Expanding the Druggable Proteome with Chemical Biology – Best Poster Awards

The 2020 conference season at the EMBL Advanced Training Centre kicked off with the EMBL Conference: Expanding the Druggable Proteome with Chemical Biology (5 – 7 February 2020). Meet the three poster prize winners from the conference – Patrick Zanon, Enric Ros and Rens de Vries.

Identification of novel antibiotic targets using covalent inhibitors and residue-specific proteomics

PHOTO: Patrick Zanon

Authors: Patrick Zanon (1), Stephan Hacker (1)

Bacterial resistance towards all marketed antibiotics poses an imminent threat to global health. In order to overcome this antibiotic crisis, drugs with novel mechanisms-of-action are desperately needed. Covalent inhibitors are especially promising in this regard as they are already prevalent as antibiotics (e.g. β-lactams and fosfomycin), allow targeting protein pockets that are hard to address with non-covalent interactions alone and hold the promise to overcome some mechanisms of resistance development.[1] Furthermore, covalent inhibitors are uniquely suited to identify new binding pockets on proteins using residue-specific proteomics and in this way to broaden the scope of targetable protein targets.
The vast majority of covalent inhibitors so far either hijack the enzymatic activity of the protein by modification of catalytic serines and tyrosines or address cysteines through their inherent outstanding nucleophilicity. Nevertheless, the number of potentially addressable proteins in the bacterial proteome is significantly limited by the requirement for these amino acids to be present in target proteins. By developing electrophilic groups that are selective for other amino acids (e.g. lysine), we strive to expand the number of exploitable interaction sites for covalent inhibitors in the bacterial proteome. Furthermore, to assess the reactivity and selectivity of covalent inhibitors and to streamline the discovery of novel antibiotic targets, we develop new methods for residue-specific activity-based protein profiling.[2,3] In this way, we are convinced, that we will be able to make important contributions to overcome the antibiotic crisis.

References:
[1] R. A. Bauer, Drug Discov. Today 2015, 20, 1061–1073.
[2] K. M. Backus et al., Nature 2016, 534, 570.
[3] P. R. A. Zanon, L. Lewald, S. M. Hacker Angew. Chem. Int. Ed., doi: 10.1002/anie.201912075.

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(1) Technical University of Munich, Germany


Incorporating 1,2,4,5-tetrazines into proteins: A method for targeted drug release

PHOTO: Enric Ros

Authors: Enric Ros (1), Antoni Riera (1), Lluís Ribas de Pouplana (1)

Bioorthogonal reactions, namely reactions that can take place under biocompatible conditions, are having a major impact in the development of new research tools and novel therapeutic strategies. In the latter case, the discovery of the reaction commonly referred to as “click-to-release” (CtR), which triggers the liberation of a given cargo (normally a drug or a fluorophore), has led to several applications in drug delivery. This reaction happens between a 1,2,4,5-Tetrazine (Tz) fragment and certain alkenes or alkynes and, in order to achieve drug delivery specifically at the site of action, one of the two reactant counterparts should be conjugated to a biomolecule acting as a carrier, ideally a protein.
We have synthetized the previously unreported 3-bromo-1,2,4,5-tetrazine and used its excellent reactivity to attain chemoselective protein labelling onto lysines. Due to the chemical features of the formed amino-Tz. The resulting labelled lysines can undergo fast CtR reactions with trans-cyclooctenes, thereby releasing a desired cargo under physiological conditions. To showcase the applicability of this approach, we have labelled the monoclonal antibody Trastuzumab (anti-Her2) and demonstrated the specific release of the cytotoxic drug doxorubicin upon reaction in a mammalian cell culture context, resulting in a decrease in cell viability.
Additionally, we have also used 3-bromo-1,2,4,5-tetrazine to synthetize an amino-Tz containing non-natural amino acid and used it to achieve protein labelling through its genetic incorporation by amber codon suppression in Escherichia coli. The resulting site-selectively labelled proteins can also trigger fast, high yielding CtR reactions.
To summarize, we have successfully applied a new compound, 3-bromo-1,2,4,5-tetrazine, as a reagent to achieve either chemoselective or site selective protein labelling. We have applied the bioconjugated proteins to demonstrate their potential use for targeted drug delivery in a relevant cellular model, opening new therapeutically useful methodologies.

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(1) IRB Barcelona, Spain


Modulation of nuclear receptors through ligand architecture

PHOTO: Rens de Vries

Authors: Rens de Vries (1), Femke Meijer (1), Luc Brunsveld (1)

Nuclear receptors (NRs) have been one of the primary drug targets over the last decades for their ability to regulate gene expression. The traditional approach of modulating NRs is to design small synthetic molecules that interact with the ligand-binding domain (LBD) of the NR. Ligands can thereby either enhance or inhibit gene transcription. Apart from the effects on transcription, recent research shows that minor changes in the ligand scaffold can have a significant impact on the behavior of the NR. In this research, we show how small-molecules can change both the dimerization behavior of NRs and the recruitment of allosteric modulators.
The Retinoic X Receptor α (RXRα) is known as a master regulator among NRs through its ability to heterodimerize with, and thereby modulate, other NRs. We show, using a novel NanoBIT complexation assay, that small directed changes in the RXR ligand scaffold can lead to selective formation of specific hetero- and homodimers. Using our structural data and focused compound library, a model was developed to help to understand this effect of the ligand. This information can serve as a blueprint to design small-molecules that selectively target specific NRs via RXR. This makes RXR as an exciting and versatile target for NR modulation, especially when classical modulation of the partner NR is not possible.
Recently, small-molecules have been found to bind to allosteric sites of NRs. Allosteric ligands are of interest since they do not compete with the endogenous ligand of the NR and often shown an increased selectivity towards their target. We show, using X-ray crystallography and biochemical assays, that there is communication between orthosteric and allosteric ligands in the RAR-related orphan receptor γ t (RORγt). We successfully solved eleven new ternary crystal structures of RORγt in the presence of both orthosteric and allosteric ligands. These structures mechanistically show how binding of the orthosteric ligand leads to positive cooperative binding of the allosteric ligand.

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(1) Eindhoven University of Technology, The Netherlands


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Using chemical biology to expand the druggable proteome

Gerard Drewes
Head of Science, GSK Cellzome, Germany

In 2020 the EMBL Resource Development team and  industry partners of the EMBL Corporate Partnership Programme will bring together academic and industrial scientists with interests in chemical biology, chemogenomic libraries, pharmacology, medicinal chemistry and bioinformatics for the EMBL Conference: Expanding the Druggable Proteome with Chemical Biology (5-7 February 2020).

We spoke to co-organiser Gerard Drewes from GSK Cellzome about how chemical biology is helping to expand the druggable proteome.

How would you define the “druggable proteome”?

This is the fraction of our >20,000 human proteins that can be functionally modulated by a drug. Drugs can be small molecules or large molecules such as therapeutic antibodies. Estimates of how many proteins are “tractable” vary widely, I think there may be around 5,000. Only a subset of these 5,000 would be “druggable” which means that modulating them with a drug will also have a therapeutic benefit.

How are advances in chemical biology helping to expand the druggable proteome?

Small molecules are still the main modality for intracellular targets. Deep pockets, typical for enzymes, are more easily tractable than shallow pockets typical for protein-protein interactions. Chemical biology has developed tools to explore different types of pockets. I am excited in particular by the potential of DNA-encoded libraries, and small fragment approaches with covalent modes of action. Some of these compounds will just be “binders” but these can be made into target degraders as PROTACs.

How can these advances help our understanding of disease biology?

If we had more chemical probes, we could use these in a standardised, controlled way to interrogate target function in cell-based models, organoids, and in some cases animal models. Yes, we have gene editing now, but that is not the same as pharmacological modulation.

We also need in vitro models that translate better to in vivo. Our old immortalised cell lines won’t do, we are going to need more work in primary cells, organoids, etc.

What are the main challenges facing scientists in this field?

Lack of standardised probe sets. Bad probe compounds, e.g. with bad selectivity, are still used and wrong conclusions drawn.

Lack of translational in vitro models.

Why is it important to bring industry and academia together to discuss this topic?

Academia brings creativity, agility, fast progress of new ideas and concepts, thinking out of the box.

Industry sometimes lacks these but knows how to develop a compound into a drug, which requires a host of technologies not readily available to academia. Also, industry requires a new generation of drug targets with better validation, and historically targets are often discovered in academia. Once a target hypothesis exists, academics and industry should ideally collaborate to figure out how to drug it.

What will be the main highlight of this conference?

I see many but still hope to be surprised!

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Best Poster Awards – Precision Health

140 researchers came together recently at the EMBL Advanced Training Centre in Heidelberg, Germany, for the EMBO Workshop: Precision Health: Molecular Basis, Technology and Digital Health (13 – 16 November 2019) to present and discuss the promises and challenges of precision health and the molecular insights necessary to enable a maintenance of wellness and prevention of disease.

Out of the posters presented, 4 were awarded a poster prize based on popular vote. Here we present the poster abstracts of four of the winners.

A computational modelling approach to characterizing postprandial glucose responses in individuals
Balazs Erdos from TiFN Wageningen and MaCSBio, Maastricht University, The Netherlands, PHOTO: Balazs Erdos

Balazs Erdos (1), (2)*, Bart van Sloun (1), (2), Shauna O’Donovan (2), Michiel Adriaens (2), Natal van Riel (3), Ellen Blaak (4), Ilja Arts (2)

The large variability in the dynamic properties of the postprandial glucose response curves in individuals suggest that it is not sufficient to use average values or single time point measures of postprandial glycemia in order to characterize individuals’ glycemic control. Instead, approaches that are capable of capturing the dynamic events are necessary. In this study, we develop personalized computational models based on ordinary differential equations, to describe the glucose and insulin dynamics of individuals in response to an oral glucose tolerance test. We observed that these personalized models are capable of capturing a wide range of glucose and insulin dynamics including normal, prediabetic and type 2 diabetic responses as well as responses from intermediate states.

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(1) TiFN, Wageningen, The Netherlands, (2) Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, Maastricht, The Netherlands, (3) Dept. of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands, (4) Dept. of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, The Netherlands

*E-mail: balazs.erdos@maastrichtuniversity.nl


Predict nephrotoxicity associated with cisplatin-based chemotherapy in testicular cancer patients

Sara Garcia (1), Jakob Lauritsen (2), Zeyu Zhang (3), Mikkel Bandak (2), Marlene Danner Dalgaard (1), Rikke Linnemann Nielsen (1), Gedske Daugaard (2), Ramneek Gupta (1)

In industrialized countries, testicular cancer (TC) is the most common solid tumor in men between 20 and 40 years old and besides being one of the most treatable types of cancer, the long-term side-effects of chemotherapy are worrisome, since they are largely irreversible. Their severity is normally related to the total amount of chemotherapy received, which makes that an important factor to a successful treatment. The standard treatment for TC is 3 cycles of cisplatin, etoposide and bleomycin (BEP), being that the number of cycles can vary between 4-5 or more if the prognosis of the patient is intermediate or poor. Some of the late side-effects include nephrotoxicity, which can be measured by the drop in glomerular filtration rate after the patient follows chemotherapy. Materials and Methods: Integrative machine learning models were built using a dataset of 400 Danish individuals in order to identify clinical and/or genomics features and classify patients at higher risk of developing nephrotoxicity given a treatment of BEP-cycles. Results: First, only clinical features, such as age at the time of treatment, dose of cisplatin, patient’s prognosis, and number of cycles, were considered, and relevant features were selected to use in the classifier (AUC 0.66, SD 0.02). The classifier was then optimized by adding genomics markers, which helped improving the prediction (AUC 0.75, SD 0.02). Conclusions: Therefore, it is proposed a machine learning algorithm which, by helping predicting nephrotoxicity in advance, can benefit to improve chemotherapy efficacy in TC patients. These data driven models can also be applicable to other cancers, such as ovarian, bladder, and lung cancer where more elderly patients are at risk of nephrotoxicity and identification upfront will have direct clinical implications.

Poster currently not available

(1) Technical University of Denmark, Denmark, (2) Copenhagen University Hospital, Denmark, (3) University of Chinese Academy of Sciences, China


Loss of N-glycanase 1 alters transcriptional and translational regulation
Petra Jakob from EMBL Heidelberg, Germany, PHOTO: Petra Jakob

Petra Jakob (1), William Mueller (1), Sandra Clauder-Münster (1), Han Sun (2), Sonja Ghidelli-Disse (3), Diana Ordonez (1), Markus Boesche (3), Markus Bantscheff (3), Paul Collier (1), Bettina Haase (1), Vladimir Benes (1), Malte Paulsen (1), Peter Sehr (1), Joe Lewis (1), Gerard Drewes (3), Lars Steinmetz (1)

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

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(1) EMBL Heidelberg, Germany, (2) Stanford University, United States of America, (3) Cellzome, Germany


Data integration for prediction of weight loss in clinically controlled dietary trials

Rikke Linnemann Nielsen (1), Marianne Helenius (1), Sara Garcia (1), Henrik Munch Roager (2), Derya Aytan (3), Lea Benedicte Skov Hansen (1), Mads Vendelbo Lind (2), Josef Vogt (1), Marlene Danner Dalgaard (1), Martin I Bahl (3), Cecilia Bang Jensen (1), Rasa Muktupavela (1), Christina Warinner (4), Vincent Appel (5), Rikke Gøbel (5), Mette B Kristensen (2), Hanne Frøkjær (6), Morten H Sparholt (7), Anders F Christensen (7), Henrik Vestergaard (5), Torben Hansen (5), Karsten Kristiansen (6), Susanne Brix Pedersen (1), Thomas Nordahl Petersen (3), Lotte Lauritzen (2), Tine Rask Licht (3), Oluf Pedersen (5), Ramneek Gupta (1)

Diet is a key strategy in weight loss management. Advances in omics technologies research allow analyses of determinants of clinical interventions outcomes. We have previously reported diet-induced weight loss in non-diabetic middle-aged Danes in two clinically controlled dietary trials where the content of whole grain or gluten was changed. However, it remains elusive how predictable weight loss is at the individual level. We here classify weight loss responders and non-responders from the whole grain and gluten trials by integrating multi-omics data (host genetics, gut microbiome, urine metabolome) together with physiology and anthropometrics into random forest models. The most predictive models for weight loss included features of diet, gut microbial species and urine metabolites (ROC-AUC:0.84-0.88, model only with diet type ROC-AUC:0.62). Furthermore, we demonstrate that a model ensemble is robust to missing information of microbiome and metabolome profiles given features of physiology (including postprandial response), host genetics and transit-time (ROC-AUC:0.72).

Poster currently not available

(1) Technical University of Denmark, Denmark, (2) University of Copenhagen, National Food Institute, Technical University of Denmark, Denmark, (3) National Food Institute, Technical University of Denmark, Denmark, (4) Harvard University, United States of America, (5) The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark, (6) University of Copenhagen, Denmark, (7) Bispebjerg University Hospital, Denmark


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Best Poster Awards – Seeing is Believing

For the 5th time, the EMBL Advanced Training Centre played host to 466 researchers and imaging specialists at the EMBO|EMBL Symposium: Seeing is Believing – Imaging the Molecular Processes of Life (9 – 12 October 2019), where cutting-edge applications illustrated how imaging can answer biological questions and capture the dynamics of life. 

Out of the 248 posters presented, 2 stood out from the rest and were awarded a poster prize based on popular vote. Here we present the abstracts and posters of the winners.

CalQTrace: Simultaneous Calculation and Quantification of 100,000 immune activation Traces at single-cell resolution using CNN

Liliana Barbieri is a doctoral student at the Biomedical Imaging Doctoral Training Centre, University of Oxford, UK. PHOTO: Liliana Barbieri

Authors: Liliana Barbieri (1), Kseniya Korobchevskaya (2), Azeem Ahmad (3), Huw Colin-York (1), Aurelien Barbotin (4), Glykeria Karanika (1), Loic Peters (5), Isabela Pedroza-Pacheco (4), Angela Lee (1), Lena Cords (1), Anish Priyadarshi (3), Dominic Waithe (6), Jana Kohler (6), Christoffer Lagerholm (6), Balpreet Singh Ahluwalia (3), Marco Fritzsche (2)

Quantification of immune cell activation is essential to the understanding of their effector function. Tracing activation signatures like cellular calcium release and the expression of surface markers in response to activation signals allows the classification of the course of immune cell activation from early triggering events to late differentiation. However, robust quantitative platforms for such measurements represent a major challenge, restricting the analysis to small single-cell population or more recently to cell ensembles with high-dimensional parameter analysis tools. Here, we introduce a combination of a convolutional neural network-based CalQTrace (Calculation and Quantification of Trace) software, together with a Graphical User Interface, and an optical high-throughput light-sheet platform, allowing the simultaneous fully automated quantitation of immune cell activation traces of >100,000 live immune cells. CalQTrace enables user-independent statistically robust classification and quantification of multiple fluorescent activation markers including calcium, CD25+/- expression, and cell viability tracking single cells in space and time within a 5 mm x 5 mm large-field-of-view, opening-up unprecedented insights into physiological activation tracing in living immune cells.

(1) MRC Human Immunology Unit, University of Oxford, United Kingdom
(2) Kennedy Institute for Rheumatology, University of Oxford, United Kingdom
(3) The Arctic University of Norway, Norway
(4) University of Oxford, United Kingdom
(5) University College London, United Kingdom
(6) Weatherall Institute of Molecular Medicine, University of Oxford, United Kingdom

Poster currently not available


Bleaching-insensitive STED microscopy with exchangeable fluorescent probes

Mike Heilemann is a Principal Investigator at Johann Wolfgang Goethe-University, Frankfurt, Germany. PHOTO: Mike Heilemann

Authors: Christoph Spahn (1), Florian Hurter (1), Mathilda Glaesmann (1), Jonathan Grimm (2), Luke Lavis (2), Hans-Dieter Barth (1), Marko Lampe (3), Mike Heilemann (1)

Photobleaching affects image quality and resolution in fluorescence microscopy, and thus limits the extractable information. This is in particular relevant for super-resolution microscopy where typically high laser intensities are used. In order to minimize photobleaching, we repurposed the use of exchangeable fluorescent probes, as used in single-molecule localisation microscopy methods such as Point Accumulation for Imaging in Nanoscale Tomography (PAINT) [1], for STED microscopy. We demonstrate pseudo-permanent labeling of target structures and constant exchange of photobleached fluorophores. This concept allows for whole-cell, 3D, multi-color and live-cell STED microscopy [2]. Using transiently binding hydrophobic dyes and fluorophore-labeled major minor groove binders [3, 4], we visualised the nanostructure of chromatin, cell membranes and organelles in bacterial and mammalian cells in 3D. To expand the range of targets, we employed oligonucleotide-labeled antibodies that transiently bind fluorophore-labeled oligonucleotides, as used in single-molecule super-resolution imaging with DNA-PAINT [5], and demonstrate multi-color STED imaging.

References:
[1] Sharanov and Hochstrasser, PNAS 103 (50), 18911-18916 (2006)
[2] Spahn et al., Nano Letters 19 (1), 500-505 (2019)
[3] Lukinavičius et al., Nature Communications 6, 8497 (2015)
[4] Spahn et al., Scientific Reports 8, 14768 (2018)
[5] Schnitzbauer et al., Nature Protocols 12(6), 1198-1228 (2017)

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(1) Johann Wolfgang Goethe-University Frankfurt, Germany
(2) HHMI – Janelia Research Campus, United States of America
(3) EMBL Heidelberg, Germany


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Creating is Understanding: Synthetic Biology Masters Complexity – Best Poster Awards

The recent EMBO Workshop: Creating is Understanding: Synthetic Biology Masters Complexity (22 – 25 Sep) covered various themes that are geared toward basic research while being at the forefront of synthetic biology.

110 researchers came together at the EMBL Advanced Training Centre in Heidelberg, Germany for 3,5 days of talks, posters and networking. Here we present the work of 4 scientists who received best poster awards at the conference by popular vote.

Engineering portability of the CcaSR light switch for the control of biofilm formation in Pseudomonas putida

Angeles Hueso-Gil is a PhD researcher at the Spanish National Centre for Biotechnology in Madrid.

Authors: Angeles Hueso-Gil (1), Ákos Nyerges (2), Csaba Pál (2), Belén Calles (1), Victor de Lorenzo (1)

Two of the technical challenges faced by contemporary microbiology involve controlling gene expression using light and regulating bacterial biofilm formation, determined by the intracellular levels of the secondary messenger c-di-GMP. CcaSR system is one of the light switches repeatedly used for transcription induction in Escherichia coli. This two-component system represented a good candidate for its adaptation to Pseudomonas putida. Previous attempts have tried to use this microorganism as chassis for the implementation of new pathways, being biofilm formation an important function to control. To this end, we unified CcaSR components in one single construct and randomly mutagenized their regulatory regions to find a clone with a balanced expression of the system key parts inside P. putida. The combination of this novel mutagenization process with a proper screening, which included a first sorting of the libraries and the later isolation of colonies, lead us to a clone with a much improved induction by green light. The selected variant had a notable capacity in response to green light. Finally, optimized CcaSR was used to control the expression of super-efficient variant of PleD, a diguanylate cyclase of Caulobacter which allowed a tight control of c-di-GMP levels, and therefore, of biofilm production.

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(1) National Centre for Biotechnology, Spain
(2) Biological Research Centre of the Hungarian Academy of Sciences, Hungary

Designer membraneless organelles enable orthogonal translation in eukaryotes

Christopher Reinkemeier is a PhD student at EMBL Heidelberg, JGU Mainz and IMB Mainz, Germany

Christopher Reinkemeier (1,2,3), Gemma Estrada Girona (3), Edward A. Lemke (1,2,3)

Genetic code expansion is a powerful tool to study and control protein function with single-residue precision. It is widely used to e.g. perform labeling for microscopy or to photocontrol proteins. This is achieved by introducing an orthogonal tRNA/synthetase suppressor pair into the host, to recode a stop codon to incorporate a noncanonical amino acid (ncAA) into the nascent chain. This technique is codon-specific, but it cannot select specific mRNAs, so naturally occurring stop codons could be suppressed leading to potential interference with housekeeping translation. Nature avoids cross-talk between cellular processes by confining specific functions into organelles. We aimed to design an organelle dedicated to protein engineering, but as translation is a complex process requiring hundreds of factors to work together, membrane-encapsulation would not be feasible. Inspired by the concept of phase separation we hypothesized that such an organelle could instead be designed membraneless. Phase separation can generate high local concentrations of proteins and RNAs in cells and has recently gained attention owing to its role in the formation of specialized organelles such as nucleoli or stress granules. Despite being membraneless and constantly exchanging with the cytoplasm/nucleoplasm, these organelles still perform complex tasks, such as transcription. We combined phase separating proteins with microtubule motor proteins to generated orthogonally translating organelles in living cells that contain an RNA-targeting system, the stop codon suppression machinery and ribosomes. These large organelles enable site- and mRNA-specific ncAA incorporation, decoding one specific codon exclusively in the mRNA of choice. Our results demonstrate a simple yet effective approach to the generation of semi-synthetic eukaryotic cells containing artificial organelles to harbor two
distinct genetic codes, providing a route towards customized orthogonal translation and protein engineering.

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(1) Johannes Gutenberg University Mainz, Germany
(2) Institute of Molecular Biology, Germany
(3) EMBL Heidelberg, Germany

Metabolic perceptrons for neural computing in biological systems

Paul Soudier is a PhD Student at the French National Institute of Agricultural Research, France

Amir Pandi (1), Mathilde Koch (1), Peter Voyvodic (2), Paul Soudier (1), Jerome Bonnet (2), Manish Kushwaha (1), Jean-Loup Faulon(1)

Synthetic biological circuits are promising tools for developing sophisticated systems for medical, industrial, and environmental applications. So far, circuit implementations commonly rely on gene expression regulation for information processing using digital logic. Here, we present a new approach for biological computation through metabolic circuits designed by computer-aided tools, implemented in both whole-cell and cell-free systems. We first combine metabolic transducers to build an analog adder, a device that sums up the concentrations of multiple input metabolites. Next, we build a weighted adder where the contributions of the different metabolites to the sum can be adjusted. Using a computational model fitted on experimental data, we finally implement two four-input of metabolite combinations by applying model-predicted weights to the metabolic perceptron. The perceptron-mediated neural computing introduced here lays the groundwork for more advanced metabolic circuits for rapid and scalable multiplex sensing.

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(1) French National Institute of Agricultural Research, France
(2) INSERM, France

Programmed uptake of biomacromolecules into protocells

Wiggert Altenburg is a PhD student at the Eindhoven University of Technology, The Netherlands

Wiggert Altenburg, Amy Yewdall, Daan Vervoort, Alex Mason, Jan van Hest

The bottom up recreation of cellular processes into synthetic compartments has, in recent years, emerged as an exciting line of research with which to study biological processes in a controlled environment. However, the interior of a living cell is a difficult milieu to mimic in bottom-up synthetic cells, as it is an environment crowded with high concentrations of many different biomacromolecules. In this work, we describe the development of a powerful new tool to more accurately emulate the cell cytosol in discrete coacervate-based protocells. The coacervate core utilized herein not only provides an inherently crowded and highly charged microenvironment, but has also been chemically modified to interact specifically with recombinantly expressed proteins. Our method leverages the well-established binding of His-tagged proteins to Ni2+-nitrilotriacetic acid, which ensures that macromolecules are taken up in a highly efficient, yet gentle manner, thus preserving biological activity. The straightforward method allowed for both control over the amount taken up and an increased local concentration. Moreover, the engineered uptake of proteins was then employed to study two key aspects: the effect of the Ni-NTA interaction on the diffusivity of incorporated proteins, and the enhancement in activity of an encapsulated two-enzyme cascade. This direct and targeted method of protein uptake into a discrete, membrane bound platform is a significant step forward for synthetic cells, and will enable the engineering of highly complex enzyme and signaling networks with increasingly life-like properties.

Poster currently not available

Eindhoven University of Technology, The Netherlands

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