Best poster prizes at ‘Recent advances in structural biology of membrane proteins’

Over 60 posters were presented at the virtual EMBO Workshop ‘Recent advances in structural biology of membrane proteins’, highlighting the importance of lipids in MP research and how they can be preserved. There were two live poster sessions and the presenters could also be contacted via chat, message or video call throughout the conference – their work was then voted for by other attendees and speakers. We are pleased to be able to share with you the research from the three winners of the best poster prizes: congratulations to Kevin, Natalie and Karthik!

Cryo-EM Snapshots of Nanodisc-Embedded Native Eukaryotic Membrane Proteins

Presenter: Kevin Janson

Kevin Janson, Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Germany
Abstract

New membrane complex purification technologies in combination with cryo electron microscopy (cryoEM) recently allowed the exploration of near native membrane protein complex architectures. Polymer nanodiscs in particular provide the basis to study overexpressed membrane proteins at high resolution while retaining protein—protein and protein—lipid interactions. However, how the majority of endogenous membrane proteins are organized remains elusive, mainly due to the inherent complexities that a hydrophobic environment poses to biochemical preparations.
In this work, we combined biochemical enrichment protocols for native membrane complexes together with amphiphilic polymers to increase the quality of recovered endogenous membrane complexes. The derived protein encapsulated nanodiscs were identified by mass spectrometry and imaged with cryoEM. This set of technologies is applied to Chaetomium thermophilum, a thermophilic fungus, that confers additional advantages for protein structure determination due to the increased thermal stability of its biomolecular assemblies.
Our results show a highly efficient recovery of protein encapsulating nanodiscs, amenable to structural and biophysical characterization with a multitude of methods. Initial mass spectrometry results reveal ~1300 proteins while multiple 2D class averages from cryoEM data show prominent nanodisc embedded structural signatures. This combined methodological approach to isolate multiple endogenous membrane protein complexes provides unprecedented opportunities for a deeper understanding of the membrane proteome of a eukaryote.

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Structural studies and inhibition of essential pathogenic secondary active transporters with synthetic nanobodies and solid supported membrane electrophysiology

Presenter: Natalie Bärland

Natalie Bärland, University of Basel, Switzerland
Abstract

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of membrane proteins. Additionally, to their traditional use in aiding structural elucidation they harbour a great potential as tools for clinical therapies. Nanobodies are specifically selected to target the protein of interest with high affinities and are capable of trapping specific states with inhibitory potential. A high throughput identification of inhibitory nanobodies is essential for subsequent studies and often challenging if a technique consumes large amounts of sample or if the required labelling for their characterisation is costly. Solid supported membrane (SSM) electrophysiology has been a fast way to screen and characterise electrogenic transport of compounds and can be expanded for the identification of putative nanobody inhibitors. Here we show a combination of structural studies assisted by synthetic nanobodies (sybodies), selected by using SSM electrophysiology to screen and identify inhibitors. As our model protein we used the secondary active transporter LicB, which is a choline importer essential for the survival of the pathogen Streptococcus pneumoniae in the host. S. pneumoniae is a highly invasive pathogen that can exhibit multiple resistances to antimicrobials and remains to be a burden for society. The methods described here can be applied for the characterization of any electrogenic transporter. It can help to screen and select with a high throughput putative inhibitory sybodies and help in the development of novel drugs and drug targets.

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Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family

Presenter: Karthik Ramanadane

Karthik Ramanadane, University of Zurich, Switzerland
Abstract

Divalent transition metals are essential nutrients whose uptake is mediated by membrane transporters of the SLC11/NRAMP family. Over the last 25 years, family members have been characterized as proton coupled divalent transition metal symporters, a property, which was later supported in structural and functional studies of prokaryotic homologues. In this study, we have combined the phylogenetic analysis of SLC11 proteins with their biochemical characterization to show that the transition metal ion transporters only represent a fraction of the SLC11/NRAMP family. After extensive screening, we have identified a family member with diverging properties, which was successfully purified and reconstituted. In transport studies, we were able to classify this protein as a prokaryotic NRAMP related magnesium transporter from Eggerthella lenta (ElenNRMT). Unlike known members of the SLC11/NRAMP family, ElenNRMT does not co transport protons along with its primary substrate. These observations are partly contradicting previous studies on the substrate selectivity within this family. To gain insight into the structural basis of the distinct substrate selectivity, ElenNRMT was characterized by single particle cryoEM in absence and presence of Mg2+ at respective resolutions of 3.5 Å and 4.1 Å. Using X ray crystallography, we were able to confirm the location of the bound substrate using the anomalous scattering properties of Mn2+. In all structures, ElenNRMT adopts an inward facing conformation revealing an ion binding site that differs from classical NRAMP transporters in its volume and the distinct residues mediating ion interactions. Together, these results define the determinants of the diverse selectivity in the SLC11/NRAMP family and provide insight into its evolution.

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Congratulations to all three winners!

The EMBO Workshop ‘Recent advances in structural biology of membrane proteins’ took place from 29 November – 1 December 2021.

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Best Poster Flash Talk winners of the Mobile Genome

This year, the virtual EMBO Workshop: “the Mobile Genome: Genetic and Physiological Impacts of Transposable Elements” centered around the broad impacts of TEs on organismal biology. It turned out to be a diverse meeting with interesting cross-disciplinary discussions, assembling experts from diverse fields including genomics, epigenetics, structural biology, developmental biology, immunology, cancer biology and neurobiology.

Over 90 posters were available on the virtual platform and three presenters stood out with their poster flash talks. An extraordinary accomplishment in the virtual format. With no further ado, we are introducing the winners and their research!

The repressive SCAN zinc-finger protein family targets transposable elements

Presenter: Wayo Matsushima

Wayo Matsushima, École Polytechnique Fédérale de Lausanne, Switzerland 
Wayo Matsushima, École Polytechnique Fédérale de Lausanne, Switzerland

Abstract

Transposable elements (TEs) contribute to genome innovations through insertions of coding and non-coding elements. KRAB zinc-finger proteins (KZFPs) function as sequence-specific repressors by recruiting KAP1/TRIM28, a scaffold protein of a heterochromatin-inducing complex, to TE-derived sequences. An ancestral KZFP gave rise to another family of transcription factors through the exonisation of the gag gene from the Gmr1-like transposon family as the SCAN domain. This SCAN zinc-finger protein (SZFP) family experienced multiple rounds of segmental duplication events, resulting in the presence of ~300 family members in diverse amniote genomes. Despite its abundance and evolutionary proximity to KZFPs, the functions of the SZFP family are still not well understood.

To analyse evolutionary conservation of SZFPs, we compared the DNA-contacting zinc-finger amino acid sequences of the human SZFPs to those of 65 other amniote species. This revealed that the zinc finger signatures of SZFPs are under rapid lineage-specific selection, and that several human SZFPs harbour primate-specific zinc finger sequences.

To characterise the genomic targets of all of the 55 human SZFPs, we performed ChIP-seq on 293T cell lines, each expressing one of the SZFPs with an HA-tag. We found that the binding sites of a number of SZFPs significantly overlap with specific TE subfamilies, and further observed that the evolutionary ages of SZFPs and their TE targets often matched, suggesting the TE-driven positive selection of this transcription factor family.

We next performed luciferase assays to study the regulatory function of SZFPs. We discovered that SZFPs act as transcriptional repressors owing to their SCAN domain with its conserved C-flanking 15 amino acid residues. Similar levels of repression were obtained in KAP1/TRIM28 knockout cells, demonstrating that, in contrast to KZFPs, SZFPs act independently of this master corepressor.

Together, this study identifies SZFPs as putative controllers of the regulatory potential of TEs, through their lineage-specific zinc finger repertoire and the repressive domain derived from the co-opted retroviral gene. Future work will explore the potential impact of SZFPs and their TE targets on species-specific gene regulatory networks.

The poster contains unpublished data and can therefore not be published. Follow Wayo Matsushima on Twitter for more information on his projects.

A natural transposon affects gene regulation and fitness related traits depending on the developmental stage and environmental conditions in D. melanogaster

Presenter: Miriam Merenciano

Miriam Merenciano, Institute of Evolutionary Biology (CSIC-UPF), Spain

Abstract

TEs have been considered a genome-wide source of regulatory elements capable of regulating nearby gene expression. In Drosophila melanogaster, the FBti0019985 natural TE insertion has been previously reported to add a transcription start site to the Lime transcription factor. In this work, we performed invivo enhancer assays and gene expression analysis with CRISPR/Cas9 mutants and natural populations to explore the effects of FBti0019985 on Lime expression under different stress conditions and different developmental stages. We found that this insertion acts as an enhancer in the adult stage under immune-stress conditions. Indeed, the deletion of predicted immune-related binding sites in the TE significantly reduces its enhancer activity in infected conditions, confirming that it harbors functional cis-regulatory elements. We also found that the TE upregulates Lime in embryos, however, in this case we could not pinpoint the molecular mechanism. Finally, we found that TE-induced Lime upregulation was associated with tolerance to bacterial infection and with increased egg-to-adult viability. Our results suggest that different developmental stages and environmental conditions should be tested in order to fully characterize the molecular and functional effects of a genetic variant.

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An ATPase filament bridge: How a transposon and CRISPR stick together

Presenter: Irma Querques

Irma Querques, University of Zurich, Switzerland

Since the discovery of bacterial adaptive immunity, CRISPR-Cas systems have been mainly regarded as a mechanism to counteract horizontal transfer of mobile genetic elements including transposons in prokaryotic genomes. Conversely, a distinct family of Tn7-like elements co-opted CRISPR-Cas RNA-guided machineries to direct transposon insertion into specific target sites. In type V CRISPR-associated transposons, RNA-directed transposition relies on the cross-talk between the pseudonuclease Cas12k, the transposase TnsB, the zinc-finger protein TniQ and the ATPase TnsC. Yet, the molecular mechanisms underpinning this interplay have remained unknown. Here we present a cryo-electron microscopy structure of DNA-associated TnsC in its ATP-bound state. The structure reveals that the AAA+ ATPase forms an ATP-dependent helical filament that encloses and remodels the underlying target DNA. One strand only of the duplex is tracked by consecutive TnsC protomers with an unexpected two-nucleotide periodicity, resulting in a DNA helix with 12 base pairs per turn. Biochemical studies show that TnsC polymerization is a critical aspect of the system that enables the coupling of RNA-guided target recognition by Cas12k with the downstream recruitment of TnsB by direct protein interactions. In turn, TnsB triggers filament disassembly upon ATP hydrolysis, establishing target immunity. We also show that TniQ directly interacts with TnsC, restricting its polymerization. A crystal structure of TniQ reveals structural diversity within the TniQ protein superfamily, further suggesting a role in TnsC regulation rather than DNA targeting. Together, our data point to a mechanistic model whereby TnsC oligomers bridge between the RNA-guided target selector Cas12k and the transpososome, promoting target DNA remodeling and ultimately transposon integration. This work discloses first mechanistic insights into targeting and regulation of type V CRISPR-associated elements and will guide the rational design of these systems as programmable, site-specific gene insertion tools.

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The EMBO Workshop: “The Mobile Genome: Genetic and Physiological Impacts of Transposable Elements” took place from 29 August – 1 September 2021. 

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Seeing is Believing Best Poster Awards

From its beginning in 2011, Seeing is Believing has embraced novel imaging technologies that open new windows for biological discovery, including single-molecule and super-resolution, light sheet and correlative light electron microscopy. This year, the EMBO| EMBL Symposium was held virtually for the first time, and it did not disappoint! We had around 640 participants, with over 130 posters presented during the digital poster sessions. We are excited to share with you the research from the five best poster prize winners, who were also given the opportunity to share their work with a talk at the end of the meeting!

 

A novel view on protein-protein interaction: Investigating protein-complex formation using correlative dual-color single particle tracking
Presenter: Tim Abel

Tim Abel, Max Delbrück Center for Molecular Medicine in the Helmholtz Association

Abstract

Characterization of protein-protein interactions is one of the central aspects, when investigating cellular mechanisms. Most of the established biochemical techniques to describe protein-interactions rely on the isolation and purification of the proteins of interest. This becomes especially problematic, when analyzing membrane-localized complexes or transient and dynamic interactions, which are difficult to isolate or easily disrupted. In this project, we use dual-color single particle tracking to investigate the formation of the ER-membrane resident multi-protein complex Hrd1 in living cells.

The Hrd1-complex is one of the central ubiquitin ligase complexes in endoplasmic reticulum-associated degradation (ERAD). During ERAD misfolded or otherwise faulty proteins are transported from the ER to the cytosol and targeted for degradation, which both involves the name-giving subunit of the Hrd1-complex Hrd1p. Biochemical analysis indicates that this protein forms homo-oligomers but the stoichiometry within the functional complex and the dynamics of its formation remains disputed.

To assess Hrd1p-oligomerization using live-cell imaging we labeled endogenous Hrd1p with either the SNAP- or the Halo-tag using CRISPR/Cas9 mediated gene integration. Both variants remained fully functional and enabled labeling using dyes compatible with SM-imaging. TIRF microscopy revealed single diffraction limited spots. When labeling competitively with differently colored halo-ligand-conjugates, Hrd1p-punctae exhibited strong correlated movement between both colors indicative of homo-oligomerization. Its degree of interaction was quantified by directly correlating single-steps extracted from the SM-trajectories, which proved to be a robust way to analyze protein-protein interactions. Additionally, PALM-imaging of a Halo-labeled Hrd1-substrate was able to directly show its interaction with Hrd1p-SNAP by correlated movement on the SM-level. In the future we will use this correlative SM-approach in combination with genetic and inhibitor based interventions to interrogate factors required for Hrd1p-complex assembly, its formation dynamics and how substrates are recruited.

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HaloTag9: An engineered protein tag for fluorescence lifetime multiplexing
Presenter: Michelle Frei

Michelle Frei, Max Planck Institute for Medical Research, Heidelberg, Germany and EPFL, Switzerland

Abstract

Self-labeling protein tags have become important tools in fluorescence microscopy. Their use in combination with fluorogenic fluorophores, which only become fluorescent when bound to their protein target, makes them particularly suitable for live-cell applications. The fluorogenic turn-on observed upon labeling as well as the photophysical properties of the fluorophore are mainly determined by the protein surface near the fluorophore binding site. However, up to now, most efforts have been invested in the development of new fluorophores and only little attention has been paid to the engineering of the self-labeling protein tag.

Here we report on the engineering of HaloTag7 to modulate the brightness and fluorescence lifetime of bound rhodamines. Specifically, we developed HaloTag9, which showed up to 40% higher brightness in cellulo and 20% higher fluorescence lifetime than HaloTag7 upon labeling with rhodamines. This makes it an ideal tag for imaging techniques such as confocal microscopy or stimulated emission depletion microscopy. In addition, combining HaloTag7 and HaloTag9 enabled us to perform live-cell fluorescence lifetime multiplexing using a single fluorophore. The difference in fluorescence lifetime was further exploited to generate a chemigenetic fluorescence lifetime based biosensor to monitor cell cycle progression. Overall, our work highlights that the combination of protein engineering and chemical synthesis can generate imaging tools with outstanding properties. We expect HaloTag9 to be beneficial for a multitude of live-cell microscopy applications.

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Asymmetric nuclear division generates sibling nuclei with different identities
Presenter: Chantal Roubinet

Chantal Roubinet, MRC-LMCB, University College London, UK and MRC-LMB, Cambridge, UK

Abstract

Although nuclei are defining features of eukaryotes, we still do not fully understand how the nuclear compartment is duplicated and partitioned during division. This is important, as the mechanism of nuclear division differs profoundly across systems. In studying this process in Drosophila neural stem cells, we recently found that the nuclear compartment persists during mitosis due to the maintenance of a mitotic nuclear lamina. This bounding spindle envelope is then asymmetrically remodelled and partitioned at division, giving rise to two daughter nuclei that profoundly differ in size (physical asymmetry) and envelope composition (molecular asymmetry). The asymmetry in the size of daughter nuclei following division results from: i) an asymmetric nuclear envelope resealing at mitotic exit that depends on the central spindle, and ii) a differential nuclear growth in early G1 that depends on the availability of ER/nuclear membranes reservoir in the cytoplasm. Furthermore, my data show that asymmetric nuclear division in this system is associated with a different chromatin organization between the two daughter nuclei as well as with an asymmetric distribution of several histone marks, before the cortical release of cell fate determinants. This suggests that the asymmetric remodelling of the nuclear envelope has profound functional consequences for these stem cell divisions. Taken together, these data make clear the importance of considering the path of nuclear re-modelling when investigating how a stem cell division generates two distinct sibling cells with different identities/fates.

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A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis
Presenter: Aline Tschanz
Aline Tschanz, EMBL Heidelberg, Germany

Abstract

Clathrin Mediated Endocytosis (CME) is one of the most trafficked endocytic pathways in a cell and is involved in a multitude of processes including cell signaling, nutrient uptake and membrane homeostasis. During CME, extracellular material becomes progressively engulfed by the plasma membrane (PM) until a vesicle is formed and released into the cytosol.

Whilst many endocytic components have been extensively studied, the underlying mechanism by which protein assembly drives membrane invagination is not entirely understood. This is in parts due to technical limitations in the visualization of the endocytic machinery in-situ.

Here, we use 3D superresolution microscopy to resolve clathrin coated pits (CCP) in fixed mammalian cells. We are able to resolve thousands of CCPs, each representing a distinct snapshot of the overall endocytic process. By applying a spherical model fit to thousands of individual CCPs, we can quantitatively describe and compare the population of endocytic sites. We further use the extracted parameters to sort individual CCPs along their endocytic progression and reconstruct the dynamic remodeling of the clathrin coat.

We find that both the curvature as well as the surface area of these structures increase during CME. This motivates a refinement of the currently proposed models used to approximate the process of membrane bending. Our findings further allow for the formulation of new physical models describing the underlying mechanism of force generation by protein assembly at the PM.

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Correlative imaging of high-speed atomic force microscopy and fluorescence microscopy revealed asymmetric closing process of endocytosis
Presenter: Yiming Yu

Yiming Yu, Kyoto University, Japan

Abstract

High-speed atomic force microscopy (HS-AFM) has been a powerful tool for visualizing various biological processes at a single-molecule level. Our group had developed a unique type of correlative imaging system by combining HS-AFM for live-cell imaging and confocal laser scanning microscopy to simultaneously visualize dynamic morphological changes of the cell membrane and protein dynamics in living cells. By using this system, we analyzed the molecular process of clathrin-mediated endocytosis (CME), in which more than 60 different proteins assemble on the plasma membrane to produce a clathrin-coated pit (CCP) with a diameter of ~100 nm by inducing a series of morphological change of the plasma membrane. A unique membrane morphology was frequently observed at the closing step of the CME; a small bulge of the plasma membrane grew besides the CCP and finally closed the pit in an asymmetric manner, which is distinct from the constricting motion induced by dynamin. After a screening of siRNA against CME-related proteins and inhibitors for actin-related proteins, we found that a strong self-assembly of a BAR-domain containing protein near the CCP recruited actin and promoted its polymerization and branching. Such an asymmetric growing of actin filaments near the CCP promoted the closing step of the CME by generating lateral force against the pit. These results demonstrated a clear advantage of the correlative imaging system of HS-AFM and fluorescence microscopy in analyzing nano-scale events on the plasma membrane.

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Multiomics to Mechanisms Best Poster Awards

The EMBO|EMBL Symposium: Multiomics to Mechanisms: Challenges in Data Integration took place virtually 15 – 17 September 2021. With over 400 participants, this was the biggest multi-omics conference since it began in 2017. We had 96 posters presented virtually, and are excited to share the research from the three best poster prize winners. 

Identification of transcription factor signaling molecules by coupling gene expression and metabolomics

A portrait picture of Daniela Ledezma‑Tejeida
Daniela Ledezma‑Tejeida, ETH Zurich, Switzerland (Photo credit: Stefania Laddage)

Abstract

Bacteria need to adapt to changes in their environment in order to survive. Transcription factors (TFs) bind metabolites that signal such changes and in turn alter gene expression. Escherichia coli has the best characterized transcriptional regulatory network involving 300 predicted TFs, of which ~75% have a metabolite‑binding domain. However, the binding partners of only 95 TFs have been identified due to low-throughput of common in vitro identification methods. Here, we combined metabolomics and gene expression data obtained in vivo across several growth conditions to identify TF‑metabolite interactions of four TFs without a known binding partner: CdaR, CsgD, FlhDC and GadX. We have validated our method by accurately predicting the known binding partners of ArgR, TyrR and CysB, three highly studied TFs. The in vivo nature of our approach can not only identify new TF‑metabolite interactions but also provide insight into the most functionally relevant.

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Towards topology‑based multi‑omics pathway enrichment and its application in toxicology

A portrait picture of Sebastian Canzler, Helmholtz‑Centre for Environmental Research
Sebastian Canzler, Helmholtz‑Centre for Environmental Research ‑ UFZ, Germany

Abstract

The call for an application of (multi‑)omics data in toxicology became highly prominent in recent years, since omics experiments are intended to generate comprehensive information on molecular changes in cells and tissues more quickly, more accurately, and with fewer resources than ever before. The associated hopes explicitly include the reduction of live animal testing and an increased number of analyzed substances that can be tested. Therein, multi‑omics data are essential to comprehensively infer mechanistic knowledge on molecular response pathways to subsequently guide and aid chemical risk assessment. However, currently available multi‑omics pathway enrichment methods struggle to cope with different aspects hampering their application in computational toxicology, e.g., the utilization of insufficient enrichment methods, missing support for time‑ and concentration resolved data, and restrictions on the pathway sources. Most approaches utilize a sequential data integration and thereby completely ignore the connections between different omics layers. With ToPaFC, we present the first step towards a consistent and simultaneous multi-omics-based pathway enrichment that accounts for those obstacles and explicitly takes the underlying pathway topology into account. Right now, we can deal with up to eight different pathway databases and two omics layers (trans/meta or prot/meta). The pathway topology is reflected in two different ways: i) the importance of a node (omics feature) is measured based on its connections and its relative localization within the pathway and ii) the influence of each node on the network is specified by the weight of its outgoing edges, whether they are inhibiting, neutral, or activating. With this integration of edge information along the pathway, our method inherently accounts for consistent molecular changes of the features. The derived node‑centered pathway representation is combined with measured multi‑omics features to calculate a topology‑based pathway fold change that accounts for consistent changes within the molecular response.

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Computational approaches to scrutinize results from spatial proteomics of operable pancreatic cancer and neighboring tissue

A portrait picture of Ábel Szkalisity, University of Helsinki, Finland
Ábel Szkalisity, University of Helsinki, Finland

Abstract

The advance of laser‑microdissection technologies coupled with proteomics enables unprecedented insights into tissue proteomes. However, the limited availability of patient materials coupled with the high dimensional output of proteomics necessitates data integration across studies to safeguard the reliability of the results. We microdissected morphologically benign and neoplastic pancreas and surrounding stromal areas from 14 patients with early pancreatic ductal adenocarcinoma and analyzed their protein compositions with nLC‑MS/MS. The results indicated downregulated digestive functions in the malignant exocrine tissue and lower metabolic activity in the stroma vs. exocrine pancreas. Intriguingly, the majority of the most significant proteins for survival originated from the morphologically benign exocrine regions, suggesting that these areas may harbor early, predisposing changes. To scrutinize this idea, we compared their proteomes to proteomics data of 12 healthy control pancreatic samples obtained from publications. The protein identification and quantification pipeline from the raw mass spectrometer files were standardized to minimize variation introduced by search engines or protein sequence databases. Altogether, we identified 7,099 proteins in 67 samples involving 5 tissue types from 2 experiments and 5 batches. We investigated two independent strategies for rendering the values comparable. First, batch effects within experiments were corrected for with ComBat and the abundances across experiments were aligned with housekeeping protein normalization. However, this approach required full observations, removing over 90% of the identified proteins from the analysis. Hence, our second approach involved applying Group Factor Analysis to directly extract factors that reveal relationships between the tissue types in our study without compromising the protein coverage. These approaches not only showed that our main results are independent of the data analysis pipeline but also implicated changes in the mRNA splicing machinery as important players in pancreatic cancer. By surveying 165 patients from The Cancer Genome Atlas we revealed that increased transcriptional complexity indeed associates with poor survival in this disease.

View Ábel’s Poster

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Best poster awards – BioMalPar XVII

This year,  the BioMalPar conference took place for the 17th time, but the second time virtually. Three poster presenters stunned their peers with their visually attractive digital posters, presentations and research insights. Out of 90 posters, they received the best poster award by popular vote. Meet the winners!

Sex-specific genetic screens identify hundreds of Plasmodium fertility genes essential for the transmission of malaria parasites

Claire Sayers, Umea University, Sweden

Abstract

Sexual reproduction of malaria parasites is essential for their transmission by mosquitoes. Biological processes required for Plasmodium fertility include the formation of gametocytes, their transformation into gametes in response to signals from the mosquito, fertilisation in the bloodmeal, meiosis, and the formation of an invasive ookinete. Stage-specific gene expression data suggest that hundreds of parasite genes are uniquely required for sexual reproduction, but previous gene knockout studies have merely scratched the surface of this important aspect of parasite biology. We have mutagenised P. berghei lines that make only fertile male or only fertile female gametocytes, with barcoded PlasmoGEM vectors to screen >1200 targetable genes for sex-specific phenotypes. Our screens identify hundreds of genes with sex-specific roles. The data recapitulate existing knowledge of Plasmodium fertility and assign functions to previously unannotated genes. For the first time, we are gaining an unbiased picture of the molecular mechanisms of Plasmodium fertility at genome-scale, which will lead to a deeper understanding of this novel biology that could serve as targets for transmission blocking drugs or vaccines.

View Claire Sayers’ poster

ABCI3 confers pleiotropic drug resistance to antimalarial compounds

Emma Carpenter, Wellcome Sanger Institute, UK

Abstract

Understanding the mechanisms available to the malaria parasite for acquiring multidrug resistance will be important for predicting which genes may become important for clinical resistance in the future.
ABC transporters are an important protein family with roles in drug resistance across a variety of organisms, and mutations in PfMDR1 modulate sensitivity to multiple antimalarials. Several other ABC transporters are encoded in the Plasmodium genome, and we have identified mutations in ABCI3 that confer resistance to several experimental antimalarial compounds.
Using in vitro drug selection regimes with a set of four chemically related compounds (SY4, 10, 11, 13), we isolated 12 drug resistant lines that were subjected to whole genome sequencing. All contained either single nucleotide variants (SNVs) or copy number amplifications of abci3. The point mutations were located in or near predicted transmembrane domains, consistent with a role in modifying the substrate specificity of the transporter, and testing of these lines against other compounds chemically-unrelated to the SY series identified a subset to which sensitivity is also affected.

In addition, natural variants of ABCI3 are observed at or near to these putative resistance SNVs, and preliminary evidence indicates differing sensitivities to the SY compounds among field isolates and common lab strains that may be driven by variation in ABCI3.

This work suggests abci3 should be among the genes monitored for changes in prevalence in longitudinal sampling of field isolates.

View  Emma Carpenters’ Poster

Characterization of a new malaria vaccine candidate against Plasmodium vivax using genetically modified rodent malaria parasites

Diana Moita, Instituto de Medicina Molecular João Lobo Antunes, Portugal

Abstract

Malaria, a mosquito-borne disease caused by Plasmodium parasites, is the most prevalent parasitic infection worldwide. Despite considerable efforts, there is still no effective vaccine against human-infective Plasmodium parasites, of which P. falciparum (Pf) and P. vivax (Pv) are the clinically most significant. Whole-sporozoite (Wsp) vaccines, which induce efficient immune responses against the pre-erythrocytic (PE) stages of Plasmodium parasites, are among the most promising immunization strategies so far. Although most malaria vaccine research has focused on Pf infection, Pv continues to be the most widespread of the human-infective Plasmodium species, imposing significant health and economic burdens on affected countries. Importantly, Pv can originate dormant parasitic liver forms – hypnozoites – which may cause malaria relapses long after mosquito transmission. Recently, our lab developed a new Wsp based on the use of transgenic rodent P. berghei (Pb) parasites as a platform to deliver immunogens of human-infective Plasmodium parasites. Since our in silico studies predict that >60% of CD8+ T cell epitopes encoded in both the Pv and Pb proteomes are shared between these two parasites, we generated a new genetically modified Pb expressing the highly immunogenic circumsporozoite (CS) protein from Pv (PvCS), in addition to its endogenous CS, Pb(PvCS@UIS4), to be used as a vaccine candidate against Pv malaria. Our immunofluorescence microscopy studies confirmed that both the endogenous PbCS and the inserted PvCS are expressed during the PE stages of this transgenic parasite, and that its infectivity is similar to that of its wild-type (WT) counterpart. Specifically, the ability of Pb(PvCS@UIS4) to infect Anopheles stephensi mosquitoes, as measured by the number of oocysts or sporozoites formed, as well as its ability to infect and develop normally in mouse hepatocytes and red blood cells showed no significant differences from those observed for WT parasites. Subsequent studies showed that mice immunization with Pb(PvCS@UIS4) elicits the production of anti-PvCS antibodies that efficiently recognize and bind to Pv sporozoites. Considering the lack of efficient strategies to tackle Pv, this study represents a crucial step on the development of a new Wsp vaccine candidate against this parasite.

View Diana Moita’s poster

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