Dokument: Structural characterization of intrinsically disordered prostate apoptosis response factor 4 (Par-4)
| Titel: | Structural characterization of intrinsically disordered prostate apoptosis response factor 4 (Par-4) | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=67137 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20241030-131736-3 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Tumeh, Aziz [Autor] | |||||||
| Dateien: |
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| Beitragende: | Labahn, Jörg [Gutachter] Weiergräber, Oliver H. [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Par-4, a tumor suppressor protein, plays a crucial role in inducing apoptosis in cancer cells, primarily through its N-terminal SAC domain. Structurally, Par-4 is recognized as an intrinsically disordered protein with two significant domains: the SAC domain responsible for selective apoptosis and the C-terminal coiled-coil (CC) domain, essential for multiple interactions. While the structure beyond the CC domain remains elusive, the crystallographic analysis of the CC domain has highlighted disorder in this region. This study aims to characterize both the CC domain and the extended region including the SAC domain. To achieve this, several approaches were employed. Phosphorylation-mimicking, salt bridge removal, and charge repulsion removal mutations were introduced into the CC domain, and an extended Par-4 variant (Par-4 CC EXS) was constructed to stabilize the conformation and analyze it. Expressions in insect cells and E. coli were utilized for Par-4 CC EXS expression. Additionally, the partner protein P62 PB1 was expressed and purified for binding studies. The Par-4 proteins, tagged at the N-terminal with strep tags, were purified using affinity chromatography and size exclusion chromatography (SEC). However, during purification, it was observed that Par-4 CC EXS underwent degradation under acidic conditions, necessitating mutation to enhance its resistance to acidolysis.
SEC analysis revealed that all CC domain mutants exhibited similar elution volumes, suggesting minimal impact of the mutations on protein oligomerization. In contrast, SEC analysis of Par-4 CC EXS under acidic conditions unveiled diverse oligomeric forms. Additionally, the presence of Sodium chloride exacerbated aggregation behavior in Par-4 CC EXS, as evidenced by SEC results. To validate the proteins' integrity and identity, western blot analysis was performed utilizing anti-strep antibodies, confirming the preservation of the N-terminal sequence. Furthermore, Mass spectrometry (MALDI TOF) and LC-Mass/mass analysis confirmed the presence of full-length proteins. Interestingly, Par-4 CC EXS expressed in insect cells exhibited no post-translational modifications, as evidenced by the results of LC-Mass/Mass and PNGase and Endo H digestions. Subsequent biophysical characterizations assessing the proteins' oligomerization state and homogeneity in solution using Dynamic Light Scattering (DLS) showed for the mutant proteins different effects on protein oligomerization and polydispersity, implying that polydispersity and oligomerization are not strictly correlated, though both depend on charge interactions. The results from DLS revealed that Par-4 CC EXS exhibited a significantly smaller particle size under acidic conditions compared to neutral conditions indicating that Asp and Glu protonation decreases aggregation. Additionally, the protein displayed high polydispersity under acidic and neutral to alkaline conditions implying the presence of different conformations with different degrees of folding and stability. Furthermore, NaCl salt was observed to have a negative effect on Par-4 CC EXS particle size even at low concentration, though at high concentrations it had a positive effect on protein homogeneity. Circular Dichroism (CD) spectroscopy was utilized to evaluate the secondary structure of the proteins: All the muations in the CC domain showed a MRE222/MRE208 ratio greater than 1 and an increase in the number of residues forming helical structure implying the conservation of coiled coil structure and induction of protein folding. CD results for the mutated Par-4 CC EXS under acidic conditions revealed improved protein folding compared to the neutral state suggesting that its structural properties are highly sensitive to pH changes. No secondary structure differences were observed between proteins purified from E. coli and Hi5 cells, indicating no additional folding in Hi5 cells. Different secondary structure contents were observed among different SEC fractions under acidic condition, suggesting the instability of secondary and tertiary structure. Varying concentrations of KF, NaF, and 50 mM MgSO4 changed the secondary structure towards more beta sheet and turn contents. Melting temperature (Tm) computations were conducted by observing alterations in CD spectra at different temperatures, with calculation of thermodynamic parameters. Compared to native CC domain, all the mutants showed a significantly higher Tm implying stabilization of the CC domain upon these mutations. In addition, All the mutants showed a significant increase in ∆G unfolding and ΔCp compared to the CC domain suggesting the native amino acid composition allows structural changes more easily than any of the mutants. This is possibly a functional feature of the CC domain. The Tm for Par-4 CC EXS fractions were almost identical, suggesting that the oligomerization state of the protein is not significantly influenced by changes in secondary structure. Though the presence of salt increases the Tm and hence the stability of Par-4 CC EXS, the nature of the salt did not have any significant effect. The structural elucidation of the mutants T248D, S249D, and S249E required integrating data in space group P1, as the N-terminal helices exhibited multiple conformations within the crystals, indicating disorder in the N-termini. The R-free values were 0.3444, 0.3056, and 0.3081 for the mutants T248D, S249D, and S249E, respectively. Conversely, attempts to resolve the structure of Par-4 CC EXS crystals were thwarted due to protein degradation, precluding diffraction despite exhaustive efforts. This degradation underscores the disorder within the region spanning the CC domain and SAC domain (linker), a finding consistent with the Alphafold model of Par-4 CC EXS, which highlighted the disorder in this region. In an effort to stabilize Par-4 CC EXS, its interaction with P62 PB1 was probed using a pull-down assay. While Par-4 CC EXS was found to bind to P62 PB1, it also exhibited complete aggregation and precipitation. This study reveals that Par-4 constructs exhibit maximum structure and minimal oligomerization under acidic conditions, indicating pH sensitivity. All single amino acid mutations introduced stabilize Par-4, suggesting its structural instability may be a biological feature which enables diverse binding modes. Despite the challenges posed by this instability, it highlights Par-4's unique biological adaptability. Given the observed variability of the protein future research should focus on single particle analysis using EM to uncover the elusive details of the Par-4 structure to enhance our understanding of its functional mechanisms. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Sonstige Einrichtungen/Externe » Institute in Zusammenarbeit mit der Heinrich-Heine-Universität Düsseldorf » Institut für Biologische Informationsvereinbarung, Forschungszentrum Jülich GmbH | |||||||
| Dokument erstellt am: | 30.10.2024 | |||||||
| Dateien geändert am: | 30.10.2024 | |||||||
| Promotionsantrag am: | 04.07.2024 | |||||||
| Datum der Promotion: | 01.10.2024 |
