-injection are not seismically appreciable.Francesco Parisio acknowledges funding from the European Union’s Horizon 2020 Research and Innovation Program through the Marie Sklowdowska-Curie Individual Fellowship ARMISTICE, Spain under Grant Agreement No. 882733

Correlations of Seismic Velocities and Elastic Moduli with Temperature in Superhot and Enhanced Geothermal Systems

This paper presents correlations derived by linear regression analysis of seismic velocities VP and VS and elastic moduli EP and ES with temperature in Los Humeros superhot (SHGS) and Acoculco enhanced (EGS) geothermal systems at depths down to 3 km below the surface and temperatures up to approximately 400 °C. In Los Humeros, the seismic velocity models were derived from the inversion of legacy active seismic survey data acquired in 1998, as well as from passive seismic monitoring and ambient seismic noise interferometry carried out during 2017–2019 by the GEMex consortium. In the Acoculco EGS, ambient seismic noise data were used. Steady-state formation temperatures were re-evaluated during and after the end of the GEMex project using measurements provided as a courtesy of the Federal Electricity Commission of Mexico (CFE). The density data needed for the calculation of elastic moduli were provided by the GEMex consortium, as derived from the inversion of regional and local gravity surveys. The analysis indicated that statistically significant correlations of seismic parameters to temperature exist in the vertical direction, namely exponential in Los Humeros superhot and logarithmic in Acoculco EGS, but no correlation was evident in the horizontal direction. This result suggests an indirect relationship among the considered variables due to interdependence on other parameters, such as pressure and vapor saturation. As the analysis was performed using only data obtained from sensing-at-surface methods, without direct geophysical calibration at depth, a distributed fiber-optic seismic and temperature sensing system at both surface and downhole is proposed for active-source and passive seismic monitoring, and seismic-while-drilling by the drill-bit source is considered for reverse vertical seismic profile (RVSP) recording whenever possible for future high-temperature geothermal applications

Correlations of Seismic Velocities and Elastic Moduli with Temperature in Superhot and Enhanced Geothermal Systems

This paper presents correlations derived by linear regression analysis of seismic velocities VP and VS and elastic moduli EP and ES with temperature in Los Humeros superhot (SHGS) and Acoculco enhanced (EGS) geothermal systems at depths down to 3 km below the surface and temperatures up to approximately 400 °C. In Los Humeros, the seismic velocity models were derived from the inversion of legacy active seismic survey data acquired in 1998, as well as from passive seismic monitoring and ambient seismic noise interferometry carried out during 2017–2019 by the GEMex consortium. In the Acoculco EGS, ambient seismic noise data were used. Steady-state formation temperatures were re-evaluated during and after the end of the GEMex project using measurements provided as a courtesy of the Federal Electricity Commission of Mexico (CFE). The density data needed for the calculation of elastic moduli were provided by the GEMex consortium, as derived from the inversion of regional and local gravity surveys. The analysis indicated that statistically significant correlations of seismic parameters to temperature exist in the vertical direction, namely exponential in Los Humeros superhot and logarithmic in Acoculco EGS, but no correlation was evident in the horizontal direction. This result suggests an indirect relationship among the considered variables due to interdependence on other parameters, such as pressure and vapor saturation. As the analysis was performed using only data obtained from sensing-at-surface methods, without direct geophysical calibration at depth, a distributed fiber-optic seismic and temperature sensing system at both surface and downhole is proposed for active-source and passive seismic monitoring, and seismic-while-drilling by the drill-bit source is considered for reverse vertical seismic profile (RVSP) recording whenever possible for future high-temperature geothermal applications

STRUCTURAL AND FUNCTIONAL STUDIES OF STF76 FROM THE SULFOLOBUS ISLANDICUS PLASMID–VIRUS PSSVX: A NOVEL PECULIAR MEMBER OF THE WINGED HELIX–TURN–HELIX TRANSCRIPTION FACTOR FAMILY

Seven families of double-stranded DNA viruses have been identified, among which the Fuselloviridae and Rudiviridae are the most well-studied specimens and therefore represent model systems for detailed studies of archaeal virus biology. Two distinct genetic elements, SSV2 and pSSVx, belong to Fuselloviridae and coexist in the same Sulfolobus islandicus REY15/4 host, thus representing one of the few known two-virus systems in Archaea (1). pSSVx is a satellite virus that generates virus particles with the help of SSV2-associated packaging mechanisms. The transcriptional pattern of pSSVx undergoes a temporal variation of gene expression during its own life cycle, thus providing a good model for studying regulation of gene expression in Archaea (2). This genetic element encodes four TFs possibly implicated in the regulation of gene expression, i.e. ORF-c68, ORF51, ORF91 and ORF76. Among these, ORF76, here named Stf76 (Sulfolobus transcription factor 76 aminoacid protein), has homologs in almost all conjugative and cryptic plasmids from Sulfolobus (3), thus suggesting a relevant role for this protein in replication and/or maintenance of the plasmid. In this study, we have performed a detailed structural and functional characterization of Stf76. The corresponding gene has been cloned, expressed in Escherichia coli and the recombinant protein purified to homogeneity. To elucidate its interaction with the identified DNA operator sequence, analyses regarding its DNA binding capabilities by means of EMSA, circular dichroism, spectrofluorimetric and isothermal titration calorimetry experiments have been performed. Moreover, a structural study has been undertaken by Nuclear Magnetic Resonance spectroscopy leading to:(i) the solution structure of Stf76 based on CS-Rosetta approach, (ii) the characterization of the Stf76-DNA interaction by chemical shift perturbation analysis, (iii) a structural model describing the interaction of a single Stf76 monomer with its DNA operator. Altogether these results contribute to elucidate the regulatory mechanism underpinning the role of this protein (4). REFERENCES: 1. ARNOLD, H.P., ET AL.(1999) THE GENETIC ELEMENT PSSVX OF THE EXTREMELY THERMOPHILIC CRENARCHAEON SULFOLOBUS IS A HYBRID BETWEEN A PLASMID AND A VIRUS. MOL MICROBIOL, 34, 217-226. 2. CONTURSI, P., ET.AL. (2010) TRANSCRIPTION TERMINATION IN THE PLASMID/VIRUS HYBRID PSSVX FROM SULFOLOBUS ISLANDICUS. EXTREMOPHILES, 14, 453-463. 3. LIPPS, G. (2006) PLASMIDS AND VIRUSES OF THE THERMOACIDOPHILIC CRENARCHAEOTE SULFOLOBUS. EXTREMOPHILES, 10, 17-28. 4. CONTURSI P., FARINA B., PIRONE L. ET AL. NUCLEIC ACIDS RES. ACCEPTED 28 FEB. 2014

FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay

In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays