Specificity Determining Features at the Interface of Biomolecular Complexes as Regulators of Biological Functions

Amino acid residues at the biomolecular interface play essential roles in many biological and cellular processes; relevant to this thesis, protein-protein interactions regulate signaling pathways and enzymatic activity, whereas protein-DNA interactions control gene expression, and protein-peptide interactions are central to the immune system. Biomolecular recognition and binding stability are largely determined by residues at the molecular interface. In this thesis, we focused on three biological datasets that are related to humans and human health: 1) dysregulated citrullination in the inflamed joints of rheumatoid arthritis patients, 2) a novel family of PRD-like transcription factors critical to the first few cell divisions in human life, and 3) epitopes that likely activate a cytotoxic T cell-mediated immune response against SARS-CoV-2 infection. For each dataset, in order to study the structural and functional consequences of molecular interactions, we applied a wide range of bioinformatics techniques to analyze sequences, structures and biological data retrieved from various databases, as well as taking into account experimental results from collaborators and from the literature. In rheumatoid arthritis, normally cytoplasmic peptidylarginine deiminase (PAD) enzymes citrullinate arginine residues in extracellular matrix (ECM) proteins. To examine specificity determining features that regulate the citrullination activity, we analyzed the sequence and structure data of the ECM proteins that were found citrullinated in chronically inflamed human joints. For citrullination, we found that an arginine side chain needs to be exposed to solvent but can arise from β-strands, α-helices, loops and β-turns. Moreover, there is no sequence motif linked to enzymatic activity. In addition, we studied the effect of citrullination on proteins important for a normal ECM, focusing on integrin binding to fibronectin and transforming growth factor-β (TGF-β). Citrullination of these proteins was found to inhibit cell attachment and spreading since PAD-treatment of the isoDGR motif in fibronectin and the RGD motif in TGF-β significantly reduced their binding with integrin αVβ3 and αVβ6, respectively. The expression of the human paired (PRD)-like transcription factors (TFs) are limited to the period of embryonic genome activation up to the 8-cell stage. We identified that one of these PRD-like TFs, LEUTX, binds to a TAATCC sequence motif. Sequence comparisons revealed that LEUTX protein is comprised of two domains: the DNA-binding homeodomain and a Leutx domain containing a transactivation domain. We identified specificity determining residues in the LEUTX homeodomain that are important for recognition of the TAATCC-containing 36 bp DNA motif enriched in genes involved in embryonic genome activation. We demonstrated using molecular models why a heterozygotic missense mutation A54V at the DNA-specificity determining position of LEUTX has significantly reduced overall transcriptional activity, as well as why the double mutant – I47T and A54V – form of LEUTX restores binding to the DNA motif similarly to that seen in the I47T mutation alone. At the onset of the COVID-19 pandemic we sought to understand the molecular factors that trigger the cytotoxic T cell-mediated immune response against the SARS-CoV-2 virus, taking advantage of binding data and 3D structures for related viruses and other pathogenic organisms. We first predicted the MHC class I (MHC-I)-specific immunogenic epitopes of length 8- to 11 amino acids from the SARS-CoV-2 proteins. Next, we predicted that the 9-mer epitopes would have the highest potential to elicit a strong immune response. For experimental validation, the predicted 9-mer epitopes were matched with the SARS-CoV-derived epitopes that are known to elicit an effective T cell response in vitro. Furthermore, our observations provide a structural explanation for the binding of SARS-CoV-2 epitopes to MHC-I molecules, identifying conserved immunogenic epitopes essential for understanding the pathogenesis of COVID-19. The three investigated datasets were made in concert with collaborative experimental studies and/or considering publicly available experimental data. The experimental studies generally provided the starting point for the in silico studies, which in turn had the objective of providing a detailed explanation of the experimental results. Furthermore, the in silico results could be used to devise novel and focused experiments, suggesting that bioinformatics predictions and wet-laboratory experimental investigations optimally take place with multiple advantages. Overall, this thesis demonstrates the synergy that is possible by applying this interdisciplinary approach to understanding the consequences of molecular interactions.Aminosyror i kontaktytan mellan olika biomolekyler spelar en viktig roll i många biologiska och cellulära processer; relevanta interaktioner för den här avhandlingen är protein-protein interaktioner som reglerar signaleringsrutter och enzymatisk aktivitet, protein-DNA interaktioner som kontrollerar genexpression, samt protein-peptid interaktioner som har en central roll i immunförsvaret. Biomolekylär igenkänning och bindningsstabilitet beror till stor del på de aminosyror som finns i den molekylära kontaktytan. I den här avhandlingen fokuserade vi på tre biologiska dataset som är relaterade till människor och människors hälsa: 1) felreglerad citrullinering i inflammerade leder hos patienter med reumatoid artrit, 2) en nyupptäckt familj av PRD (human paired)-lika transkriptionsfaktorer som är nödvändiga för de första celldelningarna i människolivet, och 3) epitoper som troligen aktiverar en cytotoxisk T-cell-förmedlad immunrespons mot SARS-CoV-2 infektioner. För att studera de strukturella och funktionella konsekvenserna av de molekylära interaktionerna i varje dataset, användes en mängd olika bioinformatiska tekniker för att analysera sekvenser, strukturer och biologiska data från olika databaser och dessutom beaktades experimentella resultat från samarbetspartners och från litteraturen. I reumatoid artrit citrullinerar vanligen PAD (cytoplasmatiska peptidyl arginin deiminas)-enzymer arginin-aminosyror i proteiner i det extracellulära matrixet (ECM). För att undersöka egenskaper som avgör specificiteten hos citrullineringsaktiviteten analyserade vi sekvens- och strukturdata för ECM-proteiner som blir citrullinerade i kroniskt inflammerade leder hos människor. Vi upptäckte att en argininsidokedja måste vara i kontakt med det omgivande lösningsmedlet för att kunna citrullineras, att de kan finnas i beta-strängar, alfa-helixar och beta-svängar, samt att det inte finns några sekvensmotiv som är kopplade till enzymatisk aktivitet. Utöver detta studerade vi effekten av citrullinering på proteiner som är viktiga för normal extracellulär matrix, med fokus på integrinbinding till fibronektin och TGF-β (transforming growth factor-β). Citrullinering av dessa proteiner upptäcktes inhibera cellvidhäftning och spridning eftersom PAD-behandling av isoDGR-motivet i fibronektin och RGD-motivet i TGF-β ordentligt reducerar deras bindning till integrin αVβ3 och αVβ6, respektive. Expressionsnivåerna av PRD-lika transkriptionsfaktorer (TF) är begränsade till perioden av zygotens genomaktivering upp till 8-cells stadiet. Vi identifierade att en av dessa PRD-lika transkriptionsfaktorer, LEUTX, binder till ett TAATCC sekvensmotiv. Sekvensjämförelser avslöjade att LEUTX proteinet består av två domäner, det DNA-bindande homeodomänet och en leutx-domän som innehåller en transaktiveringsdomän. Vi identifierade specificitetsbestämmande aminosyror i LEUTX homeodomänen som är viktiga för igenkänning av TAATCC-innehållande 36 baspars DNA-motivet som är berikad med gener involverade i zygotens genomaktivering. Vi använde molekylära modeller för att visa varför en heterozygotisk missense-mutation, A54V, i DNA-specificitetsbestämmande positionen i LEUTX har ordentligt minskad generell transkriptionsaktivitet, och varför dubbelmutanten I47T och A54V återställer bindning till DNA-motivet på samma sätt som observerats i enbart I47T mutationen. När COVID-19 pandemin inleddes försökte vi förstå de molekylära faktorer som startar den cytotoxiska T-cell-förmedlade immunresponsen mot SARS-CoV-2 viruset, genom att utnyttja bindningsdata och 3D strukturer för relaterade virus och andra patogena organismer. Vi förutspådde först MHC klass I (MHC-I)-specifika immunogena epitoper av längden 8 till 11 aminosyror från SARS-CoV-2 proteiner. Därefter förutspådde vi att epitoper bestående av 9 aminosyror hade den högsta potentialen att orsaka en stark immunrespons. För experimentell validering matchades de 9 aminosyror långa epitoperna med epitoper från SARS-CoV som man vet att orsakar en effektiv T-cell respons in vitro. Våra observationer bidrar också med en strukturell förklaring för bindningen av SARS-CoV-2 epitoper till MHC-I molekyler, vilket identifierar konserverade immunogena epitoper som är nödvändiga för att förstår patogenesen hos COVID-19. De tre undersökta dataseten gjordes i samarbete med experimentella studier och/eller genom att ta allmänt tillgängliga experimentella data i beaktande. De experimentella studierna gav en startpunkt för in silico-studierna, vilka i sin tur hade som mål att ge en detaljerad förklaring till de experimentella resultaten. In silico-resultaten kan också användas för att utveckla nya och fokuserade experiment, vilket indikerar att bioinformatiska förutspåelser och experimentella studier optimalt sker med många fördelar. Över lag visar denna avhandling synergin som är möjlig genom att använda detta interdisciplinära arbetssätt för att förstå konsekvenserna av molekylära interaktioner

Phylogenetic and mutational analyses of human LEUTX, a homeobox gene implicated in embryogenesis

Recently, human PAIRED-LIKE homeobox transcription factor (TF) genes were discovered whose expression is limited to the period of embryo genome activation up to the 8-cell stage. One of these TFs is LEUTX, but its importance for human embryogenesis is still subject to debate. We confirmed that human LEUTX acts as a TAATCC-targeting transcriptional activator, like other K50-type PAIRED-LIKE TFs. Phylogenetic comparisons revealed that Leutx proteins are conserved across Placentalia and comprise two conserved domains, the homeodomain, and a Leutx-specific domain containing putative transcriptional activation motifs (9aa TAD). Examination of human genotype resources revealed 116 allelic variants in LEUTX. Twenty-four variants potentially affect function, but they occur only heterozygously at low frequency. One variant affects a DNA-specificity determining residue, mutationally reachable by a one-base transition. In vitro and in silico experiments showed that this LEUTX mutation (alanine to valine at position 54 in the homeodomain) results in a transactivational loss-of-function to a minimal TAATCC-containing promoter and a 36 bp motif enriched in genes involved in embryo genome activation. A compensatory change in residue 47 restores function. The results support the notion that human LEUTX functions as a transcriptional activator important for human embryogenesis.Peer reviewe

Immunogenic SARS-CoV-2 Epitopes: In Silico Study Towards Better Understanding of COVID-19 Disease-Paving the Way for Vaccine Development

The emergence of the COVID-19 outbreak at the end of 2019, caused by the novel coronavirus SARS-CoV-2, has, to date, led to over 13.6 million infections and nearly 600,000 deaths. Consequently, there is an urgent need to better understand the molecular factors triggering immune defense against the virus and to develop countermeasures to hinder its spread. Using in silico analyses, we showed that human major histocompatibility complex (MHC) class I cell-surface molecules vary in their capacity for binding different SARS-CoV-2-derived epitopes, i.e., short sequences of 8-11 amino acids, and pinpointed five specific SARS-CoV-2 epitopes that are likely to be presented to cytotoxic T-cells and hence activate immune responses. The identified epitopes, each one of nine amino acids, have high sequence similarity to the equivalent epitopes of SARS-CoV virus, which are known to elicit an effective T cell response in vitro. Moreover, we give a structural explanation for the binding of SARS-CoV-2-epitopes to MHC molecules. Our data can help us to better understand the differences in outcomes of COVID-19 patients and may aid the development of vaccines against SARS-CoV-2 and possible future outbreaks of novel coronaviruses

DUX4 is a multifunctional factor priming human embryonic genome activation

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhance-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding Kith minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.Peer reviewe

Major disturbance events in terrestrial ecosystems detected using global satellite data sets. Global Change Biology

Abstract. Ecosystem scientists have yet to develop a proven methodology to monitor and understand major disturbance events and their historical regimes at a global scale. This study was conducted to evaluate patterns in an 18-year record of global satellite observations of vegetation phenology from the Advanced Very High Resolution Radiometer (AVHRR) as a means to characterize major ecosystem disturbance events and regimes. The fraction absorbed of photosynthetically active radiation (FPAR) by vegetation canopies worldwide has been computed at a monthly time interval from 1982 to 1999 and gridded at a spatial resolution of 0.5 o latitude/longitude. Potential disturbance events of large extent (>0.5 Mha) were identified in the FPAR time series by locating anomalously low values (FPAR-LO) that lasted longer than 12 consecutive months at any 0.5 o pixel. We find that nearly 400 Mha of the global land surface could be identified with at least one FPAR-LO event over the 18-year time series. The majority of these potential disturbance events occurred in tropical savanna and shrublands or in boreal forest ecosystem classes. Verification of potential disturbance events from our FPAR-LO analysis was carried out using documented records of the timing of large-scale wildfires at locations throughout the world. Disturbance regimes were further characterized by association analysis with historical climate anomalies. Assuming accuracy of the FPAR satellite record to characterize major ecosystem disturbance events, we estimate that nearly 9 Pg of carbon could have been lost from the terrestrial biosphere to the atmosphere as a result of large-scale ecosystem disturbance over this 18-year time series

Técnicas avançadas de sensoriamento remoto aplicadas ao estudo de mudanças climáticas e ao funcionamento dos ecossistemas amazônicos Advanced remote sensing techniques for global changes and Amazon ecosystem functioning studies

Este artigo se propõe a apresentar exemplos de questões científicas que puderam ser respondidas no contexto do Projeto LBA (Large Sale Biosphere-Atmosphere Experiment in Amazonia) graças à contribuição de informações derivadas de sensoriamento remoto. Os métodos de sensoriamento remoto permitem integrar informações sobre os vários processos físicos e biológicos em diferentes escalas de tempo e espaço. Nesse artigo, são enfatizados aqueles avanços de conhecimento que jamais seriam alcançados sem a concorrência da informação derivada de sensoriamento.<br>This paper aims to assess the contribution of remote sensing technology in addressing key questions raised by the Large Scale Biosphere-Atmosphere Experiment in Amazonia (LBA). The answers to these questions foster the knowledge on the climatic, biogechemical and hydrologic functioning of the Amazon, as well as on the impact of human activities at regional and global scales. Remote sensing methods allow integrating information on several processes at different temporal and spatial scales. By doing so, it is possible to perceive hidden relations among processes and structures, enhancing their teleconnections. Key advances in the remote sensing science are summarized in this article, which is particularly focused on information that would not be possible to be retrieved without the concurrence of this technology