Doctor of Philosophy / Division of Biology / Jeroen Roelofs / The ubiquitin-proteasome pathway is the major pathway of selective protein degradation in the cell. Disruption of this pathway affects cellular protein homeostasis and contributes to diseases like cancer, and neurodegeneration. The end point of this pathway is the proteasome, a complex protease formed by 66 polypeptides. Structurally, it can be subdivided into the Core Particle (CP) and the Regulatory Particle (RP). The CP harbors the proteolytic sites, whereas, the RP contains six orthologous AAA-ATPases, the Rpt proteins. These Rpt’s are essential for proteasome function and are at the interface between RP and CP. The work in this thesis focuses on the Rpt subunit Rpt5 from yeast. The C-terminal tail of Rpt5 has been shown to contribute to the binding with the CP. However, our study showed it is also essential for the interaction with Nas2, one of nine proteasome-specific chaperones. Thus, Nas2 might function as a regulator of the Rpt5-CP interaction. Further analyses suggested that Nas2 has an additional function in assembly, and that mutating the tail of Rpt5 results in increased binding of the proteasome-associated protein Ecm29 to the proteasome. We showed that Ecm29 binds Rpt5 directly, thereby inducing a closed conformation of the CP substrate entry channel, and inhibiting proteasomal ATPase activity. Consistent with these activities, several proteasome mutant strains showed Ecm29-dependent accumulation of unstable substrates. Thus, Ecm29 is an inhibitor of the proteasome in vivo and in vitro. Interestingly, besides the Rpt5 mutants, several other proteasome mutants show increased levels of Ecm29, suggesting Ecm29 has a role in quality control. Consistent with this, we observed that Ecm29 associates preferably with specific mutants and nucleotide-depleted proteasomes. Based on our data we propose a model, where early in assembly Nas2 binds to the Rpt5 tail inhibiting the Rpt5-CP interaction directly. Later in assembly Ecm29 performs a quality control function, where it recognizes and remains bound to defective proteasomes. By inhibiting these proteasomes Ecm29 prevents the aberrant degradation of proteins.

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Jeroen Roelofs / The proteasome is a large protease in the cell that contributes to the controlled degradation of proteins. This 2.5MDa 26S proteasome complex consists of a 19S regulatory particle (RP) that recognizes substrates and a 20S proteolytic core particle (CP) that hydrolyses substrates. To function optimally all 66 subunits of the proteasome complex need to assemble properly. Efficient and accurate assembly of the proteasome is achieved with the help of proteins that can monitor the quality of the proteasome during pre- and post-assembly processes. The work in this thesis described an investigation into two of such quality control mechanisms. Pba1-Pba2 dimer has been known to facilitate the CP assembly by interacting with the top of the α-ring of CP throughout CP maturation. After CP maturation, RP utilizes same surface to form a CP-RP complex. Our data showed that Pba1-Pba2 binds tightly to the immature CP and prevents RP association. Once matured CP has a reduced affinity for Pba1-Pba2 and shows a higher affinity towards RP, resulting the formation of 26S proteasome complex. Our results imply that during maturation, CP undergoes conformational changes that results in this switch in affinity. Mathematical models indicate that during assembly such an 'affinity switch' quality control mechanism is required to prevent immature CP-RP complex formations. These types of wrong dead end products prevent efficient proteasome complex formation. Proteasomes formed with post-assembly defects are enriched with the proteasome associated protein Ecm29. Here Ecm29 is proposed to function as a quality control factor that inhibits such defective proteasomes to avoid aberrant protein degradation. This would require Ecm29 to preferably bind to mutant proteasomes. While we know Ecm29 interacts with RP as well as CP, we still don’t understand well how it binds to proteasomes holoenzyme. Here, we identify that besides the Rpt5 subunit of RP, Ecm29 binds to alpha7. We showed that conserved acidic residues containing unstructured C-terminal region of the CP subunit alpha7 facilitates the Ecm29-Proteasome interactions. Further mapping revealed the importance of phosphorylation of serine residues at the alpha7 C-terminal tail for Ecm29 interaction. We anticipate that this study leads to identification of specificity of the Ecm29 for the defective proteasomes. Overall this will help us to understand the role of Ecm29 in regulation of defective proteasomes in vivo.

Indiana University-Purdue University Indianapolis (IUPUI) / Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.

Das Proteasomsystem ist die Hauptquelle von Peptiden für die MHC Klasse I Antigen-Präsentation. In Vertebraten kann dieses durch die Expression verschiedener Subtypen des 20S Proteasoms moduliert werden. Die häufigsten Subtypen sind konstitutive Proteasomen (c20S) mit den katalytischen Untereinheiten beta1, beta2 und beta5 und Immunoproteasomen (i20S) mit den Immunountereinheiten beta1i, beta2i und beta5i. Die Expression von i20S optimiert in der Regel die MHC Klasse I Antigen-Präsentation, indem die Bildung von Peptiden mit hoher Affinität zu MHC I Molekülen verstärkt wird. Die Bildung von i20S wird momentan durch ein Modell der kooperativen Assemblierung erklärt, das auf der präferentiellen Interaktion zwischen den Immunountereinheiten beruht. In dieser Arbeit wurde die Assemblierung von 20S Proteasomen in beta5i defizienten Mäusen nach Infektion mit Listeria monocytogenes analysiert. In diesem Modell konnte keine präferentielle Interaktion zwischen den Untereinheiten festgestellt werden. Stattdessen zeigen die Ergebnisse, daß die Integration von konstitutiven oder Immunountereinheiten durch direkte Kompetition reguliert wird. Des Weiteren wurde während der Infektion eine beta5i-abhängige Zunahme der zellulären Proteasommenge festgestellt und somit ein neuer Mechanismus zur Regulation des zellulären Proteasomgehaltes entdeckt. Funktionell führt die beta5i-Defizienz zu einer verringerten MHC I Expression auf antigenpräsentierenden Zellen und zu einer verminderten Prozessierung des bakteriellen Antigens LLO296-304. Bei der Analyse der LLO296-304 spezifischen CD8 T Zell Antwort konnte jedoch kein Unterschied zwischen Wildtyp- und beta5i defizienten Mäusen festgestellt werden .Die Kontrolle der Infektion in den beta5i defizienten Mäusen ist jedoch in der Leber verzögert. Dies deutet darauf hin, dass die Erkennung und Elimination infizierter Zellen durch cytotoxische CD8 T Zellen auf Grund der geringeren MHC Klasse I Präsentation bakterieller Antigene behindert wird. / The proteasome-system is the main source of peptides for MHC class I antigen presentation. In vertebrates this system can be modulated by the expression of different subtypes of the 20S proteasome. The most common subtypes are constitutive proteasomes (c20S) with the catalytic subunits beta1, beta2 and beta5 and immunoproteasomes (i20S) with the immunosubunits beta1i, beta2i and beta5i. Expression of i20S generally optimizes MHC class I antigen presentation by increasing the generation of peptides with high affinity to MHC class I molecules. Currently, the formation of i20S is explained by a model of cooperative proteasome assembly, which is based on preferential interactions among the immunosubunits. Here, the assembly of 20S proteasomes was analysed in beta5i deficient mice during an ongoing infection with Listeria monocytogenes. In this model, no preferential interactions among constitutive subunits or immunosubunits could be determined. Instead, the results show that the integration of constitutive subunits or immunosubunits is regulated by direct competition. Further, a beta5i-dependent increase in cellular proteasome quantity was observed following infection. This reveals a novel mechanism for the regulation of cellular proteasome quantity, which is based on the differential expression of beta5i. Functionally, the deficiency in beta5i results in a reduced MHC class I cell surface expression on professional antigen presenting cells and a drastically diminished processing of the bacterial antigen LLO296-304. However, the analyses of LLO296-304 specific CD8 T cells did not reveal differences in the frequencies of these T cells between wild-type and beta5i deficient mice. Still, the control of infection in the liver of beta5i deficient mice was delayed. This phenotype suggests that the recognition and elimination of infected target cells by cytotoxic CD8 T cells is constrained due to the lowered MHC class I presentation of bacterial antigens.