Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c-ABL and BCR-ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: the identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.

SummaryThe UCS (UNC-45/CRO1/She4) chaperones play an evolutionarily conserved role in promoting myosin-dependent processes, including cytokinesis, endocytosis, RNA transport, and muscle development. 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 forms linear protein chains that offer multiple binding sites for cooperating chaperones and client proteins. Accordingly, Hsp70 and Hsp90, which bind to the TPR domain of UNC-45, could act in concert and with defined periodicity on captured myosin molecules. In vivo analyses reveal the elongated canyon of the UCS domain as a myosin-binding site and show that multimeric UNC-45 chains support organization of sarcomeric repeats. In fact, expression of transgenes blocking UNC-45 chain formation induces dominant-negative defects in the sarcomere structure and function of wild-type worms. Together, these findings uncover a filament assembly factor that directly couples myosin folding with myofilament formation.

SummaryNuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery.

Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.

Graphical abstract

Muscle development requires the coordinated activities of specific protein folding and degradation factors. UFD-2, a U-box ubiquitin ligase, has been reported to play a central role in this orchestra regulating the myosin chaperone UNC-45. Here, we apply an integrative in vitro and in vivo approach to delineate the substrate-targeting mechanism of UFD-2 and elucidate its distinct mechanistic features as an E3/E4 enzyme. Using Caenorhabditis elegans as model system, we demonstrate that UFD-2 is not regulating the protein levels of UNC-45 in muscle cells, but rather shows the characteristic properties of a bona fide E3 ligase involved in protein quality control. Our data demonstrate that UFD-2 preferentially targets unfolded protein segments. Moreover, the UNC-45 chaperone can serve as an adaptor protein of UFD-2 to poly-ubiquitinate unfolded myosin, pointing to a possible role of the UFD-2/UNC-45 pair in maintaining proteostasis in muscle cells.

In eukaryotic cells, organelle-specific stress-response mechanisms are vital for maintaining cellular homeostasis. The Golgi apparatus, an essential organelle of the secretory system, is the major site of protein modification and sorting within a cell and functions as a platform for spatially regulated signaling. Golgi homeostasis mechanisms that regulate organelle structure and ensure precise processing and localization of protein substrates remain poorly understood. Using a chemical biology strategy to induce protein unfolding, we uncover a Golgi-specific transcriptional response. An RNA-sequencing profile of this stress response compared with the current state-of-the-art Golgi stressors, nigericin and xyloside, demonstrates the enhanced precision of Golgi targeting achieved with our system. The data set further reveals previously uncharacterized genes that we find to be essential for Golgi structural integrity. These findings highlight the Golgi’s ability to sense misfolded proteins and establish new aspects of Golgi autoregulation.

In eukaryotic cells, organelle-specific protein quality control (PQC) is critical for maintaining cellular homeostasis. Despite the Golgi apparatus being the major protein processing and sorting site within the secretory pathway, how it contributes to PQC has remained largely unknown. Using different chemical biology-based protein unfolding systems, we reveal the segregation of unfolded proteins from folded proteins in the Golgi. Quality control (QC) substrates are subsequently exported in distinct carriers, which likely contain unfolded proteins as well as highly oligomerized cargo that mimic protein aggregates. At an additional sorting step, oligomerized proteins are committed to lysosomal degradation, while unfolded proteins localize to the endoplasmic reticulum (ER) and associate with chaperones. These results highlight the existence of checkpoints at which QC substrates are selected for Golgi export and lysosomal degradation. Our data also suggest that the steady-state ER localization of misfolded proteins, observed for several disease-causing mutants, may have different origins.