Unraveling the functional dark matter through global metagenomics

30 pages, 4 figures, 1 table, supplementary information https://doi.org/10.1038/s41586-023-06583-7.-- Data availability: All of the analysed datasets along with their corresponding sequences are available from the IMG system (http://img.jgi.doe.gov/). A list of the datasets used in this study is provided in Supplementary Data 8. All data from the protein clusters, including sequences, multiple alignments, HMM profiles, 3D structure models, and taxonomic and ecosystem annotation, are available through NMPFamsDB, publicly accessible at www.nmpfamsdb.org. The 3D models are also available at ModelArchive under accession code ma-nmpfamsdb.-- Code availability: Sequence analysis was performed using Tantan (https://gitlab.com/mcfrith/tantan), BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), LAST (https://gitlab.com/mcfrith/last), HMMER (http://hmmer.org/) and HH-suite3 (https://github.com/soedinglab/hh-suite). Clustering was performed using HipMCL (https://bitbucket.org/azadcse/hipmcl/src/master/). Additional taxonomic annotation was performed using Whokaryote (https://github.com/LottePronk/whokaryote), EukRep (https://github.com/patrickwest/EukRep), DeepVirFinder (https://github.com/jessieren/DeepVirFinder) and MMseqs2 (https://github.com/soedinglab/MMseqs2). 3D modelling was performed using AlphaFold2 (https://github.com/deepmind/alphafold) and TrRosetta2 (https://github.com/RosettaCommons/trRosetta2). Structural alignments were performed using TMalign (https://zhanggroup.org/TM-align/) and MMalign (https://zhanggroup.org/MM-align/). All custom scripts used for the generation and analysis of the data are available at Zenodo (https://doi.org/10.5281/zenodo.8097349)Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matterWith the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

Gi/o-protein coupled receptors in the aging brain

Cells translate extracellular signals to regulate processes such as differentiation, metabolism and proliferation, via transmembranar receptors. G protein-coupled receptors (GPCRs) belong to the largest family of transmembrane receptors, with over 800 members in the human species. Given the variety of key physiological functions regulated by GPCRs, these are main targets of existing drugs. During normal aging, alterations in the expression and activity of GPCRs have been observed. The central nervous system (CNS) is particularly affected by these alterations, which results in decreased brain functions, impaired neuroregeneration, and increased vulnerability to neuropathologies, such as Alzheimer's and Parkinson diseases. GPCRs signal via heterotrimeric G proteins, such as Go, the most abundant heterotrimeric G protein in CNS. We here review age-induced effects of GPCR signaling via the Gi/o subfamily at the CNS. During the aging process, a reduction in protein density is observed for almost half of the Gi/o-coupled GPCRs, particularly in age-vulnerable regions such as the frontal cortex, hippocampus, substantia nigra and striatum. Gi/o levels also tend to decrease with aging, particularly in regions such as the frontal cortex. Alterations in the expression and activity of GPCRs and coupled G proteins result from altered proteostasis, peroxidation of membranar lipids and age-associated neuronal degeneration and death, and have impact on aging hallmarks and age-related neuropathologies. Further, due to oligomerization of GPCRs at the membrane and their cooperative signaling, down-regulation of a specific Gi/o-coupled GPCR may affect signaling and drug targeting of other types/subtypes of GPCRs with which it dimerizes. Gi/o-coupled GPCRs receptorsomes are thus the focus of more effective therapeutic drugs aiming to prevent or revert the decline in brain functions and increased risk of neuropathologies at advanced ages.This work was supported by Fundação para a Ciência e Tecnologia, Centro 2020 and Portugal 2020, the COMPETE program, QREN, and the European Union (FEDER program) via the GoBack project (PTDC/CVT-CVT/32261/2017), the pAGE program (Centro-01-0145-FEDER-000003), and Institute for Biomedicine iBiMED (UID/BIM/04501/2013; UID/BIM/04501/2019).publishe

Computational studies of protein - protein Interactions in transmembrane proteins

Biological membranes are crucial components of all cells. They are bilayer mixtures composed by various types of lipids and a large number of membrane proteins. The latter, based on their position with respect to the membrane plane, are classified into transmembrane, peripheral membrane and lipid-anchored proteins. Transmembrane proteins constitute approximately 25-30% of known proteomes and control a wide range of cell functions, ranging from signal transduction and substrate transport to maintaining cell integrity and the regulation of gene expression, cell growth and cell death. As a result, transmembrane proteins have been implicated in a wide range of diseases and constitute prime targets in drug design. An important part of transmembrane protein functionality is their capability to form protein-protein interactions. The formation of supramolecular protein-protein complexes in the membrane plane, either between two or more transmembrane proteins or between transmembrane and non-transmembrane proteins, is an integral part of their canonical function. At the same time, a large number of transmembrane protein complexes have been implicated with several diseases. Despite their importance, however, the experimental study of transmembrane proteins and their interactions is not straightforward. The aim of this dissertation is the computational study of protein-protein interactions in biological membranes and transmembrane proteins. Towards this end, an extensive study of protein – protein interactions was conducted for several transmembrane proteins, both at the structural level, through Molecular Modeling, Molecular Dynamics simulations and Free Energy calculations, and in a system-wide approach, through the application of concepts from Network Theory. Alongside protein – protein interactions, the structural and dynamic aspects of the membrane environment were also investigated, in order to identify and evaluate the structural determinants that govern the lipid bilayer’s influences upon transmembrane protein structure and biomolecular interactions. Finally, during the course of this study, a number of publicly available, computational tools were developed, which can further aid in the study of biological membranes, transmembrane proteins and their interactions. The aforementioned studies were conducted both for transmembrane proteins located at the plasma membrane of eukaryotic cells, such as G-protein coupled receptors (GPCRs) and Receptor Tyrosine Kinases (RTKs), and for proteins found in other cell components, such as the Outer Membranes of Gram-negative bacteria and transmembrane β-barrels, as well as the double membrane system of the Nuclear Envelope and its proteins. The results of this dissertation can be applicable in the further study of protein-protein interactions in transmembrane proteins, both through experimental and through computational approaches.Οι βιολογικές μεμβράνες αποτελούν απαραίτητα συστατικά όλων των κυττάρων. Είναι συνεχείς διπλοστιβάδες αποτελούμενες από διάφορα είδη λιπιδίων, στην επιφάνεια και στο εσωτερικό των οποίων ενσωματώνονται μεμβρανικές πρωτεΐνες. Οι τελευταίες διακρίνονται, ανάλογα με την τοπολογία τους σε σχέση με τη μεμβράνη, σε διαμεμβρανικές, περιφερειακές και αγκυροβολημένες πρωτεΐνες. Οι διαμεμβρανικές πρωτεΐνες υπολογίζεται ότι συνιστούν το 25-30% των γνωστών πρωτεωμάτων και ρυθμίζουν μεγάλο εύρος λειτουργιών στο κύτταρο, από τη μεταγωγή σήματος και τη μεταφορά ουσιών ως τη μηχανική στήριξη του κυττάρου, τη ρύθμιση της γονιδιακής έκφρασης, τον κυτταρικό πολλαπλασιασμό και τον κυτταρικό θάνατο. Έτσι, έχουν εμπλακεί σε μεγάλο αριθμό ασθενειών και αποτελούν κρίσιμους στόχους για το σχεδιασμό φαρμάκων. Σημαντικό κομμάτι της λειτουργίας των διαμεμβρανικών πρωτεϊνών αποτελεί η ικανότητά τους να σχηματίζουν αλληλεπιδράσεις με άλλες πρωτεΐνες. Ο σχηματισμός υπερμοριακών συμπλόκων στο επίπεδο της μεμβράνης, τόσο ανάμεσα σε δύο ή περισσότερες διαμεμβρανικές πρωτεΐνες όσο και ανάμεσα σε διαμεμβρανικές και μη διαμεμβρανικές πρωτεΐνες, διαδραματίζει σημαντικό ρόλο στη λειτουργία τους. Επιπρόσθετα, πολλά υπερμοριακά συμπλέγματα διαμεμβρανικών πρωτεϊνών έχουν συσχετιστεί με μεγάλη ποικιλία ασθενειών. Παρά τη σημασία τους, ωστόσο, η πειραματική μελέτη των αλληλεπιδράσεων πρωτεϊνών-πρωτεϊνών στις διαμεμβρανικές πρωτεΐνες είναι δύσκολη. Αντικείμενο της παρούσας διδακτορικής διατριβής είναι η μελέτη των αλληλεπιδράσεων πρωτεϊνών-πρωτεϊνών σε διαμεμβρανικές πρωτεΐνες με τη χρήση υπολογιστικών μεθόδων. Στα πλαίσια της διδακτορικής διατριβής πραγματοποιήθηκε μελέτη αυτών των αλληλεπιδράσεων τόσο σε επίπεδο δομής, μέσα από μεθόδους Μοριακής Προτυποποίησης, προσομοιώσεις Μοριακής Δυναμικής και υπολογισμούς Ελεύθερης Ενέργειας, όσο και σε επίπεδο συστήματος, με την εφαρμογή μεθόδων από το πεδίο της Θεωρίας Δικτύων. Παράλληλα με τη μελέτη των ίδιων των αλληλεπιδράσεων πρωτεϊνών-πρωτεϊνών, διερευνήθηκαν και τα χαρακτηριστικά του μεμβρανικού περιβάλλοντος, με στόχο την αποσαφήνιση του μηχανισμού με τον οποίο η λιπιδική διπλοστιβάδα επηρεάζει τη δομή και τις αλληλεπιδράσεις των διαμεμβρανικών πρωτεϊνών. Τέλος, στα πλαίσια της διδακτορικής διατριβής αναπτύχθηκαν μια σειρά από υπολογιστικά εργαλεία για την πρόγνωση και τη μελέτη της δομής, της λειτουργίας και των αλληλεπιδράσεων των βιολογικών μεμβρανών και των διαμεμβρανικών πρωτεϊνών. Οι παραπάνω μελέτες πραγματοποιήθηκαν τόσο για πρωτεΐνες της κυτταρικής μεμβράνης των ευκαρυωτικών κυττάρων, όπως οι Συζευγμένοι με G-πρωτεΐνες υποδοχείς (GPCRs) και οι υποδοχείς με ενεργότητα τυροσινικής κινάσης (RTKs) όσο και για πρωτεΐνες που εντοπίζονται σε άλλα υποκυτταρικά διαμερίσματα, όπως οι Εξωτερικές Μέμβράνες των αρνητικών κατά Gram βακτηρίων και τα διαμεμβρανικά β-βαρέλια, καθώς και η διπλή μεμβράνη του πυρηνικού φάκελου και οι πρωτεΐνες της. Τα αποτελέσματα της παρούσας διδακτορικής διατριβής μπορούν να φανούν χρήσιμα στην περαιτέρω μελέτη των πρωτεϊνικών αλληλεπιδράσεων στις διαμεμβρανικές πρωτεΐνες, τόσο με πειραματικές όσο και με υπολογιστικές μεθόδους

Υπολογιστικές Μελέτες Αλληλεπιδράσεων Πρωτεϊνών - Πρωτεϊνών σε Διαμεμβρανικές Πρωτεΐνες

Οι βιολογικές μεμβράνες αποτελούν απαραίτητα συστατικά όλων των κυττάρων. Είναι συνεχείς διπλοστιβάδες αποτελούμενες από διάφορα είδη λιπιδίων, στην επιφάνεια και στο εσωτερικό των οποίων ενσωματώνονται μεμβρανικές πρωτεΐνες. Οι τελευταίες διακρίνονται, ανάλογα με την τοπολογία τους σε σχέση με τη μεμβράνη, σε διαμεμβρανικές, περιφερειακές και αγκυροβολημένες πρωτεΐνες. Οι διαμεμβρανικές πρωτεΐνες υπολογίζεται ότι συνιστούν το 25-30% των γνωστών πρωτεωμάτων και ρυθμίζουν μεγάλο εύρος λειτουργιών στο κύτταρο, από τη μεταγωγή σήματος και τη μεταφορά ουσιών ως τη μηχανική στήριξη του κυττάρου, τη ρύθμιση της γονιδιακής έκφρασης, τον κυτταρικό πολλαπλασιασμό και τον κυτταρικό θάνατο. Έτσι, έχουν εμπλακεί σε μεγάλο αριθμό ασθενειών και αποτελούν κρίσιμους στόχους για το σχεδιασμό φαρμάκων. Σημαντικό κομμάτι της λειτουργίας των διαμεμβρανικών πρωτεϊνών αποτελεί η ικανότητά τους να σχηματίζουν αλληλεπιδράσεις με άλλες πρωτεΐνες. Ο σχηματισμός υπερμοριακών συμπλόκων στο επίπεδο της μεμβράνης, τόσο ανάμεσα σε δύο ή περισσότερες διαμεμβρανικές πρωτεΐνες όσο και ανάμεσα σε διαμεμβρανικές και μη διαμεμβρανικές πρωτεΐνες, διαδραματίζει σημαντικό ρόλο στη λειτουργία τους. Επιπρόσθετα, πολλά υπερμοριακά συμπλέγματα διαμεμβρανικών πρωτεϊνών έχουν συσχετιστεί με μεγάλη ποικιλία ασθενειών. Παρά τη σημασία τους, ωστόσο, η πειραματική μελέτη των αλληλεπιδράσεων πρωτεϊνών-πρωτεϊνών στις διαμεμβρανικές πρωτεΐνες είναι δύσκολη. Αντικείμενο της παρούσας διδακτορικής διατριβής είναι η μελέτη των αλληλεπιδράσεων πρωτεϊνών-πρωτεϊνών σε διαμεμβρανικές πρωτεΐνες με τη χρήση υπολογιστικών μεθόδων. Στα πλαίσια της διδακτορικής διατριβής πραγματοποιήθηκε μελέτη αυτών των αλληλεπιδράσεων τόσο σε επίπεδο δομής, μέσα από μεθόδους Μοριακής Προτυποποίησης, προσομοιώσεις Μοριακής Δυναμικής και υπολογισμούς Ελεύθερης Ενέργειας, όσο και σε επίπεδο συστήματος, με την εφαρμογή μεθόδων από το πεδίο της Θεωρίας Δικτύων. Παράλληλα με τη μελέτη των ίδιων των αλληλεπιδράσεων πρωτεϊνών-πρωτεϊνών, διερευνήθηκαν και τα χαρακτηριστικά του μεμβρανικού περιβάλλοντος, με στόχο την αποσαφήνιση του μηχανισμού με τον οποίο η λιπιδική διπλοστιβάδα επηρεάζει τη δομή και τις αλληλεπιδράσεις των διαμεμβρανικών πρωτεϊνών. Τέλος, στα πλαίσια της διδακτορικής διατριβής αναπτύχθηκαν μια σειρά από υπολογιστικά εργαλεία για την πρόγνωση και τη μελέτη της δομής, της λειτουργίας και των αλληλεπιδράσεων των βιολογικών μεμβρανών και των διαμεμβρανικών πρωτεϊνών. Οι παραπάνω μελέτες πραγματοποιήθηκαν τόσο για πρωτεΐνες της κυτταρικής μεμβράνης των ευκαρυωτικών κυττάρων, όπως οι Συζευγμένοι με G-πρωτεΐνες υποδοχείς (GPCRs) και οι υποδοχείς με ενεργότητα τυροσινικής κινάσης (RTKs) όσο και για πρωτεΐνες που εντοπίζονται σε άλλα υποκυτταρικά διαμερίσματα, όπως οι Εξωτερικές Μέμβράνες των αρνητικών κατά Gram βακτηρίων και τα διαμεμβρανικά β-βαρέλια, καθώς και η διπλή μεμβράνη του πυρηνικού φάκελου και οι πρωτεΐνες της. Τα αποτελέσματα της παρούσας διδακτορικής διατριβής μπορούν να φανούν χρήσιμα στην περαιτέρω μελέτη των πρωτεϊνικών αλληλεπιδράσεων στις διαμεμβρανικές πρωτεΐνες, τόσο με πειραματικές όσο και με υπολογιστικές μεθόδους.Biological membranes are crucial components of all cells. They are bilayer mixtures composed by various types of lipids and a large number of membrane proteins. The latter, based on their position with respect to the membrane plane, are classified into transmembrane, peripheral membrane and lipid-anchored proteins. Transmembrane proteins constitute approximately 25-30% of known proteomes and control a wide range of cell functions, ranging from signal transduction and substrate transport to maintaining cell integrity and the regulation of gene expression, cell growth and cell death. As a result, transmembrane proteins have been implicated in a wide range of diseases and constitute prime targets in drug design. An important part of transmembrane protein functionality is their capability to form protein-protein interactions. The formation of supramolecular protein-protein complexes in the membrane plane, either between two or more transmembrane proteins or between transmembrane and non-transmembrane proteins, is an integral part of their canonical function. At the same time, a large number of transmembrane protein complexes have been implicated with several diseases. Despite their importance, however, the experimental study of transmembrane proteins and their interactions is not straightforward. The aim of this dissertation is the computational study of protein-protein interactions in biological membranes and transmembrane proteins. Towards this end, an extensive study of protein – protein interactions was conducted for several transmembrane proteins, both at the structural level, through Molecular Modeling, Molecular Dynamics simulations and Free Energy calculations, and in a system-wide approach, through the application of concepts from Network Theory. Alongside protein – protein interactions, the structural and dynamic aspects of the membrane environment were also investigated, in order to identify and evaluate the structural determinants that govern the lipid bilayer’s influences upon transmembrane protein structure and biomolecular interactions. Finally, during the course of this study, a number of publicly available, computational tools were developed, which can further aid in the study of biological membranes, transmembrane proteins and their interactions. The aforementioned studies were conducted both for transmembrane proteins located at the plasma membrane of eukaryotic cells, such as G-protein coupled receptors (GPCRs) and Receptor Tyrosine Kinases (RTKs), and for proteins found in other cell components, such as the Outer Membranes of Gram-negative bacteria and transmembrane β-barrels, as well as the double membrane system of the Nuclear Envelope and its proteins. The results of this dissertation can be applicable in the further study of protein-protein interactions in transmembrane proteins, both through experimental and through computational approaches

Interactions of the α-subunits of heterotrimeric G-proteins with GPCRs, effectors and RGS proteins: A critical review and analysis of interacting surfaces, conformational shifts, structural diversity and electrostatic potentials

G-protein coupled receptors (GPCRs) are one of the largest families of membrane receptors in eukaryotes. Heterotrimeric G-proteins, composed of α, β and γ subunits, are important molecular switches in the mediation of GPCR signaling. Receptor stimulation after the binding of a suitable ligand leads to G-protein heterotrimer activation and dissociation into the Gα subunit and Gβγ heterodimer. These subunits then interact with a large number of effectors, leading to several cell responses. We studied the interactions between Gα subunits and their binding partners, using information from structural, mutagenesis and Bioinformatics studies, and conducted a series of comparisons of sequence, structure, electrostatic properties and intermolecular energies among different Gα families and subfamilies. We identified a number of Gα surfaces that may, in several occasions, participate in interactions with receptors as well as effectors. The study of Gα interacting surfaces in terms of sequence, structure and electrostatic potential reveals features that may account for the Gα subunit's behavior towards its interacting partners. The electrostatic properties of the Gα subunits, which in some cases differ greatly not only between families but also between subfamilies, as well as the G-protein interacting surfaces of effectors and regulators of G-protein signaling (RGS) suggest that electrostatic complementarity may be an important factor in G-protein interactions. Energy calculations also support this notion. This information may be useful in future studies of G-protein interactions with GPCRs and effectors. © 2013 Elsevier Inc

Structural characterization and molecular dynamics simulations of the caprine and bovine solute carrier family 11 A1 (SLC11A1)

Natural Resistance-Associated Macrophage Proteins are a family of transmembrane divalent metal ion transporters, with important implications in life of both bacteria and mammals. Among them, the Solute Carrier family 11 member A1 (SLC11A1) has been implicated with susceptibility to infection by Mycobacterium avium subspecies paratuberculosis (MAP), potentially causing Crohn’s disease in humans and paratuberculosis (PTB) in ruminants. Our previous research had focused on sequencing the mRNA of the caprine slc11a1 gene and pinpointed polymorphisms that contribute to caprine SLC11A1’s susceptibility to infection by MAP in PTB. Despite its importance, little is known on the structural/dynamic features of mammalian SLC11A1 that may influence its function under normal or pathological conditions at the protein level. In this work we studied the structural architecture of SLC11A1 in Capra hircus and Bos taurus through molecular modeling, molecular dynamics simulations in different, functionally relevant configurations, free energy calculations of protein-metal interactions and sequence conservation analysis. The results of this study propose a three dimensional structure for SLC11A1 with conserved sequence and structural features and provide hints for a potential mechanism through which divalent metal ion transport is conducted. Given the importance of SLC11A1 in susceptibility to PTB, this study provides a framework for further studies on the structure and dynamics of SLC11A1 in other organisms, to gain 3D structural insight into the macromolecular arrangements of SLC11A1 but also suggesting a potential mechanism which divalent metal ion transport is conducted. © 2018, Springer Nature Switzerland AG

Extended Human G-Protein Coupled Receptor Network: Cell-Type-Specific Analysis of G-Protein Coupled Receptor Signaling Pathways

G-protein coupled receptors (GPCRs) mediate crucial physiological functions in humans, have been implicated in an array of diseases, and are therefore prime drug targets. GPCRs signal via a multitude of pathways, mainly through G-proteins and β-arrestins, to regulate effectors responsible for cellular responses. The limited number of transducers results in different GPCRs exerting control on the same pathway, while the availability of signaling proteins in a cell defines the result of GPCR activation. The aim of this study was to construct the extended human GPCR network (hGPCRnet) and examine the effect that cell-type specificity has on GPCR signaling pathways. To achieve this, protein-protein interaction data between GPCRs, G-protein coupled receptor kinases (GRKs), Gα subunits, β-arrestins, and effectors were combined with protein expression data in cell types. This resulted in the hGPCRnet, a very large interconnected network, and similar cell-type-specific networks in which, distinct GPCR signaling pathways were formed. Finally, a user friendly web application, hGPCRnet (http://bioinformatics.biol.uoa.gr/hGPCRnet), was created to allow for the visualization and exploration of these networks and of GPCR signaling pathways. This work, and the resulting application, can be useful in further studies of GPCR function and pharmacology. Copyright © 2019 American Chemical Society

Hidden aggregation hot-spots on human apolipoprotein E: A structural study

Human apolipoprotein E (apoE) is a major component of lipoprotein particles, and under physiological conditions, is involved in plasma cholesterol transport. Human apolipoprotein E found in three isoforms (E2; E3; E4) is a member of a family of apolipoproteins that under pathological conditions are detected in extracellular amyloid depositions in several amyloidoses. Interestingly, the lipid-free apoE form has been shown to be co-localized with the amyloidogenic Aβ peptide in amyloid plaques in Alzheimer’s disease, whereas in particular, the apoE4 isoform is a crucial risk factor for late-onset Alzheimer’s disease. Evidence at the experimental level proves that apoE self-assembles into amyloid fibrilsin vitro, although the misfolding mechanism has not been clarified yet. Here, we explored the mechanistic insights of apoE misfolding by testing short apoE stretches predicted as amyloidogenic determinants by AMYLPRED, and we computationally investigated the dynamics of apoE and an apoE-Aβ complex. Our in vitro biophysical results prove that apoE peptide-analogues may act as the driving force needed to trigger apoE aggregation and are supported by the computational apoE outcome. Additional computational work concerning the apoE-Aβ complex also designates apoE amyloidogenic regions as important binding sites for oligomeric Aβ; taking an important step forward in the field of Alzheimer’s anti-aggregation drug development. © 2019 by the authors. Licensee MDPI, Basel, Switzerland