@phdthesis{Schlossarek2023, author = {Schlossarek, Dennis}, title = {Identification of dynamic protein-metabolite complexes in saccharomyces cerevisiae using co-fractionation mass spectrometry}, doi = {10.25932/publishup-58282}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-582826}, school = {Universit{\"a}t Potsdam}, pages = {123}, year = {2023}, abstract = {Cells are built from a variety of macromolecules and metabolites. Both, the proteome and the metabolome are highly dynamic and responsive to environmental cues and developmental processes. But it is not their bare numbers, but their interactions that enable life. The protein-protein (PPI) and protein-metabolite interactions (PMI) facilitate and regulate all aspects of cell biology, from metabolism to mitosis. Therefore, the study of PPIs and PMIs and their dynamics in a cell-wide context is of great scientific interest. In this dissertation, I aim to chart a map of the dynamic PPIs and PMIs across metabolic and cellular transitions. As a model system, I study the shift from the fermentative to the respiratory growth, known as the diauxic shift, in the budding yeast Saccharomyces cerevisiae. To do so, I am applying a co-fractionation mass spectrometry (CF-MS) based method, dubbed protein metabolite interactions using size separation (PROMIS). PROMIS, as well as comparable methods, will be discussed in detail in chapter 1. Since PROMIS was developed originally for Arabidopsis thaliana, in chapter 2, I will describe the adaptation of PROMIS to S. cerevisiae. Here, the obtained results demonstrated a wealth of protein-metabolite interactions, and experimentally validated 225 previously predicted PMIs. Applying orthogonal, targeted approaches to validate the interactions of a proteogenic dipeptide, Ser-Leu, five novel protein-interactors were found. One of those proteins, phosphoglycerate kinase, is inhibited by Ser-Leu, placing the dipeptide at the regulation of glycolysis. In chapter 3, I am presenting PROMISed, a novel web-tool designed for the analysis of PROMIS- and other CF-MS-datasets. Starting with raw fractionation profiles, PROMISed enables data pre-processing, profile deconvolution, scores differences in fractionation profiles between experimental conditions, and ultimately charts interaction networks. PROMISed comes with a user-friendly graphic interface, and thus enables the routine analysis of CF-MS data by non-computational biologists. Finally, in chapter 4, I applied PROMIS in combination with the isothermal shift assay to the diauxic shift in S. cerevisiae to study changes in the PPI and PMI landscape across this metabolic transition. I found a major rewiring of protein-protein-metabolite complexes, exemplified by the disassembly of the proteasome in the respiratory phase, the loss of interaction of an enzyme involved in amino acid biosynthesis and its cofactor, as well as phase and structure specific interactions between dipeptides and enzymes of central carbon metabolism. In chapter 5, I am summarizing the presented results, and discuss a strategy to unravel the potential patterns of dipeptide accumulation and binding specificities. Lastly, I recapitulate recently postulated guidelines for CF-MS experiments, and give an outlook of protein interaction studies in the near future.}, language = {en} } @article{PetazziKoikkarahAjiTischleretal.2021, author = {Petazzi, Roberto Arturo and Koikkarah Aji, Amit and Tischler, Nicole D. and Chiantia, Salvatore}, title = {Detection of envelope glycoprotein assembly from old world hantaviruses in the Golgi apparatus of living cells}, series = {Journal of virology}, volume = {95}, journal = {Journal of virology}, number = {4}, publisher = {American Society for Microbiology}, address = {Baltimore, Md.}, issn = {1098-5514}, doi = {10.1128/JVI.01238-20}, pages = {18}, year = {2021}, abstract = {Hantaviruses are emerging pathogens that occasionally cause deadly outbreaks in the human population. While the structure of the viral envelope has been characterized with high precision, protein-protein interactions leading to the formation of new virions in infected cells are not fully understood. We used quantitative fluorescence microscopy (i.e., number and brightness analysis and fluorescence fluctuation spectroscopy) to monitor the interactions that lead to oligomeric spike complex formation in the physiological context of living cells. To this aim, we quantified protein-protein interactions for the glycoproteins Gn and Gc from Puumala and Hantaan orthohantaviruses in several cellular models. The oligomerization of each protein was analyzed in relation to subcellular localization, concentration, and the concentration of its interaction partner. Our results indicate that, when expressed separately, Gn and Gc form, respectively, homo-tetrameric and homo-dimeric complexes, in a concentration-dependent manner. Site-directed mutations or deletion mutants showed the specificity of their homotypic interactions. When both glycoproteins were coexpressed, we observed in the Golgi apparatus clear indication of GnGc interactions and the formation of Gn-Gc multimeric protein complexes of different sizes, while using various labeling schemes to minimize the influence of the fluorescent tags. Such large glycoprotein multimers may be identified as multiple Gn viral spikes interconnected via Gc-Gc contacts. This observation provides the possible first evidence for the initial assembly steps of the viral envelope within this organelle, and does so directly in living cells.
IMPORTANCE In this work, we investigate protein-protein interactions that drive the assembly of the hantavirus envelope. These emerging pathogens have the potential to cause deadly outbreaks in the human population. Therefore, it is important to improve our quantitative understanding of the viral assembly process in infected cells, from a molecular point of view. By applying advanced fluorescence microscopy methods, we monitored the formation of viral spike complexes in different cell types. Our data support a model for hantavirus assembly according to which viral spikes are formed via the clustering of hetero-dimers of the two viral glycoproteins Gn and Gc. Furthermore, the observation of large Gn-Gc hetero-multimers provide the possible first evidence for the initial assembly steps of the viral envelope, directly in the Golgi apparatus of living cells.}, language = {en} } @article{KolyvushkoLatzkeDahmanietal.2020, author = {Kolyvushko, Oleksandr and Latzke, Juliane and Dahmani, Ismail and Osterrieder, Nikolaus and Chiantia, Salvatore and Azab, Walid}, title = {Differentially-charged liposomes interact with alphaherpesviruses and interfere with virus entry}, series = {Pathogens}, volume = {9}, journal = {Pathogens}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2076-0817}, doi = {10.3390/pathogens9050359}, pages = {9}, year = {2020}, abstract = {Exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is induced by infection with several members of the Alphaherpesvirinae subfamily. There is evidence that PS is used by the equine herpesvirus type 1 (EHV-1) during entry, but the exact role of PS and other phospholipids in the entry process remains unknown. Here, we investigated the interaction of differently charged phospholipids with virus particles and determined their influence on infection. Our data show that liposomes containing negatively charged PS or positively charged DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium) inhibited EHV-1 infection, while neutral phosphatidylcholine (PC) had no effect. Inhibition of infection with PS was transient, decreased with time, and was dose dependent. Our findings indicate that both cationic and anionic phospholipids can interact with the virus and reduce infectivity, while, presumably, acting through different mechanisms. Charged phospholipids were found to have antiviral effects and may be used to inhibit EHV-1 infection.}, language = {en} } @phdthesis{Wettstein2015, author = {Wettstein, Christoph}, title = {Cytochrome c-DNA and cytochrome c-enzyme interactions for the construction of analytical signal chains}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-78367}, school = {Universit{\"a}t Potsdam}, pages = {120}, year = {2015}, abstract = {Electron transfer (ET) reactions play a crucial role in the metabolic pathways of all organisms. In biotechnological approaches, the redox properties of the protein cytochrome c (cyt c), which acts as an electron shuttle in the respiratory chain, was utilized to engineer ET chains on electrode surfaces. With the help of the biopolymer DNA, the redox protein assembles into electro active multilayer (ML) systems, providing a biocompatible matrix for the entrapment of proteins. In this study the characteristics of the cyt c and DNA interaction were defined on the molecular level for the first time and the binding sites of DNA on cyt c were identified. Persistent cyt c/DNA complexes were formed in solution under the assembly conditions of ML architectures, i.e. pH 5.0 and low ionic strength. At pH 7.0, no agglomerates were formed, permitting the characterization of the NMR spectroscopy. Using transverse relaxation-optimized spectroscopy (TROSY)-heteronuclear single quantum coherence (HSQC) experiments, DNAs' binding sites on the protein were identified. In particular, negatively charged AA residues, which are known interaction sites in cyt c/protein binding were identified as the main contact points of cyt c and DNA. Moreover, the sophisticated task of arranging proteins on electrode surfaces to create functional ET chains was addressed. Therefore, two different enzyme types, the flavin dependent fructose dehydrogenase (FDH) and the pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH), were tested as reaction partners of freely diffusing cyt c and cyt c immobilized on electrodes in mono- and MLs. The characterisation of the ET processes was performed by means of electrochemistry and the protein deposition was monitored by microgravimetric measurements. FDH and PQQ-GDH were found to be generally suitable for combination with the cyt c/DNA ML system, since both enzymes interact with cyt c in solution and in the immobilized state. The immobilization of FDH and cyt c was achieved with the enzyme on top of a cyt c monolayer electrode without the help of a polyelectrolyte. Combining FDH with the cyt c/DNA ML system did not succeed, yet. However, the basic conditions for this protein-protein interaction were defined. PQQ-GDH was successfully coupled with the ML system, demonstrating that that the cyt c/DNA ML system provides a suitable interface for enzymes and that the creation of signal chains, based on the idea of co-immobilized proteins is feasible. Future work may be directed to the investigation of cyt c/DNA interaction under the precise conditions of ML assembly. Therefore, solid state NMR or X-ray crystallography may be required. Based on the results of this study, the combination of FDH with the ML system should be addressed. Moreover, alternative types of enzymes may be tested as catalytic component of the ML assembly, aiming on the development of innovative biosensor applications.}, language = {en} }