Publications by authors named "Alvaro Ingles-Prieto"

16 Publications

  • Page 1 of 1

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease.

PLoS Genet 2021 Apr 15;17(4):e1009479. Epub 2021 Apr 15.

Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria.

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1009479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049241PMC
April 2021

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs.

Nat Chem Biol 2020 04 9;16(4):469-478. Epub 2020 Mar 9.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-020-0483-3DOI Listing
April 2020

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding.

Biochem J 2019 12;476(23):3631-3647

Departamento de Quimica Fisica, Facultad de Ciencias, Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Universidad de Granada, 18071 Granada, Spain.

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BCJ20190739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906118PMC
December 2019

Optogenetic Control of Nodal Signaling Reveals a Temporal Pattern of Nodal Signaling Regulating Cell Fate Specification during Gastrulation.

Cell Rep 2016 07 7;16(3):866-77. Epub 2016 Jul 7.

Laboratory of Developmental Biology, Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria. Electronic address:

During metazoan development, the temporal pattern of morphogen signaling is critical for organizing cell fates in space and time. Yet, tools for temporally controlling morphogen signaling within the embryo are still scarce. Here, we developed a photoactivatable Nodal receptor to determine how the temporal pattern of Nodal signaling affects cell fate specification during zebrafish gastrulation. By using this receptor to manipulate the duration of Nodal signaling in vivo by light, we show that extended Nodal signaling within the organizer promotes prechordal plate specification and suppresses endoderm differentiation. Endoderm differentiation is suppressed by extended Nodal signaling inducing expression of the transcriptional repressor goosecoid (gsc) in prechordal plate progenitors, which in turn restrains Nodal signaling from upregulating the endoderm differentiation gene sox17 within these cells. Thus, optogenetic manipulation of Nodal signaling identifies a critical role of Nodal signaling duration for organizer cell fate specification during gastrulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.06.036DOI Listing
July 2016

A Phytochrome Sensory Domain Permits Receptor Activation by Red Light.

Angew Chem Int Ed Engl 2016 05 21;55(21):6339-42. Epub 2016 Apr 21.

Synthetic Physiology, Institute of Science and Technology Austria (IST Austria), Am Campus 1, 3400, Klosterneuburg, Austria.

Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases-the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201601736DOI Listing
May 2016

Light-assisted small-molecule screening against protein kinases.

Nat Chem Biol 2015 Dec 12;11(12):952-4. Epub 2015 Oct 12.

Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria.

High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.1933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652335PMC
December 2015

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

Electrophoresis 2015 Feb 22;36(4):518-25. Epub 2015 Jan 22.

IST Austria (Institute of Science and Technology Austria), Klosterneuburg, Austria; Faculty of Chemistry, Aalen University, Aalen, Germany; Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germany.

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/elps.201400451DOI Listing
February 2015

Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

Mol Biol Evol 2015 Feb 12;32(2):440-55. Epub 2014 Nov 12.

Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain

Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/molbev/msu312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298172PMC
February 2015

The optogenetic promise for oncology: Episode I.

Mol Cell Oncol 2014 Oct-Dec;1(4):e964045. Epub 2014 Oct 29.

Institute of Cancer Research; Department of Medicine I; Comprehensive Cancer Center Vienna; Medical University of Vienna ; Vienna, Austria.

As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial-mesenchymal transition, and angiogenic sprouting-cell behaviors central to cancer progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/23723548.2014.964045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905200PMC
June 2016

Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.

EMBO J 2014 Aug 1;33(15):1713-26. Epub 2014 Jul 1.

Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria

Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201387695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194103PMC
August 2014

Conservation of protein structure over four billion years.

Structure 2013 Sep 8;21(9):1690-7. Epub 2013 Aug 8.

Departamento de Química Física, Facultad de Ciencias, Universidad de Granada, Granada 18071, Spain.

Little is known about the evolution of protein structures and the degree of protein structure conservation over planetary time scales. Here, we report the X-ray crystal structures of seven laboratory resurrections of Precambrian thioredoxins dating up to approximately four billion years ago. Despite considerable sequence differences compared with extant enzymes, the ancestral proteins display the canonical thioredoxin fold, whereas only small structural changes have occurred over four billion years. This remarkable degree of structure conservation since a time near the last common ancestor of life supports a punctuated-equilibrium model of structure evolution in which the generation of new folds occurs over comparatively short periods and is followed by long periods of structural stasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2013.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774310PMC
September 2013

Protein folding drives disulfide formation.

Cell 2012 Nov;151(4):794-806

Department of Biological Sciences, Columbia University, New York, NY 10027, USA. Electronic address:

PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2012.09.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3506382PMC
November 2012

Highly anomalous energetics of protein cold denaturation linked to folding-unfolding kinetics.

PLoS One 2011 29;6(7):e23050. Epub 2011 Jul 29.

Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain.

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a "mirror image" of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023050PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146537PMC
December 2011

Single-molecule paleoenzymology probes the chemistry of resurrected enzymes.

Nat Struct Mol Biol 2011 May 3;18(5):592-6. Epub 2011 Apr 3.

Department of Biological Sciences, Columbia University, New York, New York, USA.

It is possible to travel back in time at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx) dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32 °C more stable than modern enzymes, and the oldest show markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy. From the force dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes. Although Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087858PMC
May 2011

Structure of the Calx-beta domain of the integrin beta4 subunit: insights into function and cation-independent stability.

Acta Crystallogr D Biol Crystallogr 2009 Aug 17;65(Pt 8):858-71. Epub 2009 Jul 17.

Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain.

The integrin alpha6beta4 is a receptor for laminins and provides stable adhesion of epithelial cells to the basement membranes. In addition, alpha6beta4 is important for keratinocyte migration during wound healing and favours the invasion of carcinomas into surrounding tissue. The cytoplasmic domain of the beta4 subunit is responsible for most of the intracellular interactions of the integrin; it contains four fibronectin type III domains and a Calx-beta motif. The crystal structure of the Calx-beta domain of beta4 was determined to 1.48 A resolution. The structure does not contain cations and biophysical data support the supposition that the Calx-beta domain of beta4 does not bind calcium. Comparison of the Calx-beta domain of beta4 with the calcium-binding domains of Na(+)/Ca(2+)-exchanger 1 reveals that in beta4 Arg1003 occupies a position equivalent to that of the calcium ions in the Na(+)/Ca(2+)-exchanger. By combining mutagenesis and thermally induced unfolding, it is shown that Arg1003 contributes to the stability of the Calx-beta domain. The structure of the Calx-beta domain is discussed in the context of the function and intracellular interactions of the integrin beta4 subunit and a putative functional site is proposed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S0907444909018745DOI Listing
August 2009