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Zusammenfassung:
AAA (+) proteins are ATPases associated with diverse cellular activities coupling ATP-hydrolysis to remodelling, disaggregation, and unfolding of a variety of substrates. The central ATPase domain functions as a molecular switch, which receives input from N-terminal substrate recognition domains, and which transfers the output to downstream effectors. AAA+ proteases recognize misfolded proteins with their N-domain, unfold and thread them through the pore of the hexameric ring, and feed them to proteases, either residing on the same polypeptide chain, or being contacted via (C-terminal) interaction motifs. We have investigated the divergent evolution of N-domains of known and putative proteasomal ATPases and their C-terminal interaction motif, which is crucial for the regulation of the proteasome, a self-compartmentalizing protease involved in the degradation of unfolded substrates within a large cylinder-shaped architecture. The first part comprises three case studies of hypothetical proteins, homologous to double-psi barrel, beta-clam and OB-fold N-domains of AAA proteins. We present the first characterization of a CTP-specific archaeal riboflavin kinase, which is homologous to the double-psi barrel of AAA proteins of the CDC48 group, sharing a duplicated beta-element in their common core. We show that archaeal riboflavin kinases provide an evolutionary bridge between highly symmetric RIFT-barrel transcription factors and ATP-specific bacterial/eukaryotic riboflavin kinases allowing us to describe an evolutionary trajectory from DNA-binding to enzymatic activity. A beta-clam domain, which is found in AAA proteins of the CDC48 and AMA groups, was detected in context of a C-terminal domain lacking significant similarity to known domains. We present the full-length structure of a member of this family, whose C-terminal domain forms a homohexameric twelve-bladed beta-propeller (HP12). Each monomer accommodates two propeller-blades that have retained traces of a duplication event, suggesting that monomeric beta-propellers evolved via oligomeric intermediates. We show that HP12 forms a ternary complex with a genetically coupled endonuclease III and DNA implying a function in base-excision DNA-repair. We identified a protein family in methanogenic archaea that contains an OB-fold, similar to the N-domain of proteasome activating nucleotidases (PAN), and a proteasome-like Ntn-hydrolase domain. Our crystal structure of a member of this family reveals a monomeric proteasome-homolog of methanogens (MPM), which acquired an OB-fold domain, probably functioning as a substrate recognition domain for the protease. The internal symmetry of the six-stranded OB-fold suggests that it evolved by duplication of an ancestral beta-meander. The beta-element of double-psi barrels and riboflavin kinases, the propeller blade of HP12, and the three-stranded beta-meander of the OB-fold of MPM shed light on the evolution of autonomously folding domains through duplication and fusion of ancestral supersecondary structure elements, presumably via oligomeric intermediates. In the second part, we trace the origins of proteasomal protein degradation. We present a systematic sequence analysis of the C-termini of archaeal AAA proteins uncovering the presence of the proteasome-interaction motif in AAA proteins of the CDC48 and AMA group in addition to known proteasome activating nucleotidases of the PAN group. Furthermore, we detect the absence of PAN proteins in major archaeal lineages supporting our hypothesis that kingdom-wide conserved CDC48 proteins function as regulatory ATPases of the proteasome. The presence of up to five putative proteasomal ATPases in certain archaea prompted us to predict a network of AAA ATPases, which regulates the archaeal proteasome. This network could increase the capabilities of proteasomal protein degradation in archaea through the participation of different N-terminal substrate recognition domains. Analysis of the genetic context of the yet uncharacterized Anbu proteasome homolog revealed a conserved operon structure, widespread in proteobacteria and cyanobacteria. The components of the operon point to a peptide tagging system, remotely resembling ubiquitylation and sampylation, which target substrates for degradation by the proteasome in eukaryotes and archaea. Finally, we describe the global distribution of proteasome-like Ntn-hydrolases and putative proteasomal ATPases on the tree of life. This analysis supports the scenario that actinobacteria acquired the proteasome through lateral gene transfer. Among the large assemblies of proteasome-like Ntn-hydrolases the highly divergent monomeric proteasome-homolog of methanogens (MPM) is an exception, because this derived group has lost its ability to self-compartmentalize. In contrast to proteasome-ATPase complexes, MPM including the OB-fold has evolved from an intricate towards a more simplified molecular phenotype.
