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Interleukin 2 (IL-2) was the first cytokine applied for cancer treatment in human history. It has been approved as monotherapy for renal cell carcinoma and melanoma by the FDA and does mediate the regression of the tumors in patients. One of the possible mechanisms is that the administration of IL-2 led to T lymphocytes expansion, including CD4+ and CD8+ T cells. In addition, a recent study demonstrated that antigen-specific T cells could also be expanded through the induction of IL-2, which plays a crucial role in mediating tumor regression. However, despite the long-term and extensive use of IL-2 in the clinic, the ratio of patients who get a complete response was still low, and only about one-fifth of patients showed objective tumor regression. Therefore, the function of IL-2 in cancer treatment should continue to be optimized and investigated. A study by Franz O. Smith et al. has shown that the combination treatment of IL-2 and tumor-associated antigen vaccine has a strong trend to increased objective responses compared to patients with melanoma receiving IL-2 alone. Peptide vaccines are anti-cancer vaccines able to induce a powerful tumor antigenspecific immune response capable of eradicating the tumors. According to the type of antigens, peptide vaccines can be classified into two distinct categories: Tumor-associated antigens (TAA) vaccine and tumor-specific neoantigens (TSA) vaccine. Currently, Peptide vaccines are mainly investigated in phase I and phase II clinical trials of human cancer patients with various advanced cancers such as lung cancer, gastrointestinal tumors, and breast cancers. Vaccinia virus (VACV) is one of the safest viral vectors, which has been wildly used in cancer treatment and pathogen prevention. As an oncolytic vector, VACV can carry multiple large foreign genes, which enable the virus to introduce diagnostic and therapeutic agents without dramatically reducing the viral replication. Meanwhile, the recombinant vaccinia virus (rVACV) can be easily generated by homologous recombination. Here, we used the vaccinia virus as the therapeutic cancer vector, expressing mouse Interleukin 2 (IL-2) and tumor-associated antigens simultaneously to investigate the combined effect of anti-tumor immune response in the 4T1 mouse tumor model. As expected, the VACV driven mIL-2 expression remarkably increased both CD4+ and CD8+ populations in vivo, and the virus-expressed tumor-associated peptides successfully elicited theantigen-specific T cell response to inhibit the growth of tumors. Furthermore, the experiments with tumor-bearing animals showed that the mIL-2 plus tumor antigens expressing VACV vector gave a better anti-cancer response than the mIL-2 alone expressing vector. The combinations did significantly more inhibit tumor growth than mIL-2 treatment alone. Moreover, the results confirmed our previous unpublished data that the mIL-2 expression driven by synthetic early/late promoter in the Lister strain VACV could enhance the tumor regression in the 4T1 mouse model.
Eukaryotische messenger-RNAs (mRNAs) müssen diverse Prozessierungsreaktionen durchlaufen, bevor sie der Translationsmaschinerie als Template für die Proteinbiosynthese dienen können. Diese Reaktionen beginnen bereits kotranskriptionell und schließen das Capping, das Spleißen und die Polyadenylierung ein. Erst nach dem die Prozessierung abschlossen ist, kann die reife mRNA ins Zytoplasma transportiert und translatiert werden. mRNAs interagieren in jeder Phase ihres Metabolismus mit verschiedenen trans-agierenden Faktoren und bilden mRNA-Ribonukleoproteinkomplexe (mRNPs) aus. Dieser „mRNP-Code“ bestimmt das Schicksal jeder mRNA und reguliert dadurch die Genexpression auf posttranskriptioneller Ebene.
Für das La-verwandte Protein LARP4B (La-related protein 4B) wurde kürzlich eine direkte Interaktion mit den Translationsfaktoren PABPC1 (poly(A) binding protein, cytoplasmic 1) und RACK1 (receptor for activated C kinase) gefunden. Diese Befunde sowie die Assoziation mit aktiv translatierenden Ribosomen lässt vermuten, dass LARP4B zum mRNP-Code beiträgt. Die Domänenstruktur des Proteins legt darüber hinaus nahe, dass LARP4B direkt mRNAs bindet.
Um einen Einblick in die Funktion von LARP4B und seiner in vivo RNA-Bindungspartner zu erhalten, wurde die mRNA-Assoziation transkriptomweit mit Hilfe von PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation)-Experimenten bestimmt. Diese Daten zeigten, dass LARP4B ein spezifisches Set an zellulären mRNAs über Sequenzbereiche in deren 3’-untranslatierten Regionen bindet. Die bioinformatische Auswertung der PAR-CLIP-Daten identifizierte ein LARP4B-Bindemotiv, welches durch in vitro Bindungsstudien validiert werden konnte. Darüber hinaus belegten pSILAC (pulsed stable isotope labeling with amino acids in cell culture)-Experimente und eine transkriptomweite Analyse der mRNA-Level, dass LARP4B die Expression der Ziel-mRNAs beeinflusst, indem es die Stabilität der gebundenen Transkripte erhöht. LARP4B konnte somit als positiver Faktor der eukaryotischen Genexpression identifiziert werden.
Galectin-1 (hGal-1) is overexpressed by numerous cancer types and previously conducted studies confirmed that the β-galactoside-binding protein mediates various molecular interactions associated with tumor growth, spread and survival. Upon interaction with carbohydrate-based binding epitopes of glycan structures on human cell surfaces galectin-1 induces proliferative, angiogenetic and migratory signals and modulates negative T cell regulation which essentially helps the tumor to evade the immune response. These findings attributed galectin-1 a pivotal role in tumor physiology and strongly suggest the protein as target for diagnostic and therapeutic applications.
Within the scope of this work a strategy was elaborated for designing tailor-made galectin-1 ligands by functionalizing selected hydroxyl groups of the natural binding partner N-acetyllactosamine (LacNAc) that are not involved in the sophisticated interplay between the disaccharide and the protein. Synthetic modifications intended to introduce chemical groups i) to address a potential binding site adjacent to the carbohydrate recognition domain (CRD) with extended hGal-1-ligand interactions, ii) to implement a tracer isotope for diagnostic detection and iii) to install a linker unit for immobilization on microarrays.
Resulting structures were investigated regarding their targeting ability towards galectin-1 by cocrystallization experiments, SPR and ITC studies. Potent binders were further probed for their diagnostic potential to trace elevated galectin-1 levels in microarray experiments and for an application in positron emission tomography (PET).
Das Spleißen von prä-mRNAs stellt in der Expression eukaryontischer Gene einen essentiellen Reifungsschritt dar. Erst durch das exakte Entfernen von nicht-kodierenden Introns und Verbinden der kodierenden Exons kann die genetische Information am Ribosom in funktionelle Proteine umgesetzt werden. Spleißen wird durch das Spleißosom katalysiert, welches sich aus den small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, U5 und U6 und einer großen Anzahl weiterer Proteinfaktoren zusammensetzt. Die snRNPs bestehen aus einer Uridin-reichen snRNA, spezifischen und generellen (Sm-)Proteinen. Die Sm-Proteine B/B`, D1, D2, D3, E, F, und G bilden einen heptameren Ring um die sog. Sm-Bindungsstelle der snRNAs. Während die Zusammenlagerung von Sm-Proteinen mit der RNA in vitro spontan ablaufen kann, wird dieser Prozess in vivo von zwei makromolekularen Proteinkomplexen assistiert, die als PRMT5- bzw. SMN-Komplex bezeichnet werden. Der PRMT5-Komplex (bestehend aus PRMT5, WD45 und pICln) agiert in der frühen Phase der Zusammenlagerung. Seine Hauptfunktion ist die symmetrische Dimethylierung der Sm-Proteine und die Stabilisierung von Sm-Proteinkomplexen durch das Chaperon pICln in zwei Intermediaten. Einhergehend mit dieser Aktivität werden auch Aggregation bzw. unspezifische Wechselwirkungen der Sm-Proteine mit RNAs verhindert. In der späten Phase der Zusammenlagerung löst der SMN-Komplex (bestehend aus SMN, Gemin2-8 und unrip) pICln-Intermediate auf, wobei dieser die Sm-Proteine en bloc übernimmt und sie auf die snRNA überträgt. Während dieser Reaktion wird pICln aus den Komplexen verdrängt. Ein Fehlen des SMN-Proteins, einer Schlüsselkomponente des SMN-Komplexes, führt zur autosomal rezessiven Erbkrankheit `Spinale Muskelatrophie` (SMA) wobei der Schweregrad der Krankheit invers mit der Menge an funktionellem SMN-Protein korreliert. Es wird vermutet, dass eine gestörte snRNP-Biogenese die Ursache der SMA ist.
In der vorliegenden Arbeit sollte die U snRNP-Zusammenlagerungsmaschinerie aus rekombinanten Bausteinen rekonstituiert werden und so funktionellen und strukturellen Studien zugänglich gemacht werden. Folgende Resultate wurden in dieser Arbeit erhalten:
1) Im ersten Teil der Arbeit wurde eine experimentelle Strategie etabliert, welche die Rekonstitution des humanen SMN-Komplexes aus rekombinanten Untereinheiten erlaubte. Entscheidend hierfür war die Definition von Subkomplexen aufgrund einer Protein-Interaktionskarte. Die Subkomplexe konnten separat hergestellt und anschließend zum Gesamtkomplex vereinigt werden.
2) Die erfolgreiche Etablierung eines rekonstitutiven Systems erlaubte eine detaillierte biochemische Charakterisierung des SMN-Komplexes. Es konnte gezeigt werden, dass der rekombinante Komplex alle für die Biogenese von U snRNPs nötigen Schritte bewerkstelligen konnte. Dies schließt sowohl die Übernahme der Sm-Proteine aus den pICln-Intermediaten als auch das Verdrängen des Chaperons pICln und die Übertragung der Sm-Proteine auf die snRNAs ein.
3) Durch die Reduzierung des SMN-Gesamtkomplexes um Gemin3-5 auf einen SMN-Pentamer konnte dieser als ein funktioneller Kernbereich identifiziert werden, der die einzelnen Schritte der U snRNP-Biogenese vergleichbar mit dem gesamten Komplex bewerkstelligen konnte. Zudem agierte dieser reduzierte Komplex als notwendiger und ausreichender Spezifitätsfaktor der RNP-Zusammenlagerung.
4) Das rekombinante System ermöglichte erstmals SMN-Komplexe mit SMA-pathogenen Mutationen herzustellen und einer eingehenden funktionellen und strukturellen Untersuchung zu unterziehen. Die detaillierte Analyse der SMA-verursachenden Punktmutation SMN(E134K) offenbarte spezifische Defekte im Zusammenlagerungsprozess und damit Einblicke in die Pathophysiologie der Krankheit.
Mit der im Rahmen dieser Arbeit etablierten Rekonstitution des rekombinanten SMN-Komplexes wurde die Grundlage für die detaillierte biochemische und strukturbiologische Untersuchung der Zusammenlagerungsmaschinerie spleißosomaler U snRNPs gelegt. Dieses experimentelle System wird auch bei der Aufdeckung der biochemischen Defekte hilfreich sein, die zur neuromuskulären Krankheit SMA führen.
An essential step in eukaryotic gene expression is splicing, i.e. the excision of non-coding sequences from pre-mRNA and the ligation of coding-sequences. This reaction is carried out by the spliceosome, which is a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs) and a large number of proteins. Spliceosomal snRNPs are composed of one snRNA (or two in case of U4/6 snRNPs), seven common Sm proteins (SmD1, D2, D3, B, E, F, G) and several particle-specific proteins. The seven Sm proteins form a ring shaped structure on the snRNA, termed Sm core domain that forms a structural framework of all spliceosomal snRNPs. In the toroidal Sm core domain, the individual Sm proteins are arranged in the sequence SmE-SmG-SmD3-SmB- SmD1-SmD2-SmF from the first to the seventh nucleotide of the Sm site, respectively. The individual positions of Sm proteins in the Sm core domain are not interchangeable.
snRNPs are formed in vivo in a step-wise process, which starts with the export of newly transcribed snRNA to the cytoplasm. Within this compartment, Sm proteins are synthesized and subsequently transferred onto the snRNA. Upon formation of the Sm core and further modifications of snRNA, the snRNP is imported into the nucleus to join the spliceosome.
Prior to assembly into snRNPs, Sm proteins exist as specific hetero-oligomers in the cytoplasm. The association of these proteins with snRNA occurs spontaneously in vitro but requires the assistance of two major units, PRMT5- and SMN- complexes, in vivo. The early phase of assembly is critically influenced by the assembly chaperone pICln. This protein pre-organizes Sm proteins to functional building blocks and enables their recruitment onto the PRMT5 complex for methylation. Sm proteins are subsequently released from the PRMT5 complex as pICln bound entities and transferred onto the SMN-complex. The SMN complex then liberates the Sm proteins from the pICln-induced kinetic trap and allows their transfer onto the snRNA. Although the principal roles of SMN- and PRMT5 complexes in the assembly of snRNPs have been established, it is still not clear how newly translated Sm proteins are guided into the assembly line.
In this thesis, I have uncovered a new facet of pICln function in the assembly of snRNPs. I have shown that newly synthesized Sm proteins are retained at the ribosome upon termination of translation. Their release is facilitated by pICln, which interacts with the cognate Sm protein hetero-oligomers at their site of synthesis on the ribosome and recruits them into the assembly pathway. Additionally, I have been able to show that the early engagement of pICln with the Sm proteins ensures the flawless oligomerization of Sm proteins and prevents any non-chaperoned release and diffusion of Sm proteins in the cytoplasm.
In a second project, I have studied the mechanism of U7 snRNP assembly. This particle is a major component of the 3’ end processing machinery of replication dependent histone mRNAs. A biochemical hallmark of U7 is its unique Sm core in which the two canonical Sm proteins D1 and D2 are replaced by so-called “like Sm proteins”. The key question I addressed in my thesis was, how this “alternative” Sm core is assembled onto U7 snRNA. I have provided experimental evidence that the assembly route of U7 snRNPs and spliceosomal snRNPs are remarkably similar: The assembly of both particles depends on the same assembly factors and the mechanistic details are similar. It appears that formation of the U7- or spliceosomal- core specific 6S complex is the decisive step in assembly.
Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie benötigt und daher mittels vielfältiger Mechanismen streng reguliert wird. Ein Beispiel hierfür stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche hauptsächlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5’-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv ermöglicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. Nährstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird.
Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation ermöglicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erwägung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP – Sequenzen identifiziert werden, welche sich für Strukturanalysen eignen würden. Als weiterer TOP-interagierender Faktor wurde über verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert.
Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. Während gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7–Konstrukt sowie 7SK-Konstrukte für Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK – Interaktion ausüben, welcher in weiteren Studien berücksichtigt werden sollte.
As central components of life, DNA and RNA encode the genetic information. However, RNA performs several functions that exceed the competences stated in the ‘central dogma of life‘. RNAs undergo extensive post-transcriptional processing like chemical modifications. Among all classes of RNA, tRNAs are the most extensively modified. Their modifications are chemically diverse and vary from simple methylations (e.g. m3C, m6A) to more complex residues, like isopentenyl group (e.g. i6A, hypermodifications: e.g. ms2i6A) or even amino acids (e.g. t6A). Depending on their location within the overall structure, modifications can have an impact on tRNA stability and structure, as well as affinity for the ribosome and translation efficiency and fidelity. Given the importance of tRNA modifications new tools are needed for their detection and to study their recognition by proteins and enzymatic transformations.
The chemical synthesis of these naturally occurring tRNA modifications as phosphoramidite building blocks is a prerequisite to incorporate the desired modification via solid-phase synthesis into oligonucleotides. With the help of the m3C, (ms2)i6A, and t6A oligonucleotides, the importance and impact of tRNA modifications was investigated in this thesis. To this end, the role of METTL8 as the methyltransferase responsible for the installation of the methyl group at C32 for mt-tRNAThr and mt-tRNASer(UCN) was resolved. Thereby, the respective adenosine modification on position 37 is essential for the effectiveness of the enzyme. Besides, by means of NMR analysis, CD spectroscopy, thermal denaturation experiments, and native page separation, the impact of m3C32 on the structure of the tRNA ASLs was shown. The modification appeared to fine-tune the tRNA structure to optimize mitochondrial translation. To investigate the regulation of the dynamic modification pathway of m3C, demethylation assays were performed with the modified tRNA-ASLs and the (α-KG)- and Fe(II)-dependent dioxygenase ALKBH1 and ALKHB3. A demethylation activity of ALKBH3 on the mt-tRNAs was observed, even though it has so far only been described as a cytoplasmic enzyme. Whether this is physiologically relevant and ALKBH3 present a mitochondrial localization needs further validation. In addition, ALKBH1 was confirmed to not be able to demethylate m3C on mt-tRNAs, but indications for a deprenylation and exonuclease activity were found. Furthermore, the aforementioned naturally occurring modifications were utilized to find analytical tools that can determine the modification levels by DNAzymes, which cleave RNA in the presence of a specific modification. Selective DNA enzymes for i6A, as well as the three cytidine isomers m3C, m4C, and m5C have been identified and characterized.
Besides the naturally occurring tRNA modifications, the investigation on artificially modified nucleosides is also part of this thesis. Nucleosides with specific properties for desired applications can be created by modifying the scaffold of native nucleosides.
During the pandemic, the potential of antiviral nucleoside analogues was highlighted for the treatment of the SARS-CoV-2 infection. For examinations of the potential drug-candidate Molnupiravir, the N4-hydroxycytidine phosphoramidite building block was synthesized and incorporated into several RNA oligonucleotides. A two-step model for the NHC-induced mutagenesis of SARS-CoV-2 was proposed based on RNA elongation, thermal denaturation, and cryo-EM experiments using the modified RNA strands with the recombinant SARS-CoV-2 RNA-dependent RNA polymerase. Two tautomeric forms of NHC enable base pairing with guanosine in the amino and with adenosine in the imino form, leading to error catastrophe after the incorporation into viral RNA. These findings were further corroborated by thermal melting curve analysis and NMR spectroscopy of the NHC-containing Dickerson Drew sequence. In conclusion, the anti-amino form in the NHC-G base pair was assigned by NMR analysis using a 15N-labeld NHC building block incorporated into the Dickerson Drew sequence.
This thesis also addressed the synthesis of a 7-deazaguanosine crosslinker with a masked aldehyde as a diol linker for investigations of DNA-protein interactions. The diol functional group can be unmasked to release the reactive aldehyde, which can specifically form a covalent bond with amino acids Lys or Arg within the protein complex condensin. The incorporation of the synthesized phosphoramidite and triphosphate building blocks were shown and the functionality of the PCR product containing the crosslinker was demonstrated by oxidation and the formation of a covalent bond with a fluorescein label.
The development of assays that detect changes in this methylation pattern of m6A could provide new insights into important biological processes. In the last project of this thesis, the influence of RNA methylation states on the structural properties of RNA was analyzed and a fluorescent nucleoside analog (8-vinyladenosine) as molecular tools for such assays was developed. Initial experiments with the fluorescent nucleoside analog N6-methyl-8-vinyladenosine (m6v8A) were performed and revealed a strong fluorescence enhancement of the free m6v8A nucleoside by the installation of the vinyl moiety at position 8.
Overall, this thesis contributes to various research topics regarding the application of naturally occurring and artificial nucleoside analogues. Starting with the chemical synthesis of RNA and DNA modifications, this thesis has unveiled several open questions regarding the dynamic (de-)methylation pathway of m3C and the mechanism of action of molnupiravir through in-depth analysis and provided the basis for further investigations of the protein complex condensin, and a new fluorescent nucleoside analog m6v8A.
Cellular proteome profiling revealed that most biomolecules do not exist in isolation, but rather are incorporated into modular complexes. These assembled complexes are usually very large, consisting of 10 subunits on an average and include either proteins alone, or proteins and nucleic acids. Consequently, such macromolecular assemblies rather than individual biopolymers perform the vast majority of cellular activities. The faithful assembly of such molecular assemblies is often aided by trans-acting factors in vivo, to preclude aggregation of complex components and/or non-cognate interactions. A paradigm for an assisted assembly of a macromolecular machine is the formation of the common Sm/LSm core of spliceosomal and histone-mRNA processing U snRNPs. The key assembly factors united in the Protein Arginine Methyltransferase 5 (PRMT5) and the Survival Motor Neuron (SMN) complexes orchestrate the assembly of the Sm/LSm core on the U snRNAs. Assembly is initiated by the PRMT5-complex subunit pICln, which pre-arranges the Sm/LSm proteins into spatial positions occupied in the mature U snRNPs. The SMN complex subsequently binds these Sm/LSm units, displaces pICln and catalyses the Sm ring closure on the Sm-site of the U snRNA.
The SMN complex consists of the eponoymous SMN protein linked in a modular network of interactions with eight other proteins, termed Gemins 2-8 and Unrip. Despite functional and structural characterisation of individual protein components and/or sub-complexes of this assembly machinery, coherent understanding of the structural framework of the core SMN complex remained elusive. The current work, employing a combined approach of biochemical and structural studies, aimed to contribute to the understanding of how distinct modules within the SMN complex coalecse to form the macromolecular SMN complex.
A novel atomic resolution (1.5 Å) structure of the human Gemin8:7:6 sub-complex, illustrates how the peripheral Gemin7:6 module is tethered to the SMN complex via Gemin8’s C-terminus. In this model, Gemin7 engages with both Gemin6 and Gemin8 via the N- and C-termini of its Sm-fold like domain. This highly conserved interaction mode is reflected in the pronounced sequence conservation and identical biochemical behaviour of similar sub-complexes from divergent species, namely S. pombe and C. elegans.
Despite lacking significant sequence similarity to the Sm proteins, the dimeric Gemin7:6 complex share structural resemblance to the Sm heteromers. The hypothesis that the dimeric Gemin7:6 functions as a Sm-surrogate during Sm core assembly could not be confirmed in this work. The functional relevance of the structural mimicry of the dimeric Gemin7:6 sub-complex with the Sm heterodimers therefore still remains unclear.
Reduced levels of functional SMN protein is the cause of the devastating neurodegenerative disease, Spinal Muscular Atrophy (SMA). The C-terminal YG-zipper motif of SMN is a major hot-spot for most SMA patient mutations. In this work, adding to the existing inventory of the human and fission yeast YG-box models, a novel 2.2 Å crystal structure of the nematode SMN’s YG-box domain adopting the glycine zipper motif has been reported. Furthermore, it could be assessed that SMA patient mutations mapping to this YG-box domain greatly influences SMN’s self-association competency, a property reflected in both the human and nematode YG-box biochemical handles. The shared molecular architecture and biochemical behaviour of the nematode SMN YG-box domain with its human and fission yeast counterparts, reiterates the pronounced conservation of this oligomerisation motif across divergent organisms.
Apart from serving as a multimerization domain, SMN’s YG-box also acts as interaction platform for Gemin8. A systematic investigation of SMA causing missense mutations uncovered that Gemin8’s incorporation into the SMN complex is influenced by the presence of certain SMA patient mutations, albeit independent of SMN’s oligomerisation status. Consequently, loss of Gemin8 association in the presence of SMA patient mutations would also affect the incorporation of Gemin7:6 sub-complex. Gemin8, therefore sculpts the heteromeric SMN complex by bridging the Gemin7:6 and SMN:Gemin2 sub-units, a modular feature shared in both the human and nematode SMN complexes.
These findings provide an important foundation and a prospective structural framework for elucidating the core architecture of the SMN complex in the ongoing Cryo-EM studies.
The human-bacterial pathogen interaction is a complex process that results from
a prolonged evolutionary arms race in the struggle for survival. The pathogen employs
virulence strategies to achieve host colonization, and the latter counteracts using defense
programs. The encounter of both organisms results in drastic physiological changes
leading to stress, which is an ancient response accompanying infection. Recent evidence
suggests that the stress response in the host converges with the innate immune pathways
and influences the outcome of infection. However, the contribution of stress and the exact
mechanism(s) of its involvement in host defense remain to be elucidated. Using the model
bacterial pathogen Shigella flexneri, and comparing it with the closely related pathogen
Salmonella Typhimurium, this study investigated the role of host stress in the outcome of
infection.
Shigella infection is characterized by a pronounced pro-inflammatory response
that causes intense stress in host tissues, particularly the intestinal epithelium, which
constitutes the first barrier against Shigella colonization. In this study, inflammatory
stress was simulated in epithelial cells by inducing oxidative stress, hypoxia, and cytokine
stimulation. Shigella infection of epithelial cells exposed to such stresses was strongly
inhibited at the adhesion/binding stage. This resulted from the depletion of sphingolipidrafts
in the plasma membrane by the stress-activated sphingomyelinases. Interestingly,
Salmonella adhesion was not affected, by virtue of its flagellar motility, which allowed the
gathering of bacteria at remaining membrane rafts. Moreover, the intracellular replication
of Shigella lead to a similar sphingolipid-raft depletion in the membrane across adjacent
cells inhibiting extracellular bacterial invasion.
Additionally, this study shows that Shigella infection interferes with the host stress
granule-formation in response to stress. Interestingly, infected cells exhibited a nuclear
depletion of the global RNA-binding stress-granule associated proteins TIAR and TIA-1
and their accumulation in the cytoplasm.
Overall, this work investigated different aspects of the host stress-response in the
defense against bacterial infection. The findings shed light on the importance of the host
stress-pathways during infection, and improve the understanding of different strategies
in host-pathogen interaction.
The degradation of poly-adenosine tails of messenger RNAs (mRNAs) in the eukaryotic cells is a determining step in controlling the level of gene expression. The highly conserved Ccr4-Not complex was identified as the major deadenylation complex in all eukaryotic organisms. Plenty of biochemical studies have shown that this complex is also involved in many aspects of the mRNA metabolism, but we are still lacking the detailed structural information about its overall architecture and conformational states that could help to elucidate its multifunction and the way it is coordinated in the cells. Such information can also provide a basis to finding a possible way of intervention since the complex is also involved in some diseases such as cancer and cardiovascular disorders in humans. Meanwhile, the single particle Cryo-EM method has been through a “resolution revolution” recently due to the use of the newly developed direct electron detectors and has since resolved the high-resolution structures of many macromolecular protein complexes in their near-native state. Therefore, it was employed as a suitable method for studying the Ccr4-Not complex here. In this work, the Falcon 3EC direct detector mounted on the 300kV Titan Krios G3i Cryo-EM was evaluated for its practical performance at obtaining high-quality Cryo-EM data from protein samples of different molecular sizes. This served as a proof of principle for this detector’s capabilities and as a data collection guidance for studying the macromolecular complexes, such as the Ccr4-Not, when using an advanced high-performance microscope system. Next, the endogenous yeast Ccr4-Not complex was also purified via the immunoaffinity purification method and evaluated using negative staining EM to assess the conditions of the complex before proceeding to sample preparation for Cryo-EM. This has shown that the complex had an unexpected inherently dynamic property in vitro and extra optimisation procedures were needed to stabilise the complex during the purification and sample preparation. In addition, by using the label-free quantitative Mass spectrometry to examine the coimmunoprecipitated complex via different tagged subunits, it was deduced that two of the subunits (Not3/Not5) that shared some sequence similarity might compete for association with the scaffold subunit of the complex. An uncharacterised protein was also identified coimmunoprecipitating with the Caf130 subunit of the yeast complex. Cryo-EM data from the purified complex provided a low-resolution map that represents a surprisingly smaller partial complex as compared to 3D structures from previous studies, although gel electrophoresis and Mass spectrometry data have identified all of the nine subunits of the Ccr4-Not core complex in the sample. It was concluded that due to the presence of many predicted unstructured regions VI in the subunits and their dynamic composition in solution, the native complex could have been spontaneously denatured at the air/water interface during the sample preparation thus limiting the resolution of the Cryo-EM reconstruction. The purified complex was also examined for its deadenylase and ubiquitin ligase activity by in vitro assays. It was shown that the native complex has a different rate of activity and possibly also a different mode of action compared to the recombinant complexes from other species under similar reaction conditions. The Not4 E3 ligase was also shown to be active in the complex and was likely auto-ubiquitinated in the absence of a substrate. Both types of assays have also shown that the conformational flexibility does not seem to affect the enzymatic reactions when using a chemically crosslinked form of the complex for the assay, which implies that there can be other underlying mechanisms coordinating its structural and functional relationship. The findings from this work have therefore moved our understanding of the Ccr4-Not complex forward by looking at the different structural and functional behaviours of the endogenous complex, especially highlighting the obstacles in sample preparation for the native complex in high-resolution Cryo-EM. This would serve as foundation for future studies on the mechanism of this complex’s catalytic functions and also for optimising the Cryo-EM sample to generate better data that could eventually resolve the structure to a high-resolution.