High level production, characterization and structural analysis of neuronal calcium-activated potassium channels

Kaliumkanäle vermitteln als reguliertes Porenprotein den selektiven Transport von Kaliumionen durch die Zellmembran. Sie sind für physiologische Prozesse wie Neurosekretion und Tonus der glatten Muskulatur von Bedeutung. Eine Familie stellen die Calcium-aktivierten Kaliumkanäle dar, welche wiederum in drei Unterfamilien aufgeteilt werden können: SK-Kanäle (geringe Leitfähigkeit), BK-Kanäle (hohe Leitfähigkeit) und IK-Kanäle (mittlere Leitfähigkeit). Sie werden durch ein Ansteigen der intrazellulären Ca2+-Konzentration, wie sie während eines Aktionspotentials auftritt, aktiviert und zeigen keine Spannungsabhängigkeit der Aktivierung. SK-Kanäle sind in physiologische Prozesse wie Lernen, Erinnerungsvermögen, Regulation des Tagesrhytmus und Unterbrechung des normalen Schlafmusters involviert. Das SK2-Protein der Ratte (rSK2) wurde in Pichia pastoris-Zellen überexprimiert, um das Protein zu reinigen und genügend Material für strukturelle Studien zu erhalten. Dazu wurde die rSK2-cDNA in das Pichia Pastoris-Expressionssystem kloniert. Elektronenmikroskopische Lokalisationsstudien wie Immunogold-Markierung und Gefrierbruch-Analyse zeigten, daß der SK2-Kanal hauptsächlich an der Oberfläche des Endoplasmatischen Retikulums oder andersartigen internen Membranstrukturen lokalisiert ist. Dies steht im Gegensatz zu neuronalen Zellen, in denen das Protein eher in der Plasmamembran zu finden ist. Die Solubilisierung des Kanalproteins mit Detergentien war besonders schwierig zu erreichen. Von 15 getesteten Detergentien konnte das Kanalprotein nur mit Digitonin aus der Membran gelöst werden. Verschiedene chromatographische Verfahren wurden zur Reinigung des Kanals eingesetzt, aber das Protein wurde nur teilweise gereinigt und die Ausbeute war gering. Eine Gelfiltrationsanalyse zeigte, daß der gereinigte SK2-Kanal wie andere Kaliumkanäle als Homotetramer vorlag (MacKinnon 1991). Um einen verbesserten Einbau in die Plasmamembran zu erreichen, wurde versucht, ein weiteres SK2-Konstrukt zu exprimieren. Dieses verfügte über eine zusätzliche sechs Aminosäuren umfassende Sequenz (FCYENE), welche sich als bedeutsam für die Membranlokalisation anderer Kaliumkanäle herausgestellt hatte (Ma et al. 2001). Die Sequenz war an den Carboxyterminus angefügt. Der Effekt dieser Sequenz auf die SK2-Expression in Pichia pastoris wurde untersucht. Dies zeigte, daß die FCYENE-Sequenz keinen Effekt auf den gesamt-Expressionslevel bindungskompetenter Kanäle hatte. Digitonin, TritonX-100, n-Dodecyl-beta-D-maltosid (DDM) und Octyl-glycosid erlaubten eine Solubilisierung des SK2-FCYENE-Proteins. Dies zeigte, daß die FCYENE-Sequenz einen bemerkenswerten Effekt auf die Löslichkeit des SK2-Proteins hatte. Im Gegensatz zum Wildtyp zeigte das SK2-FCYENE-Protein auch eine veränderte Verteilung innerhalb der Hefezellen. Es lag nicht nur im Endoplasmatischen Retikulum, sondern auch konzentriert in Vesikeln vor. Trotzdem waren wie zuvor nur geringe Mengen an Protein in der Plasmamembran zu finden. Offenbar kann die FCYENE-Sequenz zwar den Austritt aus dem Endoplasmatischen Retikulum fördern, aber die Kanäle werden nicht in die Plasmamembran translokiert. Trotz der erhöhten Menge an löslichem Ausgangsmaterial wurde die Menge an gereinigtem Kanalprotein nicht nennenswert erhöht. Die Calcium-Sensitivität der SK-Kanäle wird durch eine Wechselwirkung mit Calmodulin hervorgerufen (Xia et al. 1998; Schumacher et al. 2001). Die Bindung zwischen Calmodulin und SK2-Protein ist nicht nur für das Öffnen, sondern auch für den Transport des Kanals essentiell (Lee et al. 2003). Deshalb wurde das SK2-Protein in tandem mit Calmodulin kloniert (SK2-q-CaM), wobei ein Verbindungsstück aus zehn Glutaminresten die beiden Proteine verband. Das Calmodulin veränderte dramatisch die Verteilung des Kanalproteins in der Zelle. Dies zeigte die Immunogold-Markierung des SK2-q-CaM-Proteins in den Pichia pastoris-Zellen. Einige der SK2-q-CaM-Chimären waren in der Plasmamembran lokalisiert. Ferner war die Effektivität der Solubilisierung mit Detergenz weitaus höher als für das wildtyp- und das SK2-FCYENE-Protein. Trotz der enormen Verbesserung der Löslichkeit war die Ausbeute an SK2-q-CaM-Protein geringer als für die beiden anderen Konstrukte, was vermutlich auf eine veränderte Verfügbarkeit der His-Sequenz durch die Wechselwirkung mit Calmodulin zurückzuführen war. Das SK2-Protein wurde zudem in der Säugerzelllinie BHK (Baby Hamster Kidney) unter Verwendung des Semliki Forest Virus-Systems exprimiert. Das Protein erreichte 24 h nach Virusinfektion einen hohen Expressionslevel. Eine Immunogold-Markierung bestätigte, daß das SK-Protein in die Plasmamembran eingebaut wurde, wenngleich ein Großteil des Proteins in den Membranen des Endoplasmatischen Retikulums verblieb. Apamin band mit hoher Affinität an das Kanalprotein, wobei die Daten der Sättigungskurven auf eine einzelne Bindungstelle hinwiesen. Diese Werte und Daten aus kompetitiven Verdrängungs-Experimenten waren mit jenen Werten, die mit nativen Hirnmembranen gemessen wurden, vergleichbar (Marqueze, Seagar et al. 1989; Wadsworth, Doorty et al. 1994; Wadsworth, Torelli et al. 1997). Das Kanalprotein ließ sich erfolgreich mit verschiedenen Detergentien, vor allem mit DDM, aus der Membran lösen. Mittels Ionenaustausch-Chromatographie konnten 0,1-0,2 mg/ml reines SK-Protein erhalten werden. Gelfiltrations-Chromatographie und Blau-Nativ Gelelektrophorese (BN-PAGE) zeigten, daß das reine Kanalprotein wie erwartet als Tetramer vorlag. Bis heute gibt es keine weiteren Berichte von gereinigtem SK2-Protein aus natürlicher oder rekombinanter Quelle. Trotz der relativ geringen Proteinausbeute ist die gelungene Reinigung des Proteins daher eine zentrale Erkenntnis für die Charakterisierung von SK-Kanälen.Calcium-activated potassium channels are fundamental regulators of neuron excitability. SK channels are activated by an intracellular increase of Ca++ (such as occurs during an action potential). They have a small single channel conductance (less than 20pS) and show no voltage dependence of activation. To date, there are only a few examples of high-resolution structures of eukaryotic membrane proteins. All of them were purified from natural sources. Since no abundant natural sources of eukaryotic K+ channels are available we overexpressed rSK2 in order to produce the quantities necessary for structural analysis. Unfortunately the Pichia pastoris expression system did not yield sufficient amount of pure protein, mainly because most of the protein was retained by in the ER and was only partially soluble. Subsequently, two constructs were expressed: SK2-FCYENE (containing a specific sequence that promotes surface expression), and SK2-q-CaM a concatamer of SK2 and calmodulin. Although these proved an improvement in terms of solubilisation, little improvement was found in terms of amounts of purified material obtained. For this reason we tested the Semliki Forest virus expression system, since the protein is expressed in a mammalian system where we hoped that it would be trafficked in the same way as in vivo. Using this system it was possible to express rSK2 and solubilise it with several detergents and to achieve much better purification. However, the levels were still not sufficient for high-resolution structural studies, although sufficient for single particle electron microscopy analysis

A Resource to Infer Molecular Paths Linking Cancer Mutations to Perturbation of Cell Metabolism

Some inherited or somatically-acquired gene variants are observed significantly more frequently in the genome of cancer cells. Although many of these cannot be confidently classified as driver mutations, they may contribute to shaping a cell environment that favours cancer onset and development. Understanding how these gene variants causally affect cancer phenotypes may help developing strategies for reverting the disease phenotype. Here we focus on variants of genes whose products have the potential to modulate metabolism to support uncontrolled cell growth. Over recent months our team of expert curators has undertaken an effort to annotate in the database SIGNOR 1) metabolic pathways that are deregulated in cancer and 2) interactions connecting oncogenes and tumour suppressors to metabolic enzymes. In addition, we refined a recently developed graph analysis tool that permits users to infer causal paths leading from any human gene to modulation of metabolic pathways. The tool grounds on a human signed and directed network that connects similar to 8400 biological entities such as proteins and protein complexes via causal relationships. The network, which is based on more than 30,000 published causal links, can be downloaded from the SIGNOR website. In addition, as SIGNOR stores information on drugs or other chemicals targeting the activity of many of the genes in the network, the identification of likely functional paths offers a rational framework for exploring new therapeutic strategies that revert the disease phenotype

SIGNOR 3.0, the SIGnaling network open resource 3.0: 2022 update

The SIGnaling Network Open Resource (SIGNOR 3.0, ) is a public repository that captures causal information and represents it according to an 'activity-flow' model. SIGNOR provides freely-accessible static maps of causal interactions that can be tailored, pruned and refined to build dynamic and predictive models. Each signaling relationship is annotated with an effect (up/down-regulation) and with the mechanism (e.g. binding, phosphorylation, transcriptional activation, etc.) causing the regulation of the target entity. Since its latest release, SIGNOR has undergone a significant upgrade including: (i) a new website that offers an improved user experience and novel advanced search and graph tools; (ii) a significant content growth adding up to a total of approx. 33,000 manually-annotated causal relationships between more than 8900 biological entities; (iii) an increase in the number of manually annotated pathways, currently including pathways deregulated by SARS-CoV-2 infection or involved in neurodevelopment synaptic transmission and metabolism, among others; (iv) additional features such as new model to represent metabolic reactions and a new confidence score assigned to each interaction

MINT and IntAct contribute to the Second BioCreative challenge: serving the text-mining community with high quality molecular interaction data

In the absence of consolidated pipelines to archive biological data electronically, information dispersed in the literature must be captured by manual annotation. Unfortunately, manual annotation is time consuming and the coverage of published interaction data is therefore far from complete. The use of text-mining tools to identify relevant publications and to assist in the initial information extraction could help to improve the efficiency of the curation process and, as a consequence, the database coverage of data available in the literature. The 2006 BioCreative competition was aimed at evaluating text-mining procedures in comparison with manual annotation of protein-protein interactions

Resources and tools for rare disease variant interpretation

: Collectively, rare genetic disorders affect a substantial portion of the world's population. In most cases, those affected face difficulties in receiving a clinical diagnosis and genetic characterization. The understanding of the molecular mechanisms of these diseases and the development of therapeutic treatments for patients are also challenging. However, the application of recent advancements in genome sequencing/analysis technologies and computer-aided tools for predicting phenotype-genotype associations can bring significant benefits to this field. In this review, we highlight the most relevant online resources and computational tools for genome interpretation that can enhance the diagnosis, clinical management, and development of treatments for rare disorders. Our focus is on resources for interpreting single nucleotide variants. Additionally, we present use cases for interpreting genetic variants in clinical settings and review the limitations of these results and prediction tools. Finally, we have compiled a curated set of core resources and tools for analyzing rare disease genomes. Such resources and tools can be utilized to develop standardized protocols that will enhance the accuracy and effectiveness of rare disease diagnosis

MINT, the molecular interaction database: 2009 update

MINT (http://mint.bio.uniroma2.it/mint) is a public repository for molecular interactions reported in peer-reviewed journals. Since its last report, MINT has grown considerably in size and evolved in scope to meet the requirements of its users. The main changes include a more precise definition of the curation policy and the development of an enhanced and user-friendly interface to facilitate the analysis of the ever-growing interaction dataset. MINT has adopted the PSI-MI standards for the annotation and for the representation of molecular interactions and is a member of the IMEx consortium

The IntAct database:Efficient access to fine-grained molecular interaction data

The IntAct molecular interaction database (https://www.ebi.ac.uk/intact) is a curated resource of molecular interactions, derived from the scientific literature and from direct data depositions. As of August 2021, IntAct provides more than one million binary interactions, curated by twelve global partners of the International Molecular Exchange consortium, for which the IntAct database provides a shared curation and dissemination platform. The IMEx curation policy has always emphasised a fine-grained data and curation model, aiming to capture the relevant experimental detail essential for the interpretation of the provided molecular interaction data. Here, we present recent curation focus and progress, as well as a completely redeveloped website which presents IntAct data in a much more user-friendly and detailed way

The MIntAct project—IntAct as a common curation platform for 11 molecular interaction databases

IntAct (freely available at http://www.ebi.ac.uk/intact) is an open-source, open data molecular interaction database populated by data either curated from the literature or from direct data depositions. IntAct has developed a sophisticated web-based curation tool, capable of supporting both IMEx- and MIMIx-level curation. This tool is now utilized by multiple additional curation teams, all of whom annotate data directly into the IntAct database. Members of the IntAct team supply appropriate levels of training, perform quality control on entries and take responsibility for long-term data maintenance. Recently, the MINT and IntAct databases decided to merge their separate efforts to make optimal use of limited developer resources and maximize the curation output. All data manually curated by the MINT curators have been moved into the IntAct database at EMBL-EBI and are merged with the existing IntAct dataset. Both IntAct and MINT are active contributors to the IMEx consortium (http://www.imexconsortium.org

Tools and data services registry: a community effort to document bioinformatics resources

Life sciences are yielding huge data sets that underpin scientific discoveries fundamental to improvement in human health, agriculture and the environment. In support of these discoveries, a plethora of databases and tools are deployed, in technically complex and diverse implementations, across a spectrum of scientific disciplines. The corpus of documentation of these resources is fragmented across the Web, with much redundancy, and has lacked a common standard of information. The outcome is that scientists must often struggle to find, understand, compare and use the best resources for the task at hand. Here we present a community-driven curation effort, supported by ELIXIR—the European infrastructure for biological information—that aspires to a comprehensive and consistent registry of information about bioinformatics resources. The sustainable upkeep of this Tools and Data Services Registry is assured by a curation effort driven by and tailored to local needs, and shared amongst a network of engaged partners. As of November 2015, the registry includes 1785 resources, with depositions from 126 individual registrations including 52 institutional providers and 74 individuals. With community support, the registry can become a standard for dissemination of information about bioinformatics resources: we welcome everyone to join us in this common endeavour. The registry is freely available at https://bio.tools

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes