In silico analysis of membrane transport/permeability mechanisms

Lipid membranes are a fundamental component of living cells, mediating the physical separation of intracellular components from the external environment, as well as the different cellular organelles from cytoplasm. Transmembrane transport proteins confer permeability to lipid membranes, which is essential for nutrient translocation and energy metabolism. Crystallography of transmembrane proteins is a particularly challenging problem. Due to their natural localization and chemical properties only a limited number of structures are to date available at atomic resolution. In silico analysis can be successfully applied to address the structure and to propose testable models of transporters and pores and of their function. My PhD work focused on two main models: Pendrin (SLC26A4) and the Permeability Transition Pore (PTP). These two systems allowed me to investigate different membrane types and permeation mechanisms, i.e. the plasma membrane-specific anion exchange (SLC26A4) and the inner mitochondrial membrane (IMM) unselective PTP. Pendrin mutations are estimated to be the second most common genetic cause of human deafness, but a precise 3D structure of the protein is still missing. Aim of my work was to obviate the absence of structural information for pendrin transmembrane domain and to give a functional explanation for mutations collected in the MORL Deafness Variation Database. The human pendrin 3D model was inferred by homology with SLC26Dg and then validated analyzing the surface distribution of hydrophobic residues. The resulting high quality model was used to map 147 pathogenic human mutations. Three mutation clusters were found, while their localization suggested an innovative 14 transmembrane domain structure for pendrin. The nature of PTP has long remained a mystery. In 2013 Giorgio et. al. suggested dimers of F1FO (F)-ATP synthase to form the pore, however the exact PTP composition and how can a pore form from the energy-conserving enzyme is still matter of debate. PTP opening is triggered by an increased Ca2+ concentration in the mitochondrial matrix, and is favored by oxidative stress. To shed light on PTP function, I investigated the effect of Ca2+ binding to the Me2+ binding site of the F1 domain of F-ATP synthase through molecular dynamics (MD) simulations. A similar approach was also applied to the F-ATP synthase β subunit mutation T163S, which alters the relative affinity for Mg2+ and Ca2+. Experimental data show that Ca2+ binding stiffens the complex structure and that the T163S mutation induces resistance to PTP opening. Further, catalytic site rearrangement induced from different ion occupancy, as well as the mutation T163S, yields relevant variation of the interaction between F1 domain and OSCP subunit. I suggest that an unstructured loop between residues 82-131 of the β subunit transmits the structural rearrangement originated into catalytic site to the OSCP subunit and then to the inner membrane through the rigid lateral stalk. The critical role emerging for OSCP in the PTP regulation opens two parallel questions, i.e. (i) how the OSCP-mediated opening signal is transmitted to the trans-membrane region and (ii) what are the transmembrane PTP components. Variation in pore conductivity among species suggested that the putative pore-forming subunits may be different in different species. Sequence alignment was performed for all the subunits of F-ATP synthase, but we mainly focused on subunits e, g and b due to their localization in the complex and sequence conservation. Specific mutations affecting F-ATP synthase were collected and their functional effect is currently under analysis. In parallel, the presence and features of e, g and f subunits across eukaryotes was investigated by mean of phylogenetic analysis. Protein homologues of these specific subunits were found to be widespread in eukaryotes from yeast to plants while we found that Oomycetes lack subunits e and g and green algae subunit e. This observation suggest an ancient evolution for the F-ATP synthase dimerization subunits and possibly for the PTP. Further analysis and experimental validation are planned to clarify this aspect

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.Fil: Quaglia, Federica. Università di Padova; Italia. Consiglio Nazionale delle Ricerche; ItaliaFil: Mészáros, Bálint. European Molecular Biology Laboratory; AlemaniaFil: Salladini, Edoardo. Università di Padova; ItaliaFil: Hatos, András. Università di Padova; ItaliaFil: Pancsa, Rita. Research Centre for Natural Sciences; HungríaFil: Chemes, Lucia Beatriz. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Pajkos, Mátyás. Eötvös Loránd University; HungríaFil: Lazar, Tamas. Vlaams Instituut voor Biotechnology; Hungría. Vrije Unviversiteit Brussel; BélgicaFil: Peña Díaz, Samuel. Universitat Autònoma de Barcelona; EspañaFil: Santos, Jaime. Universitat Autònoma de Barcelona; EspañaFil: Ács, Veronika. Research Centre for Natural Sciences; HungríaFil: Farahi, Nazanin. Vlaams Instituut voor Biotechnology; Bélgica. Vrije Unviversiteit Brussel; BélgicaFil: Fichó, Erzsébet. Research Centre for Natural Sciences; HungríaFil: Aspromonte, Maria Cristina. Università di Padova; Italia. Città della Speranza Pediatric Research Institute; ItaliaFil: Bassot, Claudio. Stockholms Universitet; SueciaFil: Chasapi, Anastasia. Centre for Research & Technology Hellas; GreciaFil: Davey, Norman E.. Chester Beatty Laboratories; Reino UnidoFil: Davidović, Radoslav. University of Belgrade; SerbiaFil: Laszlo Holland, Alicia Verónica. European Molecular Biology Laboratory; Alemania. Research Centre for Natural Sciences; HungríaFil: Elofsson, Arne. Stockholms Universitet; SueciaFil: Erdős, Gábor. Eötvös Loránd University; HungríaFil: Gaudet, Pascale. Swiss Institute of Bioinformatics; SuizaFil: Giglio, Michelle. University of Maryland School of Medicine; Estados UnidosFil: Glavina, Juliana. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Iserte, Javier Alonso. 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: Iglesias, Valentín. Universitat Autònoma de Barcelona; EspañaFil: Kálmán, Zsófia. Pázmány Péter Catholic University; HungríaFil: Lambrughi, Matteo. Danish Cancer Society Research Center; DinamarcaFil: Leonardi, Emanuela. Università di Padova; Italia. Pediatric Research Institute Città della Speranza; ItaliaFil: Rodriguez Sawicki, Luciana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

DisProt: intrinsic protein disorder annotation in 2020

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the ‘dark’ proteome

Critical assessment of protein intrinsic disorder prediction

Abstract: Intrinsically disordered proteins, defying the traditional protein structure–function paradigm, are a challenge to study experimentally. Because a large part of our knowledge rests on computational predictions, it is crucial that their accuracy is high. The Critical Assessment of protein Intrinsic Disorder prediction (CAID) experiment was established as a community-based blind test to determine the state of the art in prediction of intrinsically disordered regions and the subset of residues involved in binding. A total of 43 methods were evaluated on a dataset of 646 proteins from DisProt. The best methods use deep learning techniques and notably outperform physicochemical methods. The top disorder predictor has Fmax = 0.483 on the full dataset and Fmax = 0.792 following filtering out of bona fide structured regions. Disordered binding regions remain hard to predict, with Fmax = 0.231. Interestingly, computing times among methods can vary by up to four orders of magnitude

In silico analysis of membrane transport/permeability mechanisms

Lipid membranes are a fundamental component of living cells, mediating the physical separation of intracellular components from the external environment, as well as the different cellular organelles from cytoplasm. Transmembrane transport proteins confer permeability to lipid membranes, which is essential for nutrient translocation and energy metabolism. Crystallography of transmembrane proteins is a particularly challenging problem. Due to their natural localization and chemical properties only a limited number of structures are to date available at atomic resolution. In silico analysis can be successfully applied to address the structure and to propose testable models of transporters and pores and of their function. My PhD work focused on two main models: Pendrin (SLC26A4) and the Permeability Transition Pore (PTP). These two systems allowed me to investigate different membrane types and permeation mechanisms, i.e. the plasma membrane-specific anion exchange (SLC26A4) and the inner mitochondrial membrane (IMM) unselective PTP. Pendrin mutations are estimated to be the second most common genetic cause of human deafness, but a precise 3D structure of the protein is still missing. Aim of my work was to obviate the absence of structural information for pendrin transmembrane domain and to give a functional explanation for mutations collected in the MORL Deafness Variation Database. The human pendrin 3D model was inferred by homology with SLC26Dg and then validated analyzing the surface distribution of hydrophobic residues. The resulting high quality model was used to map 147 pathogenic human mutations. Three mutation clusters were found, while their localization suggested an innovative 14 transmembrane domain structure for pendrin. The nature of PTP has long remained a mystery. In 2013 Giorgio et. al. suggested dimers of F1FO (F)-ATP synthase to form the pore, however the exact PTP composition and how can a pore form from the energy-conserving enzyme is still matter of debate. PTP opening is triggered by an increased Ca2+ concentration in the mitochondrial matrix, and is favored by oxidative stress. To shed light on PTP function, I investigated the effect of Ca2+ binding to the Me2+ binding site of the F1 domain of F-ATP synthase through molecular dynamics (MD) simulations. A similar approach was also applied to the F-ATP synthase β subunit mutation T163S, which alters the relative affinity for Mg2+ and Ca2+. Experimental data show that Ca2+ binding stiffens the complex structure and that the T163S mutation induces resistance to PTP opening. Further, catalytic site rearrangement induced from different ion occupancy, as well as the mutation T163S, yields relevant variation of the interaction between F1 domain and OSCP subunit. I suggest that an unstructured loop between residues 82-131 of the β subunit transmits the structural rearrangement originated into catalytic site to the OSCP subunit and then to the inner membrane through the rigid lateral stalk. The critical role emerging for OSCP in the PTP regulation opens two parallel questions, i.e. (i) how the OSCP-mediated opening signal is transmitted to the trans-membrane region and (ii) what are the transmembrane PTP components. Variation in pore conductivity among species suggested that the putative pore-forming subunits may be different in different species. Sequence alignment was performed for all the subunits of F-ATP synthase, but we mainly focused on subunits e, g and b due to their localization in the complex and sequence conservation. Specific mutations affecting F-ATP synthase were collected and their functional effect is currently under analysis. In parallel, the presence and features of e, g and f subunits across eukaryotes was investigated by mean of phylogenetic analysis. Protein homologues of these specific subunits were found to be widespread in eukaryotes from yeast to plants while we found that Oomycetes lack subunits e and g and green algae subunit e. This observation suggest an ancient evolution for the F-ATP synthase dimerization subunits and possibly for the PTP. Further analysis and experimental validation are planned to clarify this aspect.Le membrane lipidiche sono una componente fondamentale delle cellule viventi, separano fisicamente le componenti intracellulari dall’ambiente esterno e i diversi organelli del citoplasma. Le proteine di trasporto conferiscono permeabilità alle membrane lipidiche, proprietà essenziale per la traslocazione di nutrienti e la conservazione dell’energia. La cristallografia di proteine transmembrana è problematica a causa della loro localizzazione e proprietà chimiche, e solo un numero piuttosto ridotto di strutture è disponibile. L’analisi in silico può essere applicata con successo per investigare le strutture e il funzionamento proporre modelli testabili di trasportatori e delle loro funzioni. Il lavoro del mio dottorato sì è focalizzato su due modelli: la pendrina (SLC26A4) e il poro di transizione di permeabilità (PTP). Questi due sistemi proteici mi hanno permesso di studiare due differenti tipi di membrana e meccanismi di permeabilità: la membrana plasmatica con scambio specifico di anioni (SLC26A4) e la membrana interna mitocondriale con la permeabilità non selettiva mitocondriale (PTP). Le mutazioni della pendrina sono stimate essere la seconda causa genetica più comune della sordità umana, ma la struttura della proteina non è stata ancora determinata. Scopo del mio lavoro è stato quello di sopperire all’assenza di informazioni strutturali per il dominio transmembrana della pendrina e di dare una spiegazione funzionale per le mutazioni raccolte nel MORL Deafness Variation Database. Il modello 3D della pendrina è basato sull’omologia con SLC26Dg (3) ed è stato validato analizzando la distribuzione sulla superfice dei residui idrofobici. L’alta qualità risultante dal modello è stata usata per mappare 147 mutazioni patologiche umane. Tre cluster di mutazioni sono stati trovati e la loro localizzazione suggerisce per pendrina un innovativa struttura a 14 domini transmembrana. Anche la natura del PTP è rimasta a lungo misteriosa. Nel 2013 Giorgio et al. hanno suggerito che i dimeri di F1FO (F)-ATP sintasi formino il poro, tuttavia l’esatta composizione e il modo in cui il poro di transizione si possa formare è ancora materia di dibattito. L’apertura del PTP è innescata da un aumento della concentrazione di Ca2+ nella matrice mitocondriale ed è favorita dallo stress ossidativo. Per fare luce sul funzionamento del PTP ho studiato l’effetto del legame del Ca2+ al sito per i cationi divalenti (Me2+) nel dominio F1 attraverso la dinamica molecolare (MD). Un approccio simile è stato anche applicato alla mutazione T163S, che fa variare l’affinità relativa per Mg2+ e Ca2+. I dati sperimentali mostrano come la mutazione induca resistenza all’apertura del PTP. La MD ha dimostrato come il legame del Ca2+ irrigidisca la struttura del complesso. Il riarrangiamento del sito catalitico indotto dai differenti ioni che lo occupano, così come la mutazione T163S, causa rilevanti variazioni delle interazioni tra il dominio F1 e la subunità OSCP. Suggerisco che un loop non strutturato tra i residui 82-131 della subunità β trasmetta il riarrangiamento strutturale originato nel sito catalitico a OSCP e quindi alla membrana interna attraverso il rigido stalk laterale. Il ruolo critico che emerge per OSCP nella regolazione del PTP apre due domande collegate: (i) come il segnale di apertura mediato da OSCP venga trasmesso alla regione trans-membrana e (ii) quali siano i componenti transmembrana del PTP. Le variazioni di conduttanza del poro osservate in specie diverse suggeriscono che le subunità che formano il canale debbano avere delle differenze significative. E’ stato prodotto un allineamento di sequenze per tutte le subunità della F-ATP sintasi. I risultati preliminari ci hanno spinto a focalizzarci sulle subunità e, g e b a causa della loro localizzazione e conservazione di sequenza. Basandomi sugli allineamenti multipli ho suggerito mutazioni puntiformi per testare l’importanza di specifici residui ai fini dell’apertura del poro. In parallelo la presenza delle subunità e e g tra gli eucarioti è stata indagata attraverso un analisi filogenetica. Proteine omologhe di queste specifiche subunità sono presenti in tutti gli eucarioti: dai lieviti alle piante, tuttavia gli Oomiceti sono risultati mancanti delle subunità e e g e le alghe verdi della subunità e. Questi risultati suggeriscono un’origine antica per le subunità di dimerizzazione della F-ATP sintasi e probabilmente anche del PTP. Per chiarire questo aspetto saranno necessarie ulteriori analisi e verifiche sperimentali

Why do eukaryotic proteins contain more intrinsically disordered regions?

Intrinsic disorder is more abundant in eukaryotic than prokaryotic proteins. Methods predicting intrinsic disorder are based on the amino acid sequence of a protein. Therefore, there must exist an underlying difference in the sequences between eukaryotic and prokaryotic proteins causing the (predicted) difference in intrinsic disorder. By comparing proteins, from complete eukaryotic and prokaryotic proteomes, we show that the difference in intrinsic disorder emerges from the linker regions connecting Pfam domains. Eukaryotic proteins have more extended linker regions, and in addition, the eukaryotic linkers are significantly more disordered, 38% vs. 12-16% disordered residues. Next, we examined the underlying reason for the increase in disorder in eukaryotic linkers, and we found that the changes in abundance of only three amino acids cause the increase. Eukaryotic proteins contain 8.6% serine; while prokaryotic proteins have 6.5%, eukaryotic proteins also contain 5.4% proline and 5.3% isoleucine compared with 4.0% proline and ≈ 7.5% isoleucine in the prokaryotes. All these three differences contribute to the increased disorder in eukaryotic proteins. It is tempting to speculate that the increase in serine frequencies in eukaryotes is related to regulation by kinases, but direct evidence for this is lacking. The differences are observed in all phyla, protein families, structural regions and type of protein but are most pronounced in disordered and linker regions. The observation that differences in the abundance of three amino acids cause the difference in disorder between eukaryotic and prokaryotic proteins raises the question: Are amino acid frequencies different in eukaryotic linkers because the linkers are more disordered or do the differences cause the increased disorder

Calcium and regulation of the mitochondrial permeability transition

Recent years have seen renewed interest in the permeability transition pore, a high conductance channel responsible for permeabilization of the inner mitochondrial membrane, a process that leads to depolarization and Ca2+ release. Transient openings may be involved in physiological Ca2+ homeostasis while long-lasting openings may trigger and/or execute cell death. In this review we specifically focus (i) on the hypothesis that the PTP forms from the F-ATP synthase and (ii) on the mechanisms through which Ca2+ can reversibly switch this energy-conserving nanomachine into an energy-dissipating device. (C) 2017 Elsevier Ltd. All rights reserved