Conference CEEPC/IPM/CMSC 2022 (Day 3)
CSMS: Conference CEEPC/IPM/CMSC 2022 (Day 3)
Program CEEPC/IPM/CMSC 2022
CSMS: Conference CEEPC/IPM/CMSC 2022
Saturday October 1, 2022
8:50 - 10:50 Session V – Applications in Biomedicine 2
- Chairperson: Haci Mehmet Kayili
8:50-9:10 Proteomic Analysis of Hepatitis B Virus
- Martin Hubálek
According to the World Health Organisation, an estimated 296 million people worldwide are infected with Hepatitis B virus (HBV). HBV is a small enveloped DNA virus from Hepadnaviridae family that persists in the infected hepatocytes by establishing an episomal covalently closed circular double-stranded DNA (cccDNA) genome containing four open reading frames (C, P, S and X). The ORFs largely overlap and encode multiple proteins using different in-frame start codons. The S ORF encodes three forms of surface envelope glycoproteins S-, M-, and L- HBs). The HBV preC-C gene gives rise to two different products translated from distinct mRNAs – core protein (HBc) and precore protein (HBe). Despite their high sequence similarity, these proteins exhibit different functions and subcellular localizations. The P ORF encodes viral polymerase, and the X ORF encodes regulatory HBx protein.
Mass Spectrometry department of IOCB has established a collaboration with the groups of Iva Pichova and Jan Weber from IOCB. This collaboration on proteomics experiments lead to the discovery of several important biological and biochemical findings including identification of interacting partners of HBc, HBx and HBe proteins or posttranslational modifications of HBc protein. The collaboration resulted in several publications. The proteomic analysis of individually expressed viral proteins has also captured out-of-frame products suggesting the possibility of frameshift or alternative internal initiation of translation. The presentation will highlight the proteomic analyses of HBV at IOCB
CSMS: Conference CEEPC/IPM/CMSC 2022: Proteomic Analysis of Hepatitis B Virus (Martin Hubálek)
9:10-9:30 The single amino acid substitutions in Mason-Pfizer Monkey Virus matrix protein modulate its proteolytic cleavage rate
- Jakub Sýs
N-terminal domain of polyprotein Gag of Mason-Pfizer Monkey Virus (M-PMV) Matrix protein (MA) is naturally myristoylated on its N-terminus. When MA is a part of polyprotein Gag, the myristate moiety is buried inside hydrophobic pocket and exposed probably upon interaction with host cell plasma membrane (PM). This mechanism called myristoyl switch does not occur in M-PMV as readily as it does in HIV-1, suggesting that it may have an important role in M-PMV maturation by regulating the MA cleavage from Gag polyprotein.
To address this hypothesis, we have treated the myristoylated (myrMAPPHis) and nonmyristoylated MAPPHis (nonmyrMAPPHis) M PMV construct, bearing the cleavage site for M-PMV viral protease (Pr13), on artificial liposomes mimicking PM by Pr13 for several time points. In contrast to rapidly degraded nonmyrMAPPHis even without liposomes, the myrMAPPHis, surprisingly, become cleaved more frequently only after addition of liposomes indicating the possible exposure of myristate upon interaction with PM.
To support our findings, we have designed and examined also four mutants of MAPPHis M PMV with single amino acid substitutions with expectation to block or, conversely, facilitate the myristoyl switch by stabilization (A79V, A79L) or destabilization (I51A, I86A) of MA M PMV hydrophobic pocket. The mutants A79V and A79L were cleaved even less effectively than myrMAPPHis as well as the degradation of mutants I51A and I86A was faster compared to nonmyrMAPPHis. The different cleavage rates of proteins were confirmed also on structural level by using method of hydrogendeuterium exchange coupled with mass spectrometric detection. It shows that the protease cleavage site has different dynamics in proteins as a result of disruption/stabilisation of hydrophobic pocket.
CSMS: Conference CEEPC/IPM/CMSC 2022: The single amino acid substitutions in Mason-Pfizer Monkey Virus matrix protein modulate its proteolytic cleavage rate (Jakub Sýs)
9:30-9:50 Cardiac alarmins as residual risk markers of atherosclerosis under lipid-lowering therapy
- Viorel-Iulian Suica
Background: The major cause in the initiation and progression of atherosclerosis are the high levels of low-density lipoproteins. Although statin treatment can effectively lower these levels, there is still a residual risk of cardiovascular events. We hypothesize that a specific panel of alarmins, a family of stress-sensing molecules, could indicate the persistence of silent atherosclerosis residual risk.
Methods: New Zeeland White rabbits were divided into: the control group (C) with standard diet, a group which received a high-fat diet for 12 weeks (Au) and a treated hyperlipidemic group, with a lipid diet for 8 weeks followed by standard diet and hypolipidemic treatment (atorvastatin and PCSK9 siRNA-inhibitor) for 4 weeks (Asi). We used the LTQ Orbitrap Velos Pro mass spectrometer to analyse the left ventricle lysates. The experiments were complemented by immunologic and genomic assays to corroborate the data.
Results: The hyperlipidemic diet determined a general alarmin up-regulation tendency over C group. A significant spectral abundance increase was measured for specific heat shock proteins, S100 family members, HMGB1 and Annexin A1. The hypolipidemic treatment demonstrated a reversed regulation trend, with non-significant spectral alteration over the C group for some identified alarmins.
Conclusion: Our study highlights the discriminating potential of alarmins in hyperlipidemia or following a hypolipidemic treatment.
9:50-10:10 Cervical mucus – a non-invasive uterine biomarker source?
- Tomáš Oždian
Cervical mucus (CM) is a viscous fluid produced by cervical glands located in the myometrium of the uterine cervix. Usually, CM forms a plug between vagina and uterus. During the ovulation, CM starts to be more fluidic to allow the sperm to go through. During the ovulation, there is a good window for non-invasive sampling of the CM, originating from uterine derived tissues. The main hypothesis for CM proteomic study is to develop a method for CM sampling, processing and to answer if there are enough proteins in the CM for potential biomarker search. The resulting protocol consists of mucus dissolution, multi-enzyme digestion, and LC-MS proteomic analysis. The optimized approach allows to identify more than thousand proteins from every sample tested. The bioinformatic analysis reveals that the identified proteins are both intra and extra cellular and discovers the proteins with enriched expression in the broader female reproductive tract. To our best knowledge, we provide the most extensive proteomic characterization available.
10:10-10:30 High-Throughput µLC-MS/MS Lipidomics of 3D In Vitro Disease Models to Investigate Lipid Dysregulation
- Darshak Gadara
Microflow liquid chromatography interfaced with mass spectrometry is increasingly applied for the high-throughput profiling of biological samples, as it demonstrated an acceptable tradeoff between sensitivity and reproducibility. However, a limited number of applications developed in lipidomics. This study introduces a robust, sensitive, and high-throughput μLC-MS/MS lipidomics workflow, applied in a differential lipidome study of APOE 3 and 4 phenotypes in iPSC-derived cerebral organoids (CO). First, we optimized the microbore column gradient conditions, injecting a mixture of 15 lipid standards on the UHPLC system coupled to a 6469 QQQ. Our μLC-MS/MS method allows quantitation of 351 lipid species from the single CO. The microflow lipidomics shows an average 3.7-fold increase in response compared to a conventional high-flow method. To benchmark the quantitative performance, 303 samples were acquired over 75 hours. The median coefficient of variation (CV) of 351 lipid species was 12.95%, only 26 lipid species show CV higher than 30%. We demonstrated that microflow separation coupled to three-stage quadruple MS substantially increases the sensitivity and allows for robust multiplexed lipid profiling (including cholesterol determination) in a single cerebral organoid. As a proof of concept, lipidomics analysis was performed for the lipid extract of APOE 3/3 and APOE 4/4 (n=10) CO samples. To our knowledge, this is the first systematic report to demonstrate the microflow LC-MS/MS allows for sensitive, high-throughput, and robust measurement of global lipidome, opening an avenue for routine application of microflow separation in the field of lipidomics.
CSMS: Conference CEEPC/IPM/CMSC 2022: High-Throughput µLC-MS-MS Lipidomics of 3D In Vitro Disease Models to Investigate Lipid Dysregulation (Darshak Gadara)
10:30-10:50 Multi-omics network analysis in atherosclerosis reveals mechanisms driving the progression towords complicated lesion
- Eva Csosz
From a biological point of view, it is crucial to understand the mechanisms laying behind atherosclerosis and its complicated forms. In the process of atherosclerosis, first, an atheromatous plaque builds up, which in some conditions can lead to the appearance of complicated lesions. The complicated lesion is characterized by ruptures on the atherosclerotic plaque surface and/or hemorrhage into the plaque. According to the WHO, hemorrhaged lesions, contribute to a considerable extent of deaths worldwide.
The main goal of our project was to perform a multi-omics study involving transcriptomics and proteomics data to get more insights into the pathophysiological processes dominating the complicated lesions.
Transcriptomic (RNAseq) and proteomic (label-free quantification) data were obtained by the analysis of artery samples originating from healthy arteries, atheromatous lesions, and complicated lesions. Both data types were examined separately by Ingenuity Pathway Analysis and later an overlay of the networks was achieved.
120 differentially expressed (DE) proteins and more than 4000 DE genes were detected. The deep analysis of the data could identify the proteins and transcripts characteristic of the complicated lesions and could reveal the pathways specific to atheroma and complicated lesions, respectively. The DE genes were arranged into 25 networks, while the DE proteins were into 12 networks. The lower number of DE proteins compared to the number of DE genes did not result in loss of information, indicating the power of the proteomics experiments. In spite of providing a lower number of DE items, the biological information achievable from proteomics experiments has equal value compared to transcriptomics data.
CSMS: Conference CEEPC/IPM/CMSC 2022: Multi-omics network analysis in atherosclerosis reveals mechanisms driving the progression towords complicated lesion (Eva Csosz)
10:50 - 11:10 Coffee break
11:10 - 12:00 Plenary lecture III:: Light-mediated discovery of surfaceome nanoscale organization and inter-cellular receptor interaction networks (Chairperson: Pavel Bouchal)
- Bernd Wollscheid
The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8⁺ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.
CSMS: Conference CEEPC/IPM/CMSC 2022: Light-mediated discovery of surfaceome nanoscale organization and inter-cellular receptor interaction networks (Bernd Wollscheid)
12:00 - 12:20 Poster prize, Closing remarks
ČSHS: Conference prize CEEPC/IPM/CMSC 2022 (Aleš Kvasnička a Michael Volný)
ČSHS: EuPA travel grants: Short talk prize CEEPC/IPM/CMSC 2022 (Kalaninová Zuzana a Eva Csosz)
ČSHS: Poster prize CEEPC/IPM/CMSC 2022 (Anton Škríba a Michael Volný)
ČSHS: EuPA travel grants: Poster prize CEEPC/IPM/CMSC 2022 (Jadeja Siddharth a Eva Csosz)
12:20 - 13:30 Lunch
CSMS: Conference CEEPC/IPM/CMSC 2022: Coffe break