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Structure-function relationships in substrate binding protein dependent secondary transporters
(2023)
This work provides new insights into the relevance of SBP dependent secondary transport systems, especially in the thus far under-researched subgroup of TAXI transporters. Importantly, we identified and characterized the TAXI transport system TAXIPm-PQM from Proteus mirabilis. We demonstrated that, in contrast to previously characterized SBP dependent secondary transport systems, TAXIPm-PQM is a proton coupled system and transports the C5-dicarboxylate α- ketoglutarate. Since initially the transport of α-ketoglutarate could only be demonstrated in vivo but not in vitro using established protocols (Mulligan et al. 2009), we investigated in detail the differences between the in vivo and in vitro assay. This resulted in a bioinformatic analysis of TRAP and TAXI signal peptides, which strongly implied that TAXIPm-P requires a transmembrane anchor to allow for transport. We then provided TAXIPm-P surface tethered to the membrane in in vitro transport assays and confirmed the prediction of our bioinformatic analysis that TAXIPm-PQM deploys a membrane-anchored instead of a soluble SBP. Furthermore, the TAXI transport system TAXIMh-PQM from Marinobacter hydrocarbonoclasticus transports fumarate only if both membrane domains Q and M are present. For further characterization, Michaelis-Menten kinetics and affinities were determined for both TAXI transport systems TAXIPm-PQM from Proteus mirabilis and TAXIMh-PQM from Marinobacter hydrocarbonoclasticus. In addition, nanobodies were selected for the membrane domain TAXIPm-QM from Proteus mirabilis to stabilize different conformations which can serve in subsequent structural elucidation studies. Furthermore, the TRAP SBP TRAPHi-SiaP from Haemophilus influenzae was shown to interact not only with its corresponding membrane domain TRAPHi-SiaQM but with at least one additional transporter. It was thereby excluded that TRAPHi- SiaP transfers N-acetylneuraminic acid to the only native E. coli TRAP transporter TRAPEc-YiaMNO and suggested to rather interact with a SBP dependent ABC transport system as this protein family represents the largest SBP dependent protein group in E. coli (Moussatova et al. 2008).
This research attempts to provide for an overview of the state of co-operation between the United Nations and regional organizations like the CoE, OSCE, EU and NATO during the last Yugoslav wars, considering the 1991-2008 period. In this case, the "reconstruction" of what the organisations did in each of the countries involved in the conflicts, the country-by-country approach used in writing the research and the consideration of both headquarters and field level should facilitate the understanding of the state of things at that time. The research further includes an analysis of the co-operative trends developed by the considered international organisations since the beginning of the 1990s and is concluded by a reflection on the normative relevance of the issue of "international cooperation". In this case, the intention of the author was to go beyond the general policy level approach used for the description of UN-regional organizations interaction and propose a re-consideration of the concept of "international co-operation" as a possible normative tool in guiding the so far nebulous division of tasks of international actors in conflict-related scenarios. In this case, the concise description of the general framework for co-operation under Chapter VIII of the UN Charter, already matter of wide debate by academics and practitioners, sets the frame for a more elaborate, and hopefully innovative, consideration of the notion of "international cooperation". This, of course, is to be contextualized to the lessons learned extrapolated from the case study.
RNA research is very important since RNA molecules are involved in various gene regulatory mechanisms as well as pathways of cell physiology and disease development.1 RNAs have evolved from being considered as carriers of genetic information from DNA to proteins, with the three major types of RNA involved in protein synthesis, including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).2 In addition to the RNAs involved in protein synthesis numerous regulatory non-coding RNAs (ncRNAs) have been discovered in the transcriptome. The regulatory ncRNAs are classified into small ncRNAs (sncRNAs) with transcripts less than 200 nucleotides (nt) and long non-coding RNAs (lncRNAs) with more than 200 nt.3
LncRNAs represent the most diverse and versatile class of ncRNAs that can regulate cellular functions of chromatin modification, transcription, and post-transcription through multiple mechanisms.4 They are involved in the formation of RNA:protein, RNA:RNA and RNA:DNA complexes as part of their gene regulatory mechanism.4,5 The RNA:DNA interactions can be divided into RNA:DNA heteroduplex formation, also called R-loops, and RNA:DNA:DNA triplex formation. In triplex formation, RNA binds to the major groove of double-stranded DNA through Hoogsteen or reverse Hoogsteen hydrogen bonding, resulting in parallel or anti-parallel triplexes, respectively. In vitro studies have confirmed the formation of RNA:DNA:DNA triplexes.6 However, the extent to which these interactions occur in cells and their effects on cellular function are still not understood, which is why these structures are so exciting to study (Chapter I RNA:DNA:DNA Triplexes).
This cumulative thesis investigates several functional and regulatory important RNAs. The first project involves the improved biochemical and biophysical characterization of RNA:DNA:DNA triplex formation between lncRNAs of interest and their target genes. Triplex formation was confirmed by a series of experiments including electromobility shift assays (EMSA), thermal melting assays, circular dichroism (CD), and liquid state nuclear magnetic resonance (NMR) spectroscopy. The following is a summary of the main findings of these publications.
In research article 5.1, the oxygen-sensitive HIF1α-AS1 was identified as a functionally important triplex-forming lncRNA in human endothelial cells using a combination of bioinformatics techniques, RNA/DNA pulldown, and biophysical experiments. Through RNA:DNA:DNA triplex formation, endogenous HIF1α-AS1 decreases the expression of several genes, including EPH receptor A2 (EPHA2) and adrenomedullin (ADM), by acting as an adaptor for the repressive human silencing hub (HUSH) complex, which has been studied by our collaborators in the groups of Leisegang and Brandes.
2) Triplex formation between HIF1α-AS1 and the target genes EPHA2 and ADM was investigated in biochemical and biophysical studies. The EMSA results indicated that HIF1α-AS1 forms a low mobility RNA:DNA:DNA triplex complex with the EPHA2 DNA target sequence. The CD spectrum of the triplex showed distinct features compared to the EPHA2 DNA duplex and the RNA:DNA heteroduplex. Melting curve analysis revealed a biphasic melting transition for triplexes, with a first melting point corresponding to the dissociation of the RNA strand with melting of the Hoogsteen hydrogen bonds. The second, higher melting temperature corresponds to the melting of stronger Watson-Crick base pairing. Stabilized triplexes were formed using an intramolecular EPHA2 DNA duplex hairpin construct in which both DNA strands were attached to a 5 nucleotide (nt) thymidine linker. This approach allowed improved triplex formation with lower RNA equivalents and higher melting temperatures. By NMR spectroscopy, the triplex characteristic signals were observed in the 1H NMR spectrum, the imino signals in a spectral region between 9 and 12 ppm resulting from the Hoogsteen base pairing. To elucidate the structural and sequence specific Hoogsteen base pairs 2D 1H,1H-NOESY measurements of the EPHA2 DNA duplex and the HIF1α-AS1:EPHA2 triplex were performed. The 1H,1H-NOESY spectrum of the HIF1α-AS1:EPHA2 triplex with a 10-fold excess of RNA was semi-quantitatively analyzed for changes in the DNA duplex spectrum. We discovered, strong and moderate attenuation of cross peak intensities in the imino region of the NOESY spectrum. This attenuation was proposed to result from weakening of Watson-Crick base pairing by Hoogsteen hydrogen bonding induced by RNA binding. The Hoogsteen interactions can be mapped based on the analysis of the cross peak attenuation in the NOESY spectra, which we used to generate a structural model of the RNA:DNA:DNA triplex. These biophysical results support the physiological function of HIF1α as a triplex-forming lncRNA that recruits the HUSH-epigenetic silencing complex to specific target genes such as EPHA2 and ADM, thereby silencing their gene expression through RNA:DNA:DNA triplex formation.
Identification of new natural products from nematode-associated bacteria using mass spectrometry
(2023)
This work aims to find unknown natural products produced by bacteria, that live in close association with nematodes and to elucidate their structure by using mass spectrometry.
The first chapter of this work is dedicated to the detection of hitherto unknown natural products by using a metabolomics approach and subsequent structure elucidation of said compounds. This chapter includes metabolomics analysis of Xenorhabdus szentirmaii wild type and knockout mutants, overproduction of the target compound, identification of derivatives from other strains and MS based structure elucidation.
The second and third chapters are about natural products that protect C. elegans from B. thuringiensis infections.
The second chapter deals with natural products that protect the nematode host without killing the pathogen. I deployed molecular biology methods to generate deletion and overproduction strains of a target compound, identified it via LC-MS/MS analysis and used LC-MS/MS and lipidomics to analyse the chemical properties of the active compound.
The third chapter aims at finding natural products, which are produced by Pseudomonas strains MYb11 and MYb12, respectively. These natural products display the ability to protect C. elegans by killing B. thuringiensis. I identified said compounds via fractionation and subsequent bioactivity testing. After identification, I generated production strains of the target compounds and elucidated the structure of the bioactive derivative.
The last chapter deals with the structure elucidation of peptides produced by an unusual GameXPeptide synthetase in Xenorhabdus miraniensis. I analysed producer strains of GameXPeptides using LC-MS and elucidated the structural differences between the known GameXPeptides, produced by P. luminescens TT01, and the unusual ones produced by X. miraniensis.
Nok Eisen : zentralnigerianische Eisenverhüttung in der Mitte des ersten Jahrtausends vor Christus
(2023)
Based on excavations, excavation documentation and archaeometallurgical analyses, this thesis aims to characterise Nok iron production in central Nigeria through a contextually based investigation.
In 2010, 2011, 2013 and 2016, the Nok research project at Goethe University Frankfurt/Main in collaboration with the National Commission for Museums and Monuments, Nigeria excavated 27 iron-smelting furnaces from 8 sites 60 kilometres north of Abuja. All furnaces date around the middle of the first millennium BCE. Absolute dates, relative pottery chronology and terracotta figurine finds in furnace contexts suggest their affiliation to the Nok context. In comparison, all 27 furnaces resemble each other closely regarding their design and spacial arrangement. The numbers of furnaces per site, furnace width, furnace wall angle and thickness as well as pits beneath the furnaces are just some features with similar qualities. The similarities of the smelting sites also extend into their finds: the structure of tuyères and their position in situ as well as macroscopic slag morphology and distribution. Find morphology and distribution as well as furnace structure suggest a highly standardized way of Nok iron production. However, archaeometallurgical analyses show heterogeneous use of raw materials between sites and/or furnaces. In similarly structured furnaces different kinds of iron ore were smelted leaving a high iron content in the respective slags. This hints at an early stage of iron production in which the smelting process was limited to one operative set-up.
This work addresses the investigation of the biosynthesis mechanisms of type II polyketide synthase (PKS) and fatty acid synthase (FAS) derived specialized metabolites (SMs) from Photorhabdus laumondii.
The elucidation of the biosynthetic pathway of the bacterial 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) was one of the major topics of this thesis. IPS exhibits several bioactive characteristics as it inhibits the phenoloxidase of insects, acts antibacterial, but also influences the soluble epoxide hydrolase which is involved in inflammatory reactions. It was recently approved as a treatment against psoriasis by the FDA and is the first Photorhabdus derived drug.
The stilbene generation in Photorhabdus requires the formation of the two acyl-carrier-protein (ACP) bound 5-phenyl-2,4-pentadienoyl- and isovaleryl-β-ketoacyl-moieties. The ketosynthase (KS)/cyclase StlD catalyzes a ring formation via a Michael-addition between the two intermediates which is then further processed by an aromatase. The formation of 5-phenyl-2,4-pentadienoyl-ACP was shown via in vitro assays with purified proteins by proving the influence of the KS FabH, ketoreductase FabG and dehydratase FabA or FabZ of the fatty acid metabolism. While E. coli was able to complement most of these enzymes in attempts to produce IPS in the heterologous host, the Photorhabdus derived FabH was not replaceable despite 73 % sequence identity with the E. coli based isoenzyme, acting as a gatekeeper enzyme for cinnamic acid (CA) moieties. Furthermore, the ability to incorporate meta-substituted halogenated CA-derivatives was shown in order to produce 3-chloro- and 3-bromo-IPS. While studying the stilbene biosynthesis, the ability of Photorhabdus and Xenorhabdus to produce hydrazines was also discovered.
The second investigated biosynthesis was the formation of benzylideneacetone (BZA). BZA is produced by Photorhabdus and Xenorhabdus strains acting as a suppressor for the immune cascade of insects and has also antibiotic activities towards Gram-negative bacteria. Due to its structural similarity towards CA and the intermediates during the stilbene formation, a shared mechanism for Photorhabdus and Xenorhabdus budapestensis was proposed due to their ability to produce CA. The production of BZA was also dependent on the stilbene related CoA-ligase, the ACP and FabH. It was verified in vitro and in vivo in E. coli yielding a 150-fold increase of the BZA production compared to the Photorhabdus and Xenorhabdus wildtype (WT) strains.
The second part of this work deals with the optimization of P. laumondii strains regarding the production titers of IPS. Therefore, several deletions of other SM related genes as well as promoter exchanges in front of stilbene related genes were carried out. These approaches were combined with the upregulation of the phenylalanine by heterologous plasmid expression, since it is the precursor of CA. Another approach applied in parallel was the optimization of the cultivation conditions with different media and supplementation with XAD-resins. It was proved that media rich on fatty acids or peptides led to higher optical densities of the cultures and thus to higher titers of stilbenes. Since IPS is inhibiting the phenoloxidase, an enzyme important for the insect immunity, it was hypothesized that cultivation in media containing insects might enhance the output of this SM. Starting from 23 mg/l of IPS in the P. laumondii WT strain, it was possible to increase the production levels to more than 860 mg/l by utilizing the mentioned approaches.
The last topic of this thesis focuses on the production of epoxidated IPS (EPS) and its derivatives. Under laboratory conditions, only a low titer of EPS was observed for the wildtype strain. However, the optimized IPS strains and IPS-production conditions could also be applied for EPS which led to higher productions and also to the detection of many new derivatives. Most of the EPS derivatives were amino acid or peptide derived acting as nucleophiles to open the epoxide ring and yielding β-amino-alcohols. However, purification and chemical synthesis attempts to obtain EPS failed due to its poor stability. Epoxides were utilized in in vitro assays with amino acids, peptides and proteins to get insights whether epoxidations might act as posttranslational modification in Photorhabdus. The reactions were performed with styrene oxide and stilbene oxide replacing EPS based on their structural similarity. The modifications were executed successfully although proteomics approaches with in vivo data are required to confirm these findings. During the purification attempts of EPS, further derivatives were detected. The structures of dimerized stilbenes, a cis-isomer of IPS and another derivative that might incorporate an amino-group in the resveratrol ring were proposed on the basis of the HPLC-MS data.
N6-methyladenosine (m6A) is the most abundant and well understood modification in eukaryotic mRNA and was first identified in polyadenylated parts of the mRNA.The distinct distribution of m6A in the transcriptome with special enrichment in long internal exons, 39UTRs and around stop codons was uncovered by early biochemical work and later on antibody based sequencing techniques. The so called m6A writer, reader and eraser machinery is responsible for the dynamic and with that regulatory nature of the m6A modification. As m6A writer, the human N6-methyltransferase complex (MTC) cotranscriptionally methylates the central adenine within a RRACH (preferably GGACU) sequence context to form m6A in the nascent RNA chain.9–15 The catalytic core of the complex is formed by the two proteins METTL3 and METTL14, with the active site located in the methyltransferase domain (MTD) of METTL3.16–18 The DPPW motif near the methyl donor S-adenosylmethionine (SAM) binding site in this MTD was postulated to bind the target adenine during catalysis. Moreover, a positively charged groove in the METTL3-METTL14 interface, the C-terminal RGG domain in METTL14 and the zinc finger motifs in METTL3 were identified as important domains for RNA binding. However, to date there are no full-length or substrate-RNA-bound structures of the catalytic METTL3-METTL14 complex.
In addition, a set of accessory proteins assembles to the METTL3-METTL14 heterodimer to form the full MTC, mediated by WTAP that firmly binds to the N-terminal leader helix in METTL3.20 WTAP was shown to locate the whole complex to the nuclear speckles and can modulate m6A deposition to specific sites in the RNA. Moreover, WTAP acts as binding platform for other accessory proteins including VIRMA, RBM15, ZC3H13 and HAKAI that are mostly identified to mediate position specific methylation. For example, RBM15 was shown to mediates region-selective methylation in a WTAP dependent manner, directing specificity towards U-rich sequences.
The observed specificity of the methyltransferase complex to methylate only site specific DRACH sequenced is still poorly understood. Some possible modulators like the role of the accessory proteins are under investigation, however, the structural context of the RNA methylation sites or a structural preference of the complex have been mainly neglected so far. Moreover, the structural dynamics of this methylation process still remain elusive. This thesis contributes to the afore-mentioned aspects by analysis of the methylation process regarding RNA structure sensitivity with enzymatic activity assays and its dynamic nature by implementing a smFRET approach.
We hypothesized the target RNA secondary structure to be an additional important modulator of methylation efficiency, based on the RNA binding elements of the complex (positively charged binding groove, zinc finger domain, RGG domain) and the supposed target adenine binding in the active site. Here, we postulated the possibility for a flipped-out adenine to be of special relevance, which is closely related to the local stability of the target adenine containing structure. Moreover, efficient binding of the protein complex to the RNA should require the ability to anchor the RNA on both sides of the target sequence.
Restructuration des répertoires langagiers de migrant·e·s de la République du Congo en Lorraine
(2023)
Cette thèse étudie la complexité du plurilinguisme des migrant·e·s d’origine de la République du Congo en Lorraine à travers le prisme de la restructuration des répertoires langagiers. En affûtant la conceptualisation de la restructuration des répertoires langagiers par l’étude du plurilinguisme des migrant·e·s d’origine congolaise, cette recherche ouvre de nouvelles perspectives pour les recherches portant sur le plurilinguisme, notamment concernant les mobilités transgénérationnelles et la diversité des processus de restructuration façonnant les répertoires langagiers. En se focalisant sur les biographies langagières et migratoires de 15 individus migrants, sur leurs réseaux sociaux et sur leurs ressources langagières, cette étude révèle la diversité des processus et des facteurs au cœur des restructurations des répertoires langagiers à travers une étude ethnographique multi-située en Lorraine et au Congo. La compréhension de la diversité des dynamiques restructurant les connaissances langagières des enquêté·e·s passe par l’étude des situations de socialisation langagière au Congo dans leur historicité, des itinéraires de migration et des restructurations des réseaux sociaux ainsi que des répertoires langagiers dans l’installation en Lorraine. Les participations à la société lorraine et ses groupes sociaux imprègnent les identifications, les orientations sociales et les positionnements dans les réseaux sociaux et vice-versa.
Les répertoires langagiers apparaissent comme des enregistrements de la mobilité des individus et de celle des générations antérieures ainsi que de leur entourage. Les restructurations concernent entre autres les ressources associées au français, aux langues congolaises et à d’autres langues appropriées par la migration. Les ressources du français sont restructurées par les migrant·e·s en s’appropriant les ressources courantes dans différentes situations sociales en Lorraine, en marquant et/ou en dissimulant les ressources appropriées ailleurs et inappropriées dans ces situations. En même temps, un savoir de différenciation des ressources, dont font aussi partie les schémas de catégorisation et les stratégies communicatives, est développé et une (in)sécurité langagière se manifeste. Les ressources associées aux langues congolaises, leurs fonctions sociales et leurs représentations sont restructurées dans des processus d’attrition, d’actualisation, de transformation et d’élaboration langagière. Les ressources associées à d’autres langues européennes appropriées par la migration sont reléguées au second plan et se perdent lentement par manque d’usage. Enfin, les connaissances liées à la gestion du plurilinguisme, de la diversité culturelle et de l’altérité, appropriées dans les mêmes situations de diversité, aident au traitement interne des expériences des mobilités spatiales et sociales ainsi que des restructurations des répertoires langagiers.
This thesis comprises the usage of two commonly known hinge-binding moieties in drug discovery. First, the quinazoline scaffold of gefitinib (5) was utilized in a macrocyclization strategy to introduce selectivity. In general, the quinazoline hinge-binding moiety is a commonly used scaffold which can be found in 14% of approved kinase inhibitors. The most familiar applications are EGFR inhibitors such as gefitinib (5), erlotinib (6), afatinib, or dacomitinib for the treatment of NSCLC. But other kinases like CDK2, CDK4, or p38 are reported targets as well.
The N-phenylquinazolin-4-amine moiety of gefitinib (5) was conserved however, the residues at the aromatic ring in the linker were modified, the residue targeting the solvent-exposed region was varied, and the linker at the C6 position of the quinazoline was adjusted to enable the macrocyclization. An overview of the structural modifications is shown in Figure 35A.
Kinome-wide screening of gefitinib (5) revealed several off-targets besides EGFR (Figure 35B), making it an excellent starting point for a macrocyclization strategy. Introducing a linker to the N phenylquinazoline-4-amine scaffold and retaining the residues on the aromatic ring as well as the methoxy group targeting the solvent-exposed region improved the selectivity profile and the efficacy towards EGFR WT and its mutants. Truncation of the linker moiety led to the mutant selective macrocycle 26f with an excellent kinome-wide selectivity profile (Figure 35B). An inhibitor that is effective on EGFR mutations while ineffective on the EGFR WT could represent an enhancement of patient treatment, as it potentially causes less side effects. Further studies could determine the effect of the most promising macrocycles in lung cancer cell lines. Additionally, the pharmacokinetic properties could be optimized, e.g. by introducing solubilizing groups, targeting the solvent-exposed region.
The second scaffold comprises the 3-aminopyrazole-based hinge-binding moiety. It is a privileged scaffold in medicinal chemistry for the development of kinase inhibitors. Previous publications report the anti-proliferative and anti-cancer potential of pyrazole-based molecules. They play a crucial role in the treatment of various diseases and cancer types like inflammation disorders, lymphoma, or breast cancer. This scaffold can be found e.g. in the aurora kinase inhibitor tozasertib or in the promiscuous kinase inhibitor 23, published by Statsuk et. al. Rescreening compound 23 in a comprehensive kinase panel against 468 human protein kinases confirmed the unselective behavior with a selectivity score of S35 = 0.56 (Figure 36B), making it a great starting point for further optimizations. The N-(1H-pyrazol-3-yl)pyrimidin-4-amine scaffold was conserved however, the residues targeting the solvent-exposed region were varied and different linkers were attached.
The introduction of different residues at the pyrazole dramatically influenced the selectivity profile of the desired kinases. Ester moieties caused to a favorable combination of selectivity and potency towards the kinase of interest CDK16. The removal of additional residues at the pyrimidine, targeting the solvent-exposed region, increased the efficiency towards CDK16. Further optimization led to the highly potent and selective CDK16 inhibitor 98d (IC50 = 33 nM). NanoBRETTM screening against the complete CDK family revealed a preferred inhibition of the PCTAIRE and PFTAIRE subfamily with cellular IC50 values of 20 nM – 120 nM and 50 nM – 180 nM, respectively. A FUCCI cell cycle assay and viability assessment of 98d confirmed previously published results, reporting a G2/M cell cycle arrest followed by apoptosis and accumulation of p27 through knockout of CDK16 in SCC cells. Consequently, further studies could evaluate the anti-tumor activity of 98d in SCC and NSCLC or elucidate the effect of 98d in AMPK-related macroautophagy. 98d represents a novel tool compound to investigate the understudied kinases of the PCTAIRE family and enable to enlighten the biological role of those kinases.
Macrocyclization of the N-(1H-pyrazol-3-yl)pyrimidin-4-amine core resulted in the selective BMPR2 inhibitor 110a. It showed a good binding affinity towards BMPR2 with a KD value of 205 nM as well as a good potency with an IC50 value of 506 nM. A comprehensive selectivity screen against 468 kinases revealed an excellent selectivity profile with S35 = 0.01. As no BMPR2 inhibitors have been published so far, 110a represents a novel compound that may provide further insights into the canonical BMP pathway, noncanonical signaling, or its impact on BMPR2-associated diseases like PAH.
The introduction of additional residues targeting the solvent-exposed region shifted the selectivity towards the MST kinases. The exchange from the pyrimidine to a quinazoline moiety resulted in the highly potent and selective macrocyclic MST3 inhibitor 113c. NanoBRETTM measurements demonstrated the preferred inhibition of MST3 with IC50 values of 210 nM and 30 nM for intact and lysed cells, respectively. A weaker activity could be seen for MST4 with 1.8 µM and 510 nM, while MST1 and MST2 were not affected. To date, no selective MST3 inhibitors have been published, making 113c a valuable tool compound for further functional studies. As MST3 is influencing the cell cycle progression, 113c could be tested in a further cell cycle assay to elucidate the inhibitory effect of 113c on MST3 and consequently on the cell cycle. Furthermore, the anti-tumor activity of 113c in breast cancer could be determined, as Madsen et. al. reported a high MST3 and MST4 activity triggered by FAM40B mutations.
Lysosomes are major degradative organelles that contain enzymes capable of breaking down proteins, nucleic acids, carbohydrates, and lipids. In the last decade, new discoveries have traced also important roles for lysosomes as signalling hubs, affecting metabolism, autophagy and pathogenic infections. Therefore, maintenance of a healthy lysosome population is of utmost importance to the cell to respond to both stress conditions and also homeostatic signalling. For example, for minor perturbations to the lysosomal membrane, the cell activates repair processes which seal membrane nicks. For more extensive damage, autophagy is activated to remove damaged organelles from the cell. on the other hand, during pathogen invasion host cells have also evolved mechanisms to hijack the endolysosomal pathway to facilitate their own growth and replication in host cells.
The first part of the thesis work focuses on a lysosomal regeneration program which is activated under conditions where the entire lysosomal pool of the cell is damaged. Upon extensive membrane damage induced by the lysosomotropic drug LLOMe, the cell activates a regeneration pathway which helps in the formation of new functional lysosomes by recycling damaged membranes. I have identified the molecules important for this novel pathway of lysosomal regeneration and showed how the protein TBC1D15 orchestrates this process to regenerate functional organelles from completely damaged membrane masses in the first 2 hours following lysosomal membrane damage. This process resembles the process of auto- lysosomal reformation (ALR)- involving the formation of lysosomal tubules which are extended along microtubules and cleaved in a dynamin2 dependent manner to form proto-lysosomes which develop into fully functional mature lysosomes. These lysosomal tubules are closely associated with ATG8 positive autophagosomal membranes and require ATG8 proteins to bind to the lysophagy receptor LIMP2 on damaged membranes. This process is physiologically important under conditions of crystal nephropathy where calcium oxalate crystals induce damage to lysosomal membranes in nephrons in kidney disease.
The second part of the thesis shows how the endolysosomal system of the cell is hijacked by the bacteriaLegionella pneumophila. During Legionella infection the formation of conventional ATG8 positive autophagosomes are blocked due to the protease activity of the bacterial effector protein RavZ which cleaves lipidated ATG8 proteins from autophagosomal membranes. The SidE effectors of Legionella modify STX17 and SNAP29 by the process of non-canonical ubiquitination called phosphoribose-linked serine ubiquitination (PR-Ub). These proteins are essential for the formation of the autophagosomal SNARE complex which is used for fusion of the autophagosome with the lysosome. Upon Legionella infection, PR-UB of STX17 aids in formation of autophagosome-like replication vacuoles. ThesevacuolesdonotfusewiththelysosomebecauseSNAP29isalsoPR-Ubmodified. PR-UbofSTX17 and SNAP29 sterically blocks the formation of the autophagosomal-SNARE complex thereby preventing fusion of the autophagosome with the lysosome. As a result, Legionella can replicate in autophagosome- like vacuoles which do not undergo lysosomal degradation. In absence of PR-Ub modified STX17, bacterial replication is compromised when measured by bacterial replication assays in lung epithelial (A549) cells.
Taken together, this thesis highlights two important aspects of the autophagy-lysosomal system- how it responds to extensive membrane damage and its importance in Legionella pneumophila infection. Extensive damage to lysosomal membranes triggers a rapid regeneration process to partially restore lysosomal function before the effects of TFEB dependent lysosomal biogenesis becomes apparent. On the other hand, Legionella pneumophila infection segregates the lysosomes from the rest of the endo-lysosomal system by blocking autophagosome-lysosome fusion. Though lysosomes remain active, they are incapable of degrading pathogens since pathogen containing vacuoles do not fuse with the lysosome.