High resolution analysis of proteolytic substrate processing.

Members of the widely conserved HtrA family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates. To gain a deeper understanding of how folded proteins are initially processed and subsequently degraded into short peptides by human HTRA1, we established an integrated and quantitative approach using time-resolved mass spectrometry, circular dichroism spectroscopy and bioinformatics. The resulting data provide high-resolution information on up to 178 individual proteolytic sites within folded ANXA1 (consisting of 346 amino acids), the relative frequency of cuts at each proteolytic site, the preferences of the protease for the amino acid sequence surrounding the scissile bond, as well as the degrees of sequential structural relaxation and unfolding of the substrate that occur during progressive degradation. Our workflow provides precise molecular insights into protease-substrate interactions, which could be readily adapted to address other post-translational modifications such as phosphorylation in dynamic protein complexes.

Ubiquitin-mediated degradation at the Golgi apparatus.

The Golgi apparatus is an essential organelle of the secretory pathway in eukaryotic cells. It processes secretory and transmembrane proteins and orchestrates their transport to other endomembrane compartments or the plasma membrane. The Golgi apparatus thereby shapes the cell surface, controlling cell polarity, cell-cell communication, and immune signaling. The cytosolic face of the Golgi hosts and regulates signaling cascades, impacting most notably the DNA damage response and mitosis. These essential functions strongly depend on Golgi protein homeostasis and Golgi integrity. Golgi fragmentation and consequent malfunction is associated with neurodegenerative diseases and certain cancer types. Recent studies provide first insight into the critical role of ubiquitin signaling in maintaining Golgi integrity and in Golgi protein quality control. Similar to well described pathways at the endoplasmic reticulum, ubiquitin-dependent degradation of non-native proteins prevents the accumulation of toxic protein aggregates at the Golgi. Moreover, ubiquitination regulates Golgi structural rearrangements in response to cellular stress. Advances in elucidating ubiquitination and degradation events at the Golgi are starting to paint a picture of the molecular machinery underlying Golgi (protein) homeostasis.

Targeted Protein Unfolding at the Golgi Apparatus.

Maintaining protein homeostasis (proteostasis) is vital to cellular and organismal health. How the Golgi apparatus, the central protein maturation and sorting station in the cell, manages misfolded proteins to maintain proteostasis is still poorly understood. Here we present a strategy for targeted protein unfolding at the Golgi that enables studying Golgi-related protein quality control and stress-signaling pathways. Targeted protein unfolding is induced by small molecule-based chemical biology approaches-hydrophobic tagging and the use of a destabilization domain. Imaging studies allow visualizing quality control (QC) phenotypes, such as the formation of QC carriers and Golgi-to-endoplasmic reticulum trafficking, and correlating these phenotypes with other trafficking processes.

Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin.

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.

Protein folding state-dependent sorting at the Golgi apparatus.

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.

Targeted protein unfolding uncovers a Golgi-specific transcriptional stress response.

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.

UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins.

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.

Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL.

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.

The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression.

Nuclear 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 chaperones.

The folding and assembly of myosin motor proteins is essential for most movement processes at the cellular, but also at the organism level. Importantly, myosins, which represent a very diverse family of proteins, require the activity of general and specialized folding factors to develop their full motor function. The activities of the myosin-specific UCS (UNC-45/Cro1/She4) chaperones range from assisting acto-myosin dependent transport processes to scaffolding multi-subunit chaperone complexes, which are required to assemble myofilaments. Recent structure-function studies revealed the structural organization of TPR (tetratricopeptide repeat)-containing and TPR-less UCS chaperones. The observed structural differences seem to reflect the specialized and remarkably versatile working mechanisms of myosin-directed chaperones, as will be discussed in this review.