Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome.

Regulation of translation during human development is poorly understood, and its dysregulation is associated with Rett syndrome (RTT). To discover shifts in mRNA ribosomal engagement (RE) during human neurodevelopment, we use parallel translating ribosome affinity purification sequencing (TRAP-seq) and RNA sequencing (RNA-seq) on control and RTT human induced pluripotent stem cells, neural progenitor cells, and cortical neurons. We find that 30% of transcribed genes are translationally regulated, including key gene sets (neurodevelopment, transcription and translation factors, and glycolysis). Approximately 35% of abundant intergenic long noncoding RNAs (lncRNAs) are ribosome engaged. Neurons translate mRNAs more efficiently and have longer 3' UTRs, and RE correlates with elements for RNA-binding proteins. RTT neurons have reduced global translation and compromised mTOR signaling, and >2,100 genes are translationally dysregulated. NEDD4L E3-ubiquitin ligase is translationally impaired, ubiquitinated protein levels are reduced, and protein targets accumulate in RTT neurons. Overall, the dynamic translatome in neurodevelopment is disturbed in RTT and provides insight into altered ubiquitination that may have therapeutic implications.

Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10.

Post-translational modification of the p53 signaling pathway plays an important role in cell cycle progression and stress-induced apoptosis. Indeed, a large body of work has shown that dysregulation of p53 and its E3 ligase MDM2 by the ubiquitin-proteasome system (UPS) promotes carcinogenesis and malignant transformation. Thus, drug discovery efforts have focused on the restoration of wild-type p53 activity or inactivation of oncogenic mutant p53 by targeted inhibition of UPS components, particularly key deubiquitinases (DUBs) of the ubiquitin-specific protease (USP) class. However, development of selective small-molecule USP inhibitors has been challenging, partly due to the highly conserved structural features of the catalytic sites across the class. To tackle this problem, we devised a protein engineering strategy for rational design of inhibitors for DUBs and other UPS proteins. We employed a phage-displayed ubiquitin variant (UbV) library to develop inhibitors targeting the DUBs USP7 and USP10, which are involved in regulating levels of p53 and MDM2. We were able to identify UbVs that bound USP7 or USP10 with high affinity and inhibited deubiquitination activity. We solved the crystal structure of UbV.7.2 and rationalized the molecular basis for enhanced affinity and specificity for USP7. Finally, cell death was increased significantly by UbV.7.2 expression in a colon cancer cell line that was treated with the chemotherapy drug cisplatin, demonstrating the therapeutic potential of inhibiting USP7 by this approach.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.