Controlled ubiquitination of the myosine chaperone Unc45

Ubiquitination refers to the post-translational modification of a protein by the covalent attachment of one or more ubiquitin (Ub) monomers and represents a central regulatory mechanism in eukaryotes. Attachment of at least four Ub monomers (poly-ubiquitination) most commonly results in the degradation of the substrate protein via the 26S proteasome. However, ubiquitination is not always a "kiss of death". Addition of either one or a few Ub molecules might modify protein activity, protein-protein interaction or subcellular localization rather than signalling degradation. Therefore the ubiquitination-proteasome system has to be tightly regulated to allow triage decisions between protein activation and protein degradation. Ub attachment proceeds by an enzymatic cascade that is constituted by 3 enzymes, which are termed E1, E2 and E3 and represent the Ub-activating enzyme, the Ub- conjugating enzyme and the Ub ligase, respectively. Dependent on the E2/E3 couple, different lysines can be utilized to connect successive Ub entities resulting in different linkage types and polyubiquitin chain lengths. Recently, it was shown that some target proteins require an additional factor, a processivity factor, for efficient polyubiquitination that was termed E4. The Chn1/Ufd2 complex from Caenorhabditis elegans represents the probably best understood E3/E4 multiubiquitination system. Chn1 is the orthologue of CHIP (C-terminus of Hsp70 Interacting Protein), whereas Ufd2 (Ubiquitin Fusion Degradation) is the orthologue of yeast Ufd2 that is crucial to deliver ubiquitinated proteins to the proteasome. Both CHIP and Ufd2 have been reported to have E3 ligase activity that depends on a C-terminal U-box domain. Interestingly, Chn1 and Ufd2 have to form a heterooligomeric complex in order to polyubiquitinate Unc45, the regulator of myosin folding and assembly. Furthermore, Ufd2 was shown to bind to an E2 enzyme (Let70) and to Cdc48, an AAA ATPase that is intimately linked to Ub pathways enabling the segregation of ubiquitinated substrates from unmodified target proteins. As outlined in this proposal, we plan to perform a structure-function analysis of the Chn1/Ufd2 system to gain insight, on a molecular level, in the polyubiquitination reaction and its regulation by various modulating proteins. Based on detailed sequence analyses, we generated different constructs of Chn1, Ufd2 and interacting proteins (Unc45, Let70, Cdc48) and established purification procedures allowing production of large amounts of recombinant protein. As it was possible to obtain all components in sufficient amounts, we are in the unique position to study the interaction between E2, E3/E4 and potential modulators in vitro and to assay their cooperation in the polyubiquitination reaction. Interaction and activity profiling will aid the setup of crystallization trials. Structure-function analysis of the Chn1/Ufd2/Unc45 complex combined with in vitro ubiquitylation studies should give detailed insights into complex formation, Ub chain assembly, and myofibre differentiation. Since both Chn1 and Unc45 interact with molecular chaperones, our studies on myosin assembly might have important implications for understanding the interplay between degradation and folding of proteins. Moreover, we presume that combining the structural and biochemical data will shed light on the essential role of Unc45 in muscle formation and its impact for protein folding diseases including various myopathies.

Muscle development is a tightly regulated process depending on the correct assembly of sarcomeric repeats that are mainly composed of aligned thick (myosin) and thin (actin) filaments. In contrast to actin filament formation, the mechanism of how myosin thick filaments are assembled is still poorly understood. A major factor in this process is the UCS (UNC-45/Cro1/She4p) protein UNC-45 that functions as a myosin specific chaperone. To investigate the protein machinery orchestrating myosin folding and assembly, we performed a comprehensive analysis of Caenorhabditis elegans UNC-45. Our structural and biochemical data demonstrate that UNC-45 can form a linear protein chain offering multiple binding sites for co-working chaperones and client proteins. Accordingly, Hsp70 and Hsp90, could act in concert and with defined periodicity on captured myosin molecules. This myosin assembly line is also crucial in vivo to organize muscle sarcomeres indicating that UNC-45 chains provide the structural framework to couple myosin folding with myofilament formation. Our data also show that the UNC-45 chaperone functions beyond simple protein folding. It represents a novel type of filament assembly factor that provides the molecular scaffold for general chaperones to work at regularly spaced positions on captured client proteins. Of note, aberrant UNC-45 function is associated with severe muscle defects resulting in skeletal and cardiac myopathies. The molecular mechanisms uncovered in the present study will thus help to address the pathophysiology of diseases connected with myosin mis-folding and mis-assembly.One major regulator of UNC-45 is the UFD-2 ubiquitin ligase. UFD2 is a central component of the ubiquitin-proteasome system functioning as a specialized ubiquitination enzyme, an E4 ligase required to elongate pre-assembled ubiquitin chains on substrate proteins. Despite its important biological role and its unique ubiquitination activity, the molecular mechanism underlying UFD2 ligase activity is largely unknown. To better understand how UFD2 combines its general function as part of the unfolded protein response with its specific role in degrading the UNC-45 myosin chaperone, we addressed its substrate targeting mechanism. In contrast to previous studies, our in vitro and in vivo data showed that UFD-2 is not involved in regulating the protein levels of UNC-45 in muscle cells. Instead UFD-2 employs the UNC-45 chaperone as an adaptor to ubiquitinate the motor domain of muscle myosin. These data reveal a central role for the UNC-45/UFD-2 couple in setting up a sophisticated triage system monitoring the functionality of myosin in the cell. Moreover, our data illustrate how muscle myosin is selectively targeted for degradation, providing first mechanistic insight into the quality control of one of the most fundamental proteins for locomotion.