@article{SchmidtDenkWiegering2020,
  author    = {Schmidt, Stefanie and Denk, Sarah and Wiegering, Armin},
  title     = {Targeting protein synthesis in colorectal cancer},
  series = {Cancers},
  volume    = {12},
  journal   = {Cancers},
  number    = {5},
  issn      = {2072-6694},
  doi       = {10.3390/cancers12051298},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206014},
  year      = {2020},
  abstract  = {Under physiological conditions, protein synthesis controls cell growth and survival and is strictly regulated. Deregulation of protein synthesis is a frequent event in cancer. The majority of mutations found in colorectal cancer (CRC), including alterations in the WNT pathway as well as activation of RAS/MAPK and PI3K/AKT and, subsequently, mTOR signaling, lead to deregulation of the translational machinery. Besides mutations in upstream signaling pathways, deregulation of global protein synthesis occurs through additional mechanisms including altered expression or activity of initiation and elongation factors (e.g., eIF4F, eIF2α/eIF2B, eEF2) as well as upregulation of components involved in ribosome biogenesis and factors that control the adaptation of translation in response to stress (e.g., GCN2). Therefore, influencing mechanisms that control mRNA translation may open a therapeutic window for CRC. Over the last decade, several potential therapeutic strategies targeting these alterations have been investigated and have shown promising results in cell lines, intestinal organoids, and mouse models. Despite these encouraging in vitro results, patients have not clinically benefited from those advances so far. In this review, we outline the mechanisms that lead to deregulated mRNA translation in CRC and highlight recent progress that has been made in developing therapeutic strategies that target these mechanisms for tumor therapy.},
  language  = {en}
}
@phdthesis{Steinmetzger2020,
  author    = {Steinmetzger, Christian},
  title     = {Fluorogenic Aptamers and Fluorescent Nucleoside Analogs as Probes for RNA Structure and Function},
  doi       = {10.25972/OPUS-20760},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207604},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {RNA plays a key role in numerous cellular processes beyond the central dogma of molecular biology. Observing and understanding this wealth of functions, discovering new ones and engineering them into purpose-built tools requires a sensitive means of observation. Over the past decade, fluorogenic aptamers have emerged to fill this niche. These short oligonucleotides are generated by in vitro selection to specifically interact with small organic fluorophores and can be utilized as genetically encoded tags for RNAs of interest. The most versatile class of fluorogenic aptamers is based on derivatives of hydroxybenzylidene imidazolone (HBI), a conditional fluorophore mimicking the chromophore structure found in green and red fluorescent proteins. The respective aptamers are well-known by the "vegetable" nomenclature, including Spinach, Broccoli and Corn, and have found numerous applications for studying RNA function in vitro and in cells. Their success, however, is somewhat overshadowed by individual shortcomings such as a propensity for misfolding, dependence on unphysiologically high concentrations of magnesium ions or, in the case of Corn, dimerization that might affect the function of the tagged RNA. Moreover, most fluorogenic aptamers exhibit limited ligand promiscuity by design, thereby restricting their potential for spectral tuning to a narrow window of wavelengths. This thesis details the characterization of a new fluorogenic aptamer system nicknamed Chili. Chili is derived from an aptamer that was originally selected to bind 4-hydroxy-3,5-dimethoxy¬hydroxy-benzylidene imidazolone (DMHBI), resulting in a green fluorescent complex. Unlike other aptamers of its kind, Chili engages in a proton transfer cycle with the bound ligand, resulting in a remarkably large Stokes shift of more than 130 nm. By means of an empirical ligand optimization approach, several new DMHBI derivatives were found that bind to Chili with high affinity, furnishing complexes up to 7.5 times brighter compared to the parent ligand. In addition, Chili binds to π-extended DMHBI derivatives that confer fluorescence in the yellow-red region of the visible spectrum. The highest affinity and degree of fluorescence turn-on for both green and red fluorogenic ligands were achieved by the incorporation of a unique, positively charged substituent into the HBI scaffold. Supplemented by NMR spectroscopy, kinetic and thermodynamic studies showed that the binding site of Chili is loosely preorganized in the absence of ligand and likely forms a G-quadruplex upon ligand binding. To showcase future applications, Chili was incorporated into a FRET sensor for monitoring the cleavage of an RNA substrate by a 10-23 DNAzyme. Besides aptamers as macromolecular fluorescent complexes, fluorescent nucleobase analogs are powerful small isomorphic components of RNA suitable for studying structure and folding. Here, the highly emissive nucleobase analog 4-cyanoindole (4CI) was developed into a ribonucleoside (r4CI) for this purpose. A new phosphoramidite building block was synthesized to enable site-specific incorporation of 4CI into RNA. Thermal denaturation experiments confirmed that 4CI behaves as a universal nucleobase, i.e. without bias towards any particular hybridization partner. Photophysical characterization established r4CI as a generally useful fluorescent ribonucleoside analog. In this work, it was employed to gain further insight into the structure of the Chili aptamer. Using several 4CI-modified Chili-HBI complexes, a novel base-ligand FRET assay was established to obtain a set of combined distance and orientation restraints for the tertiary structure of the aptamer. In addition to their utility for interrogating structure and binding, supramolecular FRET pairs comprising a fluorescent nucleobase analog donor and an innately fluorogenic acceptor hold great promise for the construction of color-switchable RNA aptamer sensor devices.},
  subject      = {Aptamer},
  language  = {en}
}
@phdthesis{Kauk2018,
  author    = {Kauk, Michael},
  title     = {Investigating the Molecular Mechanism of Receptor Activation at Muscarinic Receptors by Means of Pathway-Specific Dualsteric Ligands and Partial Agonists},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173729},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {G protein-coupled receptors (GPCRs) form the biggest receptor family that is encoded in the human genome and represent the most druggable target structure for modern therapeutics respectively future drug development. Belonging to aminergic class A GPCRs muscarinic Acetylcholine receptors (mAChRs) are already now of clinical relevance and are also seen as promising future drug targets for treating neurodegenerative diseases like Alzheimer or Parkinson. The mAChR family consist of five subtypes showing high sequence identity for the endogenous ligand binding region and thus it is challenging until now to selectively activate a single receptor subtype. A well accepted method to study ligand binding, dynamic receptor activation and downstream signaling is the fluorescence resonance energy transfer (FRET) application. Here, there relative distance between two fluorophores in close proximity (<10 nm) can be monitored in a dynamic manner. The perquisite for that is the spectral overlap of the emission spectrum of the first fluorophore with the excitation spectrum of the second fluorophore. By inserting two fluorophores into the molecular receptor structure receptor FRET sensors can serve as a powerful tool to study dynamic receptor pharmacology. Dualsteric Ligands consist of two different pharmacophoric entities and are regarded as a promising ligand design for future drug development. The orthosteric part interacts with high affinity with the endogenous ligand binding region whereas the allosteric part binds to a different receptor region mostly located in the extracellular vestibule. Both moieties are covalently linked. Dualsteric ligands exhibit a dynamic ligand binding. The dualsteric binding position is characterized by a simultaneous binding of the orthosteric and allosteric moiety to the receptor and thus by receptor activation. In the purely allosteric binding position no receptor activation can be monitored. In the present work the first receptor FRET sensor for the muscarinic subtype 1 (M1) was generated and characterized. The M1-I3N-CFP sensor showed an unaltered physiological behavior as well as ligand and concentration dependent responses. The sensor was used to characterize different sets of dualsteric ligands concerning their pharmacological properties like receptor activation. It was shown that the hybrids consisting of the synthetic full agonist iperoxo and the positive allosteric modulator of BQCA type is very promising. Furthermore, it was shown for orthosteric as well as dualsteric ligands that the degree of receptor activation is highly dependent on the length of and the chemical properties of the linker moiety. For dualsteric ligands a bell-shaped activation characteristic was reported for the first time, suggesting that there is an optimal linker length for dualsteric ligands. The gained knowledge about hybrid design was then used to generate and characterize the first photo-switchable dualsteric ligand. The resulting hybrids were characterized with the M1-I3N-CFP sensor and were described as photo-inactivatable and dimmable. In addition to the ligand characterization the ligand application methodology was further developed and improved. Thus, a fragment-based screening approach for dualsteric ligands was reported in this study for the first time. With this approach it is possible to investigate dualsteric ligands in greater detail by applying either single ligand fragments alone or in a mixture of building blocks. These studies revealed the insights that the effect of dualsteric ligands on a GPCR can be rebuild by applying the single building blocks simultaneously. The fragment-based screening provides high potential for the molecular understanding of dualsteric ligands and for future screening approaches. Next, a further development of the standard procedure for measuring FRET by sensitized emission was performed. Under normal conditions single cell FRET is measured on glass coverslips. After coating the coverslips surface with a 20 nm thick gold layer an increased FRET efficiency up to 60 \% could be reported. This finding was validated in different approaches und in different configurations. This FRET enhancement by plasmonic surfaces was until yet unreported in the literature for physiological systems and make FRET for future projects even more powerful.},
  subject      = {G-Protein gekoppelte Rezeptoren},
  language  = {en}
}
@phdthesis{Rydzek2019,
  author    = {Rydzek, Julian},
  title     = {NF-κB/NFAT Reporter Cell Platform for Chimeric Antigen Receptor (CAR)-Library Screening},
  doi       = {10.25972/OPUS-17918},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179187},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Immunotherapy with engineered T cells expressing a tumor-specific chimeric antigen receptor (CAR) is under intense preclinical and clinical investigation. This involves a rapidly increasing portfolio of novel target antigens and CAR designs that need to be tested in time- and work-intensive screening campaigns in primary T cells. Therefore, we anticipated that a standardized screening platform, similar as in pharmaceutical small molecule and antibody discovery, would facilitate the analysis of CARs by pre-selecting lead candidates from a large pool of constructs that differ in their extracellular and intracellular modules. Because CARs integrate structural elements of the T cell receptor (TCR) complex and engage TCR-associated signaling molecules upon stimulation, we reasoned that the transcription factors nuclear factor-κB (NF-κB) and nuclear factor of activated T cells (NFAT) could serve as surrogate markers for primary T cell function. The nuclear translocation of both transcription factors in primary T cells, which we observed following CAR stimulation, supported our rationale to use NF-κB and NFAT as indicators of CAR-mediated activation in a screening platform. To enable standardized and convenient analyses, we have established a CAR-screening platform based on the human T cell lymphoma line Jurkat that has been modified to provide rapid detection of NF-κB and NFAT activation. For this purpose, Jurkat cells contained NF-κB and NFAT-inducible reporter genes that generate a duplex output of cyan fluorescent protein (CFP) and green fluorescent protein (GFP), respectively. Upon stimulation of NF-κB/NFAT reporter cells, the expression of both fluorophores could be readily quantified in high-throughput screening campaigns by flow cytometry. We modified the reporter cells with CD19-specific and ROR1-specific CARs, and we co-cultured them with antigen-positive stimulator cells to analyze NF-κB and NFAT activation. CAR-induced reporter signals could already be detected after 6 hours. The optimal readout window with high-level reporter activation was set to 24 hours, allowing the CAR-screening platform to deliver results in a rapid turnaround time. A reporter cell-screening campaign of a spacer library with CARs comprising a short, intermediate or long IgG4-Fc domain allowed distinguishing functional from non-functional constructs. Similarly, reporter cell-based analyses identified a ROR1-CAR with 4-1BB domain from a library with different intracellular signal modules due to its ability to confer high NF-κB activation, consistent with data from in vitro and in vivo studies with primary T cells. The results of both CAR screening campaigns were highly reproducible, and the time required for completing each testing campaign was substantially shorter with reporter cells (6 days) compared to primary T cells (21 days). We further challenged the reporter cells in a large-scale screening campaign with a ROR1 CAR library comprising mutations in the VH CDR3 sequence of the R11 scFv. This region is crucial for binding the R11 epitope of ROR1, and we anticipated that mutations here would cause a loss of specificity and affinity for most of the CAR variants. This provided the opportunity to determine whether the CAR screening platform was able to retrieve functional constructs from a large pool of CAR variants. Indeed, using a customized pre enrichment and screening strategy, the reporter cells identified a functional CAR variant that was present with a frequency of only 6 in 1.05x10^6. As our CAR-screening platform enabled the analysis of activating signal modules, it encouraged us to also evaluate inhibitory signal modules that change the CAR mode of action. Such an inhibitory CAR (iCAR) can be used in logic gates with an activating CAR to interfere with T cell stimulation. By selecting appropriate target antigens for iCAR and CAR, this novel application aims to improve the selectivity towards tumor cells, and it could readily be studied using our screening platform. Accordingly, we tested CD19-specific iCARs with inhibitory PD-1 signal module for their suppressive effect on reporter gene activation. In logic gates with CAR or TCR stimulation, a decrease of NF-κB and NFAT signals was only observed when activating and inhibitory receptors were forced into spatial proximity. These results were further verified by experiments with primary T cells. In conclusion, our reporter cell system is attractive as a platform technology because it is independent of testing in primary T cells, exportable between laboratories, and scalable to enable small- to large-scale screening campaigns of CAR libraries. The pre-selection of appropriate lead candidates with optimal extracellular and intracellular modules can reduce the number of CAR constructs to be investigated in further in vitro and in vivo studies with primary T cells. We are therefore confident that our CAR-screening platform based on NF-κB/NFAT reporter cells will be useful to accelerate translational research by facilitating the evaluation of CARs with novel design parameters.},
  subject      = {Antigenrezeptor},
  language  = {en}
}
@phdthesis{Kaiser2020,
  author    = {Kaiser, Sebastian},
  title     = {A RecQ helicase in disguise: Characterization of the unconventional Structure and Function of the human Genome Caretaker RecQ4},
  doi       = {10.25972/OPUS-16041},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160414},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {From the simplest single-cellular organism to the most complex multicellular life forms, genetic information in form of DNA represents the universal basis for all biological processes and thus for life itself. Maintaining the structural and functional integrity of the genome is therefore of paramount importance for every single cell. DNA itself, as an active and complex macromolecular structure, is both substrate and product of many of these biochemical processes. A cornerstone of DNA maintenance is thus established by the tight regulation of the multitude of reactions in DNA metabolism, repressing adverse side reactions and ensuring the integrity of DNA in sequence and function. The family of RecQ helicases has emerged as a vital class of enzymes that facilitate genomic integrity by operating in a versatile spectrum of nucleic acid metabolism processes, such as DNA replication, repair, recombination, transcription and telomere stability. RecQ helicases are ubiquitously expressed and conserved in all kingdoms of life. Human cells express five different RecQ enzymes, RecQ1, BLM, WRN, RecQ4 and RecQ5, which all exhibit individual as well as overlapping functions in the maintenance of genomic integrity. Dysfunction of three human RecQ helicases, BLM, WRN and RecQ4, causes different heritable cancer susceptibility syndromes, supporting the theory that genomic instability is a molecular driving force for cancer development. However, based on their inherent DNA protective nature, RecQ helicases represent a double-edged sword in the maintenance of genomic integrity. While their activity in normal cells is essential to prevent cancerogenesis and cellular aging, cancer cells may exploit this DNA protective function by the overexpression of many RecQ helicases, aiding to overcome the disadvantageous results of unchecked DNA replication and simultaneously gaining resistance against chemotherapeutic drugs. Therefore, detailed knowledge how RecQ helicases warrant genomic integrity is required to understand their implication in cancerogenesis and aging, thus setting the stage to develop new strategies towards the treatment of cancer. The current study presents and discusses the first high-resolution X-ray structure of the human RecQ4 helicase. The structure encompasses the conserved RecQ4 helicase core, including a large fraction of its unique C- terminus. Our structural analysis of the RecQ4 model highlights distinctive differences and unexpected similarities to other, structurally conserved, RecQ helicases and permits to draw conclusions about the functional implications of the unique domains within the RecQ4 C-terminus. The biochemical characterization of various RecQ4 variants provides functional insights into the RecQ4 helicase mechanism, suggesting that RecQ4 might utilize an alternative DNA strand separation technique, compared to other human RecQ family members. Finally, the RecQ4 model permits for the first time the analysis of multiple documented RecQ4 patient mutations at the atomic level and thus provides the possibility for an advanced interpretation of particular structure-function relationships in RecQ4 pathogenesis.},
  subject      = {Helikasen},
  language  = {en}
}
@phdthesis{PrietoGarcia2022,
  author    = {Prieto Garc{\´i}a, Cristian},
  title     = {USP28 regulates Squamous cell oncogenesis and DNA repair via ΔNp63 deubiquitination},
  doi       = {10.25972/OPUS-27033},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270332},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer resistance toward therapies inducing DNA damage. SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited, and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities, the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a potential alternative to improve the therapeutic response and clinical outcomes of SCC patients. However, ∆Np63 is considered "undruggable." As is commonly observed in transcription factors, ∆Np63 does not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of different pathways and cellular processes, making it difficult to counteract its function by targeting downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin-proteasome system (UPS), the development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target ∆Np63 in SCC treatment. This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability, and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time, ∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in preclinical mouse models. Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance, thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28 inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such as Cisplatin.},
  language  = {en}
}
@phdthesis{Nelke2022,
  author    = {Nelke, Johannes},
  title     = {Entwicklung multi-funktioneller TNFRSF Rezeptorspezifischer Antik{\"o}rper-Fusionsproteine mit FcγR-unabh{\"a}ngiger Aktivit{\"a}t},
  doi       = {10.25972/OPUS-27985},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-279855},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Antik{\"o}rper, die gegen eine klinisch relevante Gruppe von Rezeptoren innerhalb der Tumornekrosefaktor-Rezeptor-Superfamilie (TNFRSF) gerichtet sind, darunter CD40 und CD95 (Fas/Apo-1), ben{\"o}tigen ebenfalls eine Bindung an Fc-Gamma-Rezeptoren (FcγRs), um eine starke agonistische Wirkung zu entfalten. Diese FcγR-Abh{\"a}ngigkeit beruht weitgehend auf der bloßen zellul{\"a}ren Verankerung durch die Fc-Dom{\"a}ne des Antik{\"o}rpers und ben{\"o}tigt dabei kein FcγR-Signalling. Ziel dieser Doktorarbeit war es, das agonistische Potenzial von αCD40- und αCD95-Antik{\"o}rpern unabh{\"a}ngig von der Bindung an FcγRs durch die Verankerung an Myelomzellen zu entfalten. Zu diesem Zweck wurden verschiedene Antik{\"o}rpervarianten (IgG1, IgG1-N297A, Fab2) gegen die TNFRSF-Mitglieder CD40 und CD95 genetisch mit einem einzelkettig kodierten B-Zell-aktivierenden Faktor (scBaff) Trimer als C-terminale myelom-spezifische Verankerungsdom{\"a}ne fusioniert, welche die Fc-Dom{\"a}ne-vermittelte FcγR-Bindung ersetzt. Diese bispezifischen Antik{\"o}rper-scBaff-Fusionsproteine wurden in Bindungsstudien und funktionellen Assays mit Tumorzelllinien untersucht, die einen oder mehrere der drei Baff-Rezeptoren exprimieren: BaffR, Transmembran-Aktivator und CAML-Interaktor (TACI) und B-Zell-Reifungsantigen (BCMA). Zellul{\"a}re Bindungsstudien zeigten, dass die Bindungseigenschaften der verschiedenen Dom{\"a}nen innerhalb der Antik{\"o}rper-scBaff-Fusionen gegen{\"u}ber der Zielantigene vollst{\"a}ndig intakt blieben. In Ko-Kulturversuchen von CD40- und CD95-responsiven Zellen mit BaffR-, BCMA- oder TACI-exprimierenden Verankerungszellen zeigten die Antik{\"o}rper-Fusionsproteine einen starken Agonismus, w{\"a}hrend in Ko-Kulturen mit Zellen ohne Expression von Baff-interagierenden Rezeptoren nur eine geringe Rezeptorstimulation beobachtet wurde. Die hier vorgestellten αCD40- und αCD95-Antik{\"o}rper-scBaff-Fusionsproteine zeigen also Myelom-spezifische Aktivit{\"a}t und versprechen im Vergleich zu herk{\"o}mmlichen CD40- und CD95-Agonisten geringere systemische Nebenwirkungen.},
  subject      = {Antigen CD40},
  language  = {de}
}
@phdthesis{Ries2020,
  author    = {Ries, Lena Kerstin},
  title     = {From recognition to reaction: Mechanistic analysis of the interactions of the HECT ligase E6AP with ubiquitin},
  doi       = {10.25972/OPUS-17960},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179609},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The ubiquitination of proteins controls a multitude of physiological processes. This versatility of ubiquitin as a molecular signal arises from the diverse ways by which it can be attached to target proteins. Different ubiquitination patterns are then translated into different downstream consequences. Due to the enormous complexity of possible ubiquitin modifications, the ubiquitination machinery must be highly specific and tightly controlled. Ubiquitination proceeds through an enzymatic cascade, the last step of which is catalyzed by the E3 enzyme family. E3 enzymes are the crucial regulators since they dictate the specificity of substrate selection and modification. Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity, regulation and specificity in this crucial ligase are incompletely understood. One aim of this study was to unravel the role of the a1'-helix N-terminal to the HECT domain that was found to be a key element mediating regulation and oligomerization in other HECT ligases. I found that most N-terminally extended HECT domain constructs were insoluble when expressed in E. coli, indicating that additional regions N-terminal to the tested fragments may be essential to protect this highly hydrophobic helix from causing aggregation. Another question addressed in this study was how E6AP builds ubiquitin chains. Using single-turnover experiments, I showed that ubiquitin-loaded E6AP is unable to transfer an additional ubiquitin molecule onto a stably linked ubiquitin-E6AP complex. This indicates that E6AP cannot assemble chains on its active site and may instead follow a sequential addition mechanism in which one ubiquitin molecule is transferred at a time to the target protein. Using NMR spectroscopy and extensive mutational analyses, the determinants of ubiquitin recognition by the C-lobe of E6AP were unraveled and assigned to particular steps in the catalytic cycle. A functionally critical interface was identified that is specifically required during thioester formation between the C-terminus of ubiquitin and the ligase active site. This interface resembles the one utilized by NEDD4-type enzymes, suggesting a conserved ubiquitin binding mode across HECT ligases, independent of their linkage specificities. Moreover, I identified critical surface patches on ubiquitin and in the N- and C-terminal portions of the catalytic domain of E6AP that are important for the subsequent step of isopeptide bond formation. I also uncovered key determinants of the Lys48-linkage specificity of E6AP, both in the E6AP HECT domain and ubiquitin itself. This includes the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site, Lys48. It is thus tempting to speculate that ubiquitin linkage formation by E6AP is substrate-assisted. Taken together, my results improve our mechanistic understanding of the structure-function relationship between E6AP and ubiquitin, thus providing a basis for ultimately manipulating the functions of this HECT ligase for therapeutic applications.},
  subject      = {Ubiquitin},
  language  = {en}
}
@phdthesis{Koelmel2020,
  author    = {K{\"o}lmel, Wolfgang},
  title     = {Structural and functional characterization of TFIIH from \(Chaetomium\) \(thermophilum\)},
  doi       = {10.25972/OPUS-16176},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161769},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Gene expression and transfer of the genetic information to the next generation forms the basis of cellular life. These processes crucially rely on DNA, thus the preservation, transcription and translation of DNA is of fundamental importance for any living being. The general transcription factor TFIIH is a ten subunit protein complex, which consists of two subcomplexes: XPB, p62, p52, p44, p34, and p8 constitute the TFIIH core, CDK7, CyclinH, and MAT1 constitute the CAK. These two subcomplexes are connected via XPD. TFIIH is a crucial factor involved in both, DNA repair and transcription. The central role of TFIIH is underlined by three severe disorders linked to failure of TFIIH in these processes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Only limited structural and functional data of TFIIH are available so far. Here, the model organism Chaetomium thermophilum was utilized with the aim to structurally and functionally characterize TFIIH. By combining the expression and purification of single TFIIH subunits with the co-expression and co-purification of dual complexes, a unique and powerful modular system of the TFIIH core subunits could be established, encompassing all proteins in high quality and fully functional. This system permits the step-wise assembly of TFIIH core, thereby making it possible to assess the influence of the intricate interaction network within TFIIH core on the overall enzymatic activities of TFIIH, which has not been possible so far. Utilizing the single subunits and dual complexes, a detailed interaction network of TFIIH core was established, revealing the crucial role of the p34 subunit as a central scaffold of TFIIH by linking the two proteins p44 and p52. Our studies also suggest that p62 constitutes the central interface of TFIIH to the environment rather than acting as a scaffold. TFIIH core complexes were assembled and investigated via electron microscopy. Preliminary data indicate that TFIIH adopts different conformational states, which are important to fulfill its functions in transcription and DNA repair. Additionally, a shortened construct of p62 was used to develop an easy-to-use, low cost strategy to overcome the crystallographic phase problem via cesium derivatization.},
  subject      = {Transkriptionsfaktor},
  language  = {en}
}
@article{WanzekSchwindtCapraetal.2017,
  author    = {Wanzek, Katharina and Schwindt, Eike and Capra, John A. and Paeschke, Katrin},
  title     = {Mms1 binds to G-rich regions in Saccharomyces cerevisiae and influences replication and genome stability},
  series = {Nucleic Acids Research},
  volume    = {45},
  journal   = {Nucleic Acids Research},
  number    = {13},
  doi       = {10.1093/nar/gkx467},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170577},
  pages     = {7796-7806},
  year      = {2017},
  abstract  = {The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and replication fork progression at particular G-rich motifs. This motif can form G-quadruplex (G4) structures in vitro. G4 are stable DNA structures that are known to impede replication fork progression. In the absence of Mms1, genome stability is at risk at these G-rich/G4 regions as demonstrated by gross chromosomal rearrangement assays. Mms1 binds throughout the cell cycle to these G-rich/G4 regions and supports the binding of Pif1 DNA helicase. Based on these data we propose a mechanistic model in which Mms1 binds to specific G-rich/G4 motif located on the lagging strand template for DNA replication and supports Pif1 function, DNA replication and genome integrity.},
  language  = {en}
}
@phdthesis{Kalb2021,
  author    = {Kalb, Jacqueline},
  title     = {The role of BRCA1 and DCP1A in the coordination of transcription and replication in neuroblastoma},
  doi       = {10.25972/OPUS-24871},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248711},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The deregulation of the MYC oncoprotein family plays a major role in tumorigenesis and tumour maintenance of many human tumours. Because of their structure and nuclear localisation, they are defined as undruggable targets which makes it difficult to find direct therapeutic approaches. An alternative approach for targeting MYC-driven tumours is the identification and targeting of partner proteins which score as essential in a synthetic lethality screen. Neuroblastoma, an aggressive entity of MYCN-driven tumours coming along with a bad prognosis, are dependent on the tumour suppressor protein BRCA1 as synthetic lethal data showed. BRCA1 is recruited to promoter regions in a MYCN-dependent manner. The aim of this study was to characterise the role of BRCA1 in neuroblastoma with molecular biological methods. BRCA1 prevents the accumulation of RNA Polymerase II (RNAPII) at the promoter region. Its absence results in the formation of DNA/RNA-hybrids, so called R-loops, and DNA damage. To prevent the accumulation of RNAPII, the cell uses DCP1A, a decapping factor known for its cytoplasmatic and nuclear role in mRNA decay. It is the priming factor in the removal of the protective 5'CAP of mRNA, which leads to degradation by exonucleases. BRCA1 is necessary for the chromatin recruitment of DCP1A and its proximity to RNAPII. Cells showed upon acute activation of MYCN a higher dependency on DCP1A. Its activity prevents the deregulation of transcription and leads to proper coordination of transcription and replication. The deregulation of transcription in the absence of DCP1A results in replication fork stalling and leads to activation of the Ataxia telangiectasia and Rad3 related (ATR) kinase. The result is a disturbed cell proliferation to the point of increased apoptosis. The activation of the ATR kinase pathway in the situation where DCP1A is knocked down and MYCN is activated, makes those cells more vulnerable for the treatment with ATR inhibitors. In summary, the tumour suppressor protein BRCA1 and the decapping factor DCP1A, mainly known for its function in the cytoplasm, have a new nuclear role in a MYCN-dependent context. This study shows their essentiality in the coordination of transcription and replication which leads to an unrestrained growth of tumour cells if uncontrolled.},
  subject      = {Neuroblastom},
  language  = {en}
}
@article{VeepaschitViswanathanBordonneetal.2021,
  author    = {Veepaschit, Jyotishman and Viswanathan, Aravindan and Bordonne, Remy and Grimm, Clemens and Fischer, Utz},
  title     = {Identification and structural analysis of the Schizosaccharomyces pombe SMN complex},
  series = {Nucleic Acids Research},
  volume    = {49},
  journal   = {Nucleic Acids Research},
  number    = {13},
  doi       = {10.1093/nar/gkab158},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259880},
  pages     = {7207-7223},
  year      = {2021},
  abstract  = {The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.},
  language  = {en}
}
@article{AndelovicWinterJakobetal.2021,
  author    = {Andelovic, Kristina and Winter, Patrick and Jakob, Peter Michael and Bauer, Wolfgang Rudolf and Herold, Volker and Zernecke, Alma},
  title     = {Evaluation of plaque characteristics and inflammation using magnetic resonance imaging},
  series = {Biomedicines},
  volume    = {9},
  journal   = {Biomedicines},
  number    = {2},
  issn      = {2227-9059},
  doi       = {10.3390/biomedicines9020185},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-228839},
  year      = {2021},
  abstract  = {Atherosclerosis is an inflammatory disease of large and medium-sized arteries, characterized by the growth of atherosclerotic lesions (plaques). These plaques often develop at inner curvatures of arteries, branchpoints, and bifurcations, where the endothelial wall shear stress is low and oscillatory. In conjunction with other processes such as lipid deposition, biomechanical factors lead to local vascular inflammation and plaque growth. There is also evidence that low and oscillatory shear stress contribute to arterial remodeling, entailing a loss in arterial elasticity and, therefore, an increased pulse-wave velocity. Although altered shear stress profiles, elasticity and inflammation are closely intertwined and critical for plaque growth, preclinical and clinical investigations for atherosclerosis mostly focus on the investigation of one of these parameters only due to the experimental limitations. However, cardiovascular magnetic resonance imaging (MRI) has been demonstrated to be a potent tool which can be used to provide insights into a large range of biological parameters in one experimental session. It enables the evaluation of the dynamic process of atherosclerotic lesion formation without the need for harmful radiation. Flow-sensitive MRI provides the assessment of hemodynamic parameters such as wall shear stress and pulse wave velocity which may replace invasive and radiation-based techniques for imaging of the vascular function and the characterization of early plaque development. In combination with inflammation imaging, the analyses and correlations of these parameters could not only significantly advance basic preclinical investigations of atherosclerotic lesion formation and progression, but also the diagnostic clinical evaluation for early identification of high-risk plaques, which are prone to rupture. In this review, we summarize the key applications of magnetic resonance imaging for the evaluation of plaque characteristics through flow sensitive and morphological measurements. The simultaneous measurements of functional and structural parameters will further preclinical research on atherosclerosis and has the potential to fundamentally improve the detection of inflammation and vulnerable plaques in patients.},
  language  = {en}
}
@article{TolayBuchberger2022,
  author    = {Tolay, Nazife and Buchberger, Alexander},
  title     = {Role of the ubiquitin system in stress granule metabolism},
  series = {International Journal of Molecular Sciences},
  volume    = {23},
  journal   = {International Journal of Molecular Sciences},
  number    = {7},
  issn      = {1422-0067},
  doi       = {10.3390/ijms23073624},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284061},
  year      = {2022},
  abstract  = {Eukaryotic cells react to various stress conditions with the rapid formation of membrane-less organelles called stress granules (SGs). SGs form by multivalent interactions between RNAs and RNA-binding proteins and are believed to protect stalled translation initiation complexes from stress-induced degradation. SGs contain hundreds of different mRNAs and proteins, and their assembly and disassembly are tightly controlled by post-translational modifications. The ubiquitin system, which mediates the covalent modification of target proteins with the small protein ubiquitin ('ubiquitylation'), has been implicated in different aspects of SG metabolism, but specific functions in SG turnover have only recently emerged. Here, we summarize the evidence for the presence of ubiquitylated proteins at SGs, review the functions of different components of the ubiquitin system in SG formation and clearance, and discuss the link between perturbed SG clearance and the pathogenesis of neurodegenerative disorders. We conclude that the ubiquitin system plays an important, medically relevant role in SG biology.},
  language  = {en}
}
@article{TolayBuchberger2021,
  author    = {Tolay, Nazife and Buchberger, Alexander},
  title     = {Comparative profiling of stress granule clearance reveals differential contributions of the ubiquitin system},
  series = {Life Science Alliance},
  volume    = {4},
  journal   = {Life Science Alliance},
  number    = {5},
  doi       = {10.26508/lsa.202000927},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259810},
  pages     = {e202000927},
  year      = {2021},
  abstract  = {Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslationalmodifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immuno-fluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.},
  language  = {en}
}
@phdthesis{Reil2023,
  author    = {Reil, Lucy Honor},
  title     = {The role of WASH complex subunit Strumpellin in platelet function},
  doi       = {10.25972/OPUS-24207},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-242077},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Strumpellin is a member of the highly conserved pentameric WASH complex, which stimulates the Arp2/3 complex on endosomes and induces the formation of a branched actin network. The WASH complex is involved in the formation and stabilisation of endosomal retrieval subdomains and transport carriers, into which selected proteins are packaged and subsequently transported to their respective cellular destination, e.g. the plasma membrane. Up until now, the role of Strumpellin in platelet function and endosomal trafficking has not been researched. In order to examine its role, a conditional knockout mouse line was generated, which specifically lacked Strumpellin in megakaryocytes and platelets. Conditional knockout of Strumpellin resulted in only a mild platelet phenotype. Loss of Strumpellin led to a decreased abundance of the αIIbβ3 integrin in platelets, including a reduced αIIbβ3 surface expression by approximately 20\% and an impaired αIIbβ3 activation after platelet activation. The reduced surface expression of αIIbβ3 was also detected in megakaryocytes. The expression of other platelet surface glycoproteins was not affected. Platelet count, size and morphology remained unaltered. The reduction of αIIbβ3 expression in platelets resulted in a reduced fibrinogen binding capacity after platelet activation. However, fibrinogen uptake under resting conditions, although slightly delayed, as well as overall fibrinogen content in Strumpellin-deficient platelets were comparable to controls. Most notably, reduced αIIbβ3 expression did not lead to any platelet spreading and aggregation defects in vitro. Furthermore, reduced WASH1 protein levels were detected in the absence of Strumpellin. In conclusion, loss of Strumpellin does not impair platelet function, at least not in vitro. However, the data demonstrates that Strumpellin plays a role in selectively regulating αIIbβ3 surface expression. As a member of the WASH complex, Strumpellin may regulate αIIbβ3 recycling back to the platelet surface. Furthermore, residual WASH complex subunits may still assemble and partially function in the absence of Strumpellin, which could explain the only 20\% decrease in αIIbβ3 surface expression. Nonetheless, the exact mechanism still remains unclear.},
  language  = {en}
}
@phdthesis{Klingler2023,
  author    = {Klingler, Philipp},
  title     = {Exploration of proteasome interactions with human platelet function},
  doi       = {10.25972/OPUS-32108},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-321089},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research. Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition. Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 \%, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 \%. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments. Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation. As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Huber2023,
  author    = {Huber, Hannes},
  title     = {Biochemical and functional characterization of DHX30, an RNA helicase linked to neurodevelopmental disorder},
  doi       = {10.25972/OPUS-28050},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-280505},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {RNA helicases are key players in the regulation of gene expression. They act by remodeling local RNA secondary structures as well as RNA-protein interactions to enable the dynamic association of RNA binding proteins to their targets. The putative RNA helicase DHX30 is a member of the family of DEAH-box helicases with a putative role in the ATP-dependent unwinding of RNA secondary structures. Mutations in the DHX30 gene causes the autosomal dominant neuronal disease "Neurodevelopmental Disorder with severe Motor Impairment and Absent Language" (NEDMIAL;OMIM\#617804). In this thesis, a strategy was established that enabled the large-scale purification of enzymatically active DHX30. Through enzymatic studies performed in vitro, DHX30 was shown to act as an ATP-dependent 3' → 5' RNA helicase that catalyzes the unwinding of RNA:RNA and RNA:DNA substrates. Using recombinant DHX30, it could be shown that disease-causing missense mutations in the conserved helicase core caused the disruption of its ATPase and helicase activity. The protein interactome of DHX30 however, was unchanged indicating that the pathogenic missense-mutations do not cause misfolding of DHX30, but rather specifically affect its catalytic activity. DHX30 localizes predominantly in the cytoplasm where it forms a complex with ribosomes and polysomes. Using a cross-linking mass spectrometry approach, a direct interaction of the N-terminal double strand RNA binding domain of DHX30 with sites next to the ribosome's mRNA entry channel and the subunit interface was uncovered. RNA sequencing of DHX30 knockout cells revealed a strong de-regulation of mRNAs involved in neurogenesis and nervous system development, which is in line with the NEDMIAL disease phenotype. The knockdown of DHX30 results in a decreased 80S peak in polysome gradients, indicating that DHX30 has an effect on the translation machinery. Sequencing of the pool of active translating mRNAs revealed that upon DHX30 knockout mainly 5'TOP mRNAs are downregulated. These mRNAs are coding for proteins of the translational machinery and translation initiation factors. This study identified DHX30 as a factor of the translation machinery that selectively impacts the expression of a subset of proteins and provides insight on the etiology of NEDMIAL.},
  language  = {en}
}
@article{BenhalevyGuptaDananetal.2017,
  author    = {Benhalevy, Daniel and Gupta, Sanjay K. and Danan, Charles H. and Ghosal, Suman and Sun, Hong-Wei and Kazemeier, Hinke G. and Paeschke, Katrin and Hafner, Markus and Juranek, Stefan A.},
  title     = {The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation},
  series = {Cell Reports},
  volume    = {18},
  journal   = {Cell Reports},
  number    = {12},
  doi       = {10.1016/j.celrep.2017.02.080},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171122},
  pages     = {2979-2990},
  year      = {2017},
  abstract  = {The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcriptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.},
  language  = {en}
}
@article{OthmanFathyBekhitetal.2021,
  author    = {Othman, Eman M. and Fathy, Moustafa and Bekhit, Amany Abdlrehim and Abdel-Razik, Abdel-Razik H. and Jamal, Arshad and Nazzal, Yousef and Shams, Shabana and Dandekar, Thomas and Naseem, Muhammad},
  title     = {Modulatory and toxicological perspectives on the effects of the small molecule kinetin},
  series = {Molecules},
  volume    = {26},
  journal   = {Molecules},
  number    = {3},
  issn      = {1420-3049},
  doi       = {10.3390/molecules26030670},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223064},
  year      = {2021},
  abstract  = {Plant hormones are small regulatory molecules that exert pharmacological actions in mammalian cells such as anti-oxidative and pro-metabolic effects. Kinetin belongs to the group of plant hormones cytokinin and has been associated with modulatory functions in mammalian cells. The mammalian adenosine receptor (A2a-R) is known to modulate multiple physiological responses in animal cells. Here, we describe that kinetin binds to the adenosine receptor (A2a-R) through the Asn253 residue in an adenosine dependent manner. To harness the beneficial effects of kinetin for future human use, we assess its acute toxicity by analyzing different biochemical and histological markers in rats. Kinetin at a dose below 1 mg/kg had no adverse effects on the serum level of glucose or on the activity of serum alanine transaminase (ALT) or aspartate aminotransferase (AST) enzymes in the kinetin treated rats. Whereas, creatinine levels increased after a kinetin treatment at a dose of 0.5 mg/kg. Furthermore, 5 mg/kg treated kinetin rats showed normal renal corpuscles, but a mild degeneration was observed in the renal glomeruli and renal tubules, as well as few degenerated hepatocytes were also observed in the liver. Kinetin doses below 5 mg/kg did not show any localized toxicity in the liver and kidney tissues. In addition to unraveling the binding interaction between kinetin and A2a-R, our findings suggest safe dose limits for the future use of kinetin as a therapeutic and modulatory agent against various pathophysiological conditions.},
  language  = {en}
}