Proteome-scale discovery of protein degradation and stabilization effectors.

Targeted protein degradation and stabilization are promising therapeutic modalities because of their potency, versatility and their potential to expand the druggable target space1,2. However, only a few of the hundreds of E3 ligases and deubiquitinases in the human proteome have been harnessed for this purpose, which substantially limits the potential of the approach. Moreover, there may be other protein classes that could be exploited for protein stabilization or degradation3-5, but there are currently no methods that can identify such effector proteins in a scalable and unbiased manner. Here we established a synthetic proteome-scale platform to functionally identify human proteins that can promote the degradation or stabilization of a target protein in a proximity-dependent manner. Our results reveal that the human proteome contains a large cache of effectors of protein stability. The approach further enabled us to comprehensively compare the activities of human E3 ligases and deubiquitinases, identify and characterize non-canonical protein degraders and stabilizers and establish that effectors have vastly different activities against diverse targets. Notably, the top degraders were more potent against multiple therapeutically relevant targets than the currently used E3 ligases cereblon and VHL. Our study provides a functional catalogue of stability effectors for targeted protein degradation and stabilization and highlights the potential of induced proximity screens for the discovery of new proximity-dependent protein modulators.

PGP-14 establishes a polar lipid permeability barrier within the C. elegans pharyngeal cuticle.

The cuticles of ecdysozoan animals are barriers to material loss and xenobiotic insult. Key to this barrier is lipid content, the establishment of which is poorly understood. Here, we show that the p-glycoprotein PGP-14 functions coincidently with the sphingomyelin synthase SMS-5 to establish a polar lipid barrier within the pharyngeal cuticle of the nematode C. elegans. We show that PGP-14 and SMS-5 are coincidentally expressed in the epithelium that surrounds the anterior pharyngeal cuticle where PGP-14 localizes to the apical membrane. pgp-14 and sms-5 also peak in expression at the time of new cuticle synthesis. Loss of PGP-14 and SMS-5 dramatically reduces pharyngeal cuticle staining by Nile Red, a key marker of polar lipids, and coincidently alters the nematode's response to a wide-range of xenobiotics. We infer that PGP-14 exports polar lipids into the developing pharyngeal cuticle in an SMS-5-dependent manner to safeguard the nematode from environmental insult.

Pervasive mislocalization of pathogenic coding variants underlying human disorders.

Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease-causing. This creates a new bottleneck: determining the functional impact of each variant - largely a painstaking, customized process undertaken one or a few genes or variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,547 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of unknown significance. Our publicly available resource will likely accelerate the understanding of coding variation in human diseases.

A central chaperone-like role for 14-3-3 proteins in human cells.

14-3-3 proteins are highly conserved regulatory proteins that interact with hundreds of structurally diverse clients and act as central hubs of signaling networks. However, how 14-3-3 paralogs differ in specificity and how they regulate client protein function are not known for most clients. Here, we map the interactomes of all human 14-3-3 paralogs and systematically characterize the effect of disrupting these interactions on client localization. The loss of 14-3-3 binding leads to the coalescence of a large fraction of clients into discrete foci in a client-specific manner, suggesting a central chaperone-like function for 14-3-3 proteins. Congruently, the engraftment of 14-3-3 binding motifs to nonclients can suppress their aggregation or phase separation. Finally, we show that 14-3-3s negatively regulate the localization of the RNA-binding protein SAMD4A to cytoplasmic granules and inhibit its activity as a translational repressor. Our work suggests that 14-3-3s have a more prominent role as chaperone-like molecules than previously thought.

Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains.

Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.

A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6.

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson's disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

Research in Rare Disease: From Genomics to Proteomics.

Jessica Lacoste from the Donnelly Centre at the University of Toronto was awarded best poster at the annual Society of Biomolecular Imaging and Informatics meeting held in San Diego, September 2017. Her work focuses on characterizing the protein localization of variants involved in rare disease. The current works and future directions of research in rare disease are summarized in the following overview.

The yeast 2-µm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins.

The yeast 2-µm plasmid is a remarkable genetic parasite, managing efficient maintenance at high-copy number with minimal impact on the host. Equal partitioning of the plasmid upon host cell division requires plasmid proteins Rep1 and Rep2 and the plasmid STB locus. The Rep proteins and the plasmid-encoded Raf protein also regulate plasmid gene transcription. In this study, protein interaction assays, sequence analyses and mutational approaches were used to identify domains and residues in Rep2 and Raf required for association with Rep1 and Rep2 and to delineate the Rep2 DNA-binding domain. Rep2 and Raf displayed similarities in interactions with Rep1 and Rep2, in having Rep1 promote their STB association in vivo, and in stabilizing Rep protein levels. Rep2 mutants impaired for self-association were competent for transcriptional repression while those deficient for Rep1 association were not. Surprisingly, Rep2 mutants impaired for either Rep1 interaction or self-association were able to maintain efficient plasmid inheritance provided Raf was present and competent for Rep protein interaction. Our findings provide insight into the Rep protein complexes required for partitioning and transcriptional repression, and suggest that in addition to its transcriptional function, Raf stabilization of Rep partitioning proteins contributes to the remarkable persistence of the 2-µm plasmid.