cells Review Metal Ions Induce Liquid Condensate Formation by the F Domain of Aedes aegypti Ecdysteroid Receptor. New Perspectives of Nuclear Receptor Studies Anna Wi˛ech †, Aneta Tarczewska †, Andrzej Ozyhar˙ and Marek Orłowski * Laboratory of Biochemistry and Molecular Biology, Department of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeze˙ Wyspia´nskiego27, 50-370 Wrocław, Poland; [email protected] (A.W.); [email protected] (A.T.); [email protected] (A.O.) * Correspondence: [email protected]; Tel.: +48-713-206-286 † These authors contributed equally to this work. Abstract: The superfamily of nuclear receptors (NRs), composed of ligand-activated transcription factors, is responsible for gene expression as a reaction to physiological and environmental changes. Transcriptional machinery may require phase separation to fulfil its role. Although NRs have a similar canonical structure, their C-terminal domains (F domains) are considered the least conserved and known regions. This article focuses on the peculiar molecular properties of the intrinsically disordered F domain of the ecdysteroid receptor from the Aedes aegypti mosquito (AaFEcR), the vector of the world’s most devastating human diseases such as dengue and Zika. The His-Pro-rich segment of AaFEcR was recently shown to form the unique poly-proline helix II (PPII) in the presence of Citation: Wi˛ech,A.; Tarczewska, A.; Cu2+. Here, using widefield microscopy of fluorescently labeled AaFEcR, Zn2+- and Cu2+-induced Ozyhar,˙ A.; Orłowski, M. Metal Ions liquid-liquid phase separation (LLPS) was observed for the first time for the members of NRs. The Induce Liquid Condensate Formation perspectives of this finding on future research on the F domain are discussed, especially in relation to by the F Domain of Aedes aegypti other NR members. Ecdysteroid Receptor. New Perspectives of Nuclear Receptor Keywords: intrinsically disordered proteins; intrinsically disordered regions; nuclear receptors; Aedes Studies. Cells 2021, 10, 571. https:// aegypti; F domain; EcR; liquid-liquid phase separation; Cu2+-induced LLPS doi.org/10.3390/cells10030571 Academic Editors: Natalie Borg and David A. Jans 1. Characteristics of NRs Received: 11 December 2020 Nuclear receptors (NRs) are a specific superfamily of the most abundant class of Accepted: 3 March 2021 ligand-activated transcription factors. These transcription factors play a critical role in the Published: 5 March 2021 modulation of the expression of target genes by selective binding to appropriate genomic DNA response elements. They are responsible for the regulation of fundamental biological Publisher’s Note: MDPI stays neutral processes such as development, tissue and metabolic homeostasis, cell proliferation, meta- with regard to jurisdictional claims in morphosis, and reproduction in animals [1,2]. The activities of most NRs are regulated published maps and institutional affil- endogenously by small lipophilic ligands such as retinoids, steroids, thyroid hormones, or iations. dietary lipids [3]. NRs are also the targets of other cellular signaling pathways that modify post-translationally receptors, or their co-modulators, in turn affecting their activities and functionality [4]. Their remarkable properties of transducing extracellular signals via the binding of specific ligands to directly modulate gene expression make them critical phar- Copyright: © 2021 by the authors. maceutical and therapeutic drug targets [5]. Disrupted NR signaling pathways contribute Licensee MDPI, Basel, Switzerland. to numerous diseases such as cancer or diabetes [5–7]. Although diversity in the overall This article is an open access article shape, size, and plurality of specific ligands can be observed, the majority of the known distributed under the terms and members of NRs share a common modular structure (Figure1) that generally consists of conditions of the Creative Commons four domains [8,9]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cells 2021, 10, 571. https://doi.org/10.3390/cells10030571 https://www.mdpi.com/journal/cells Cells 2021, 10, x FOR PEER REVIEW 2 of 14 Cells 2021, 10, 571 2 of 14 FigureFigure 1. 1.Scheme Scheme of of the the overalloverall structural and and functional functional organization organization of nuclear of nuclear receptors receptors (NRs). (NRs). Nuclear Nuclear receptors receptors con- containtain at least four distinct distinct domains: domains: a aN N-terminal-terminal domain domain (NTD (NTD),), a DNA a DNA-binding-binding domain domain (DBD (DBD),), a hinge a hinge region, region, and anda a ligand-binding domain (LBD). Some NRs possess an additional C-terminal F domain. The contribution of each domain to ligand-binding domain (LBD). Some NRs possess an additional C-terminal F domain. The contribution of each domain to the NR’s activities is different. The most important functions and properties located in the individual domains are listed thebelow NR’s activitiesthe scheme is [1] different.. The F domain The most is marked important in red functions [10,11]. and properties located in the individual domains are listed below the scheme [1]. The F domain is marked in red [10,11]. The N-terminal domain (NTD) is responsible for ligand-independent transactivation (AFThe-1). The N-terminal most characterized domain (NTD) NTDs is to responsible date exhibit for features ligand-independent of intrinsicallytransactivation disordered (AF-1).proteins The (IDPs most) [12 characterized–19]. Such a property NTDs to is dateadvantageous exhibit features for their of inter intrinsically- and intramolecu- disordered proteinslar interactions (IDPs) [ 12[20]–19, as]. SuchNTDs a can property undergo is advantageous disorder-to-order for their (or partia inter-lly and order intramolecular) transi- interactionstions as a result [20], of as co NTDs-activator/co can undergo-repressor disorder-to-order binding, interaction (or partially with DNA order) or posttrans- transitions as alational result of modifications co-activator/co-repressor (PTMs) [21–23]. binding, An NTD interactionis followed withby a highly DNA orconserved posttranslational DNA- modificationsbinding domain (PTMs) (DBD [)21. Next,–23]. the An disordered NTD is followed and flexible by a hinge highly region conserved servesDNA-binding as a linker domainbetween (DBD). the DBD Next, and the the disordered evolutionarily and flexibleconserved hinge ligand region-binding serves domain as a linker (LBD between), which the DBDis responsible and the evolutionarily for the ligand conserved molecule binding ligand-binding and ligand domain-dependent (LBD), activation which is responsibleof tran- forscription the ligand (AF- molecule2), but also binding mediates and homo ligand-dependent- or heterodimerization activation with of another transcription NR super- (AF-2), butfamily also member mediates. Some homo- NR ors own heterodimerization an additional hypervariable with another (in terms NR superfamily of length and member. se- Somequence NRs) C own-terminal an additional F domain hypervariable(Figure 1) [1,3,10,11,24] (in terms. The of length C-terminal and sequence) F domains C-terminal of some F domainNRs are (Figure not clearly1)[ 1 ,defined.3,10,11, 24Amino]. The acid C-terminal residues, F following domains the of somelast helix NRs (helix are not 12) clearly of defined.LBDs, define Amino the acidF domains, residues, and followingdue to their the short last sequence helix (helix, in many 12) ofmammalian LBDs, define NRs, the Fthey domains, are often and considered due to their as a shortpart of sequence, the LBDs. in many mammalian NRs, they are often considered as a part of the LBDs. 2. Transcription Factors in the Cellular Response to Mosquito-Borne Infections 2. TranscriptionTropical and Factors subtropical in the areas Cellular stand Response out in terms to Mosquito-Borne of their lush vegetation, Infections various animalTropical species, and and subtropical changing weather areas stand conditions. out in Most terms of of the their world’s lush population vegetation, live various in animalthese areas species, and and struggle changing with weatherthe health conditions. problems that Most fre ofquently the world’s occur there. population Environ- live in thesemental areas conditions and struggle are favorable with the for health insect reproduction, problems that and frequently therefore occurfor insect there.-depend- Environ- ent disease transmission. mental conditions are favorable for insect reproduction, and therefore for insect-dependent Caused by one of the 4 serotypes of the dengue virus, dengue is one of the greatest disease transmission. threats in the world. Annually, about 390 million people get infected, with 96 million of Caused by one of the 4 serotypes of the dengue virus, dengue is one of the greatest them developing symptoms [24,25]. Most people suffer from a characteristic rash, head- threats in the world. Annually, about 390 million people get infected, with 96 million ache, and joint and muscle pain. Dengue fever may develop into much more severe states: ofdengue them developinghemorrhagic symptomsfever (DHF) [ 24and,25 dengue]. Most shock people syndrome suffer ( fromDSS), awhich characteristic require im- rash, headache,mediate hospitalization and joint and and muscle specifi pain.c treatment Dengue [26] fever. Another may develop pathogen, into like much dengue more virus, severe states:belonging dengue to the