TY - THES A1 - Turakhiya, Ankit T1 - Functional characterization of the role of ZFAND1 in stress granule turnover T1 - Funktionelle Charakterisierung der Rolle von ZFAND1 im Umsatz von Stressgranula N2 - Protein quality control systems are critical for cellular proteostasis and survival under stress conditions. The ubiquitin proteasome system (UPS) plays a pivotal role in proteostasis by eliminating misfolded and damaged proteins. However, exposure to the environmental toxin arsenite results in the accumulation of polyubiquitylated proteins, indicating an overload of the UPS. Arsenite stress induces the rapid formation of stress granules (SGs), which are cytoplasmic assemblies of mRNPs stalled in translation initiation. The mammalian proteins ZFAND2A/B (also known as AIRAP and AIRAPL, respectively) bind to the 26S proteasome, and ZFAND2A has been shown to adapt proteasome activity to arsenite stress. They belong to a small subfamily of AN1 type zinc finger containing proteins that also comprises the unexplored mammalian member ZFAND1 and its yeast homolog Cuz1. In this thesis, the cellular function of Cuz1 and ZFAND1 was investigated. Cuz1/ZFAND1 was found to interact with the ubiquitin-selective, chaperone-like ATPase Cdc48/p97 and with the 26S proteasome. The interaction between Cuz1/ZFAND1 and Cdc48/p97 requires a predicted ubiquitin-like domain of Cuz1/ZFAND1. In vivo, this interaction was strongly dependent on acute arsenite stress, suggesting that it is a part of the cellular arsenite stress response. Lack of Cuz1/ZFAND1 caused a defect in the clearance of arsenite induced SG clearance. ZFAND1 recruits both, the 26S proteasome and p97, to arsenite-induced SGs for their normal clearance. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products and become aberrant. These aberrant SGs persist after arsenite removal and undergo degradation via autophagy. ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder, inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS). N2 - Systeme zur Sicherung der Proteinqualität sind von essentieller Bedeutung für die zelluläre Proteostase und das Überleben unter Stressbedingungen. Dabei spielt das Ubiquitin-Proteasom-System (UPS) eine entscheidende Rolle: Es beseitigt fehlgefaltete und beschädigte Proteine. Sind Zellen dem Umweltgift Arsenit ausgesetzt, kommt es zu einer Akkumulation von polyubiquitinierten Proteinen, was auf eine Überlastung des UPS hinweist. Dieser durch Arsenit verursachte Stress bewirkt eine schnelle Bildung von Stressgranula (SGs), einer cytoplasmatischen Ansammlung von mRNPs, die in der Initiation der Translation blockiert sind. Die Säuger Proteine ZFAND2A/B (auch bekannt als AIRAP und AIRAPL) binden an das 26S Proteasom. Zusätzlich wurde gezeigt, dass ZFAND2A die Aktivität des Proteasoms an durch Arsenit verursachten Stress, anpasst. Diese Proteine gehören zu einer kleinen Unterfamilie von Zinkfinger von AN1-type enthaltenden Proteinen, zu der auch das bislang nicht erforschte Säuge Protein ZFAND1 und sein Hefehomolog Cuz1 gehören. In dieser Arbeit wurde die zelluläre Funktion von Cuz1 und ZFAND1 untersucht. Es zeigte sich, dass Cuz1/ZFAND1 mit dem 26S Proteasom und der Ubiquitin-selektiven, Chaperon-ähnlichen ATPase Cdc48/p97 interagiert. Die Interaktion zwischen Cuz1/ZFAND1 und Cdc48/p97 benötigt eine vorhergesagte Ubiquitin-ähnliche Domäne von Cuz1/ZFAND1. Diese Interaktion ist in vivo stark von akutem Arsenitstress abhängig, was darauf hindeutet, dass sie Teil der zellulären Stressantwort gegen Arsenit ist. Fehlt Cuz1/ZFAND1, so kommt es zu einer Störung bei der Beseitigung der von Arsenit verursachten SGs. Normalerweise rekrutiert ZFAND1 sowohl das 26S Proteasom als auch p97 zu diesen SGs, um sie zu entfernen. Wenn ZFAND1 jedoch fehlt, sind auch p97 und das 26S Proteasom nicht an den SGs lokalisiert. Dadurch sammeln sich dort defekte ribosomale Produkte an, und die SGs werden abnormal. Auch nach Entfernung des Arsenitstresses bestehen diese abnormalen SGs fort und werden schließlich über Autophagie abgebaut. Bei der Beseitigung der SGs ist das Fehlen von ZFAND1 epistatisch zu der Expression einer pathogenen p97-Mutante, was darauf hinweirt, dass ZFAND1 bei der degenerativen Multisystemerkrankung Einschlusskörper-Myopathie assoziiert mit Pagets Erkrankung der Knochen und frontotemporaler Demenz und Amyotrophe Lateralsklerose (IBMPFD/ALS) eine Rolle spielt. KW - ubiquitin KW - ZFAND1 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-163751 ER - TY - JOUR A1 - Deol, Kirandeep K. A1 - Lorenz, Sonja A1 - Strieter, Eric R. T1 - Enzymatic logic of ubiquitin chain assembly JF - Frontiers in Physiology N2 - Protein ubiquitination impacts virtually every biochemical pathway in eukaryotic cells. The fate of a ubiquitinated protein is largely dictated by the type of ubiquitin modification with which it is decorated, including a large variety of polymeric chains. As a result, there have been intense efforts over the last two decades to dissect the molecular details underlying the synthesis of ubiquitin chains by ubiquitin-conjugating (E2) enzymes and ubiquitin ligases (E3s). In this review, we highlight these advances. We discuss the evidence in support of the alternative models of transferring one ubiquitin at a time to a growing substrate-linked chain (sequential addition model) versus transferring a pre-assembled ubiquitin chain (en bloc model) to a substrate. Against this backdrop, we outline emerging principles of chain assembly: multisite interactions, distinct mechanisms of chain initiation and elongation, optimal positioning of ubiquitin molecules that are ultimately conjugated to each other, and substrate-assisted catalysis. Understanding the enzymatic logic of ubiquitin chain assembly has important biomedical implications, as the misregulation of many E2s and E3s and associated perturbations in ubiquitin chain formation contribute to human disease. The resurgent interest in bifunctional small molecules targeting pathogenic proteins to specific E3s for polyubiquitination and subsequent degradation provides an additional incentive to define the mechanisms responsible for efficient and specific chain synthesis and harness them for therapeutic benefit. KW - ubiquitin KW - E2 conjugating enzyme KW - E3 ligating enzyme KW - sequential addition KW - en bloc transfer Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201731 VL - 10 IS - 835 ER - TY - JOUR A1 - Ries, Lena K. A1 - Sander, Bodo A1 - Deol, Kirandeep K. A1 - Letzelter, Marie-Annick A1 - Strieter, Eric Robert A1 - Lorenz, Sonja T1 - Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity JF - Journal of Biological Chemistry N2 - 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 and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin-conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases. KW - ubiquitin KW - ubiquitin ligase KW - ubiquitylation (ubiquitination) KW - post-translational modification KW - enzyme mechanism Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226207 VL - 294 IS - 15 ER -