Loss of stability and unfolding cooperativity in hPGK1 upon gradual structural perturbation of its N‑terminal domain hydrophobic core

Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype–phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00Junta de Andalucia P18-RT-2413ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities B-BIO-84-UGR20Department of Science & Technology (India)Science Engineering Research Board (SERB), India MTR/2019/000392Horizon 2020 EU_FT-ICR_MS project 731077EU/MEYS projects BioCeV CZ.1.05/1.1.00/02.0109CIISB LM201812

Exploring Highly Conserved Regions of SARS-CoV-2 Spike S2 Subunit as Targets for Fusion Inhibition Using Chimeric Proteins

Since the beginning of the COVID-19 pandemic, considerable efforts have been made to develop protective vaccines against SARS-CoV-2 infection. However, immunity tends to decline within a few months, and new virus variants are emerging with increased transmissibility and capacity to evade natural or vaccine-acquired immunity. Therefore, new robust strategies are needed to combat SARS-CoV-2 infection. The viral spike composed of S1 and S2 subunits mediates viral attachment and membrane fusion to infect the host cell. In this process, interaction between the highly conserved heptad repeat 1 and 2 regions (HR1 and HR2) of S2 is crucial and for this reason; these regions are promising targets to fight SARS-CoV-2. Here, we describe the design and characterization of chimeric proteins that structurally imitate the S2 HR1 region in a trimeric coiled-coil conformation. We biophysically characterized the proteins and determined their capacity to bind the HR2 region, as well as their inhibitory activity of SARS-CoV-2 infection in vitro. HR1 mimetic proteins showed conformational heterogeneity and a propensity to form oligomers. Moreover, their structure is composed of subdomains with varied stability. Interestingly, the full HR1 proteins showed high affinity for HR2-derived peptides and SARS-CoV-2 inhibitory activity, whereas smaller proteins mimicking HR1 subdomains had a decreased affinity for their complementary HR2 region and did not inhibit the virus. The results provide insight into effective strategies to create mimetic proteins with broad inhibitory activity and therapeutic potential against SARS-CoV-2.Junta de AndaluciaSpain's State Research Agency CV20.26565 ERDF/ESF PID2019.107515RB.C21ANRSFrench National Research Agency (ANR) French National Research Agency (ANR)EHVA ANR-10-LABX-77 68103

Folic acid supplementation: some practical aspects

Since 1956, when Harman first postulated the free radical theory of aging, numerous studies have been carried out to test the protective action of antioxidants. One of these protective compounds used in antioxidant therapy is folic acid (FA). Folate deficiency can lead to several pathologies and its protective role is very well known. Because the negative effects of the synthetic form on the metabolism of folates and the controversy about the role of folic acid in cancer, the question is whether or not folic acid is good for everyone. In this paper we summarize some aspects of the biochemistry of folic acid and we show some precautions that should be taken into consideration when supplementing with this compound

Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up NewWays of Inhibiting HIV-1 Fusion

Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23052794/s1.Acknowledgments: M.C.-M. acknowledges a grant from Youth Employment Operative Program of the Andalusia Government and the European Social Fund (ESF). S.C. acknowledges an exchange studentship from the ERASMUS+ program of the European Union. The results shown are included as part of M.C.-M. doctoral thesis.Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region.Grants BIO2016-76640-R and PID2019.107515RB.C21 from the Spain’s State Research AgencySRA/10.13039/501100011033, co-funded by ERDF/ESF, “A way to make Europe”/“Investing in your future

Naturally-Occurring Rare Mutations Cause Mild to Catastrophic Effects in the Multifunctional and Cancer-Associated NQO1 Protein

The functional and pathological implications of the enormous genetic diversity of the human genome are mostly unknown, primarily due to our unability to predict pathogenicity in a high-throughput manner. In this work, we characterized the phenotypic consequences of eight naturally-occurring missense variants on the multifunctional and disease-associated NQO1 protein using biophysical and structural analyses on several protein traits. Mutations found in both exome-sequencing initiatives and in cancer cell lines cause mild to catastrophic e ects on NQO1 stability and function. Importantly, some mutations perturb functional features located structurally far from the mutated site. These e ects are well rationalized by considering the nature of the mutation, its location in protein structure and the local stability of its environment. Using a set of 22 experimentally characterized mutations in NQO1, we generated experimental scores for pathogenicity that correlate reasonably well with bioinformatic scores derived from a set of commonly used algorithms, although the latter fail to semiquantitatively predict the phenotypic alterations caused by a significant fraction of mutations individually. These results provide insight into the propagation of mutational e ects on multifunctional proteins, the implementation of in silico approaches for establishing genotype-phenotype correlations and the molecular determinants underlying loss-of-function in genetic diseases.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00 SAF2015-69796Junta de Andalucía P18-RT-241

Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity.

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.This work was supported by grants CV20.26565 from the Consejería de Economía y Conocimiento, Junta de Andalucía (Spain), PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033), and co-funded by ERDF/ESF, “A way to make Europe”/“Investing in your future. The work performed in C.M.’s laboratory was supported by grants from ANRS (Agence Nationale de Recherches sur le SIDA et les h´epatites virales), the Investissements d’Avenir program managed by the ANR under reference ANR-10-LABX-77 and EHVA (No. 681032, Horizon 2020). Work in S.B.’s laboratory was supported by grants from the Agence Nationale de la Recherche (ANR) (ANR-11-LABX-0070_TRANSPLANTEX), the INSERM (UMR_S1109), the Institut Universitaire de France (IUF), all the University of Strasbourg (IDEX UNISTRA), the European Regional Development Fund (European Union) INTERREG V program (project no. 3.2 TRIDIAG) and MSD-Avenir grant AUTOGEN

Improving electrochemical hydrogen evolution of Ag@CN nanocomposites by synergistic effects with α-rich proteins

A graphitic carbon nitride nanostructure has been successfully functionalized by incorporation of different silver contents and subsequent modification with an α-rich protein, namely hemoglobin. Mechanochemistry has been employed, as an efficient and sustainable procedure, for the incorporation of the protein. A complete characterization analysis has been performed following a multitechnique approach. Particularly, XPS data exhibited considerable differences in the C 1s region for the Hb/xAg@CN, ensuring the successful protein anchorage on the surface of the graphitic carbon nitride-based materials. The as-synthesized nanomaterials delivered impressive performance toward hydrogen evolution reactions with an overpotential of 79 mV at a current density of 10 mA/cm2 for Hb/20Ag@CN nanohybrids, which is comparable with the most efficient HER electrocatalysts reported in the literature. The outstanding HER properties were associated with the unique synergistic interactions, quantitatively measured, between AgNPs, Hb tertiary architecture, and the graphitic carbon nitride networks

Treatment with mesenchymal stem cells in an animal model of parkinson´s disease

Consejería de Economía, Innovación y Ciencia, Junta de Andalucía P10-CTS-649

Probing vulnerability of the gp41 C-terminal heptad repeat as target for miniprotein HIV inhibitors

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.Spanish Ministry of Economy and Competitiveness (grant: BIO2016-76640-R), ANRS and the Vaccine Research Institute for the Investissements d'Avenir program to C.M. and by the European Fund for Research and Development from the European Union.Departamento de Química Física, Facultad de Ciencias, Universidad de Granada. Grupo FQM-171 "Biofísica y Biotecnología Molecular