Development and Applications of Chimera Platforms for Tyrosine Phosphorylation.

Chimeric small molecules that induce post-translational modification (PTM) on a target protein by bringing it into proximity to a PTM-inducing enzyme are furnishing novel modalities to perturb protein function. Despite recent advances, such molecules are unavailable for a critical PTM, tyrosine phosphorylation. Furthermore, the contemporary design paradigm of chimeric molecules, formed by joining a noninhibitory binder of the PTM-inducing enzyme with the binder of the target protein, prohibits the recruitment of most PTM-inducing enzymes as their noninhibitory binders are unavailable. Here, we report two platforms to generate phosphorylation-inducing chimeric small molecules (PHICS) for tyrosine phosphorylation. We generate PHICS from both noninhibitory binders (scantily available, platform 1) and kinase inhibitors (abundantly available, platform 2) using cysteine-based group transfer chemistry. PHICS triggered phosphorylation on tyrosine residues in diverse sequence contexts and target proteins (e.g., membrane-associated, cytosolic) and displayed multiple bioactivities, including the initiation of a growth receptor signaling cascade and the death of drug-resistant cancer cells. These studies provide an approach to induce biologically relevant PTM and lay the foundation for pharmacologic PTM editing (i.e., induction or removal) of target proteins using abundantly available inhibitors of PTM-inducing or -erasing enzymes.

Proximity-inducing modalities: the past, present, and future.

Living systems use proximity to regulate biochemical processes. Inspired by this phenomenon, bifunctional modalities that induce proximity have been developed to redirect cellular processes. An emerging example of this class is molecules that induce ubiquitin-dependent proteasomal degradation of a protein of interest, and their initial development sparked a flurry of discovery for other bifunctional modalities. Recent advances in this area include modalities that can change protein phosphorylation, glycosylation, and acetylation states, modulate gene expression, and recruit components of the immune system. In this review, we highlight bifunctional modalities that perform functions other than degradation and have great potential to revolutionize disease treatment, while also serving as important tools in basic research to explore new aspects of biology.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

Phosphorylation and Pin1 binding to the LIC1 subunit selectively regulate mitotic dynein functions.

The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the C-terminal domain (CTD) of its light intermediate chain 1 subunit (LIC1), which binds directly with cargo adaptors. Here we show that mitotic phosphorylation of LIC1-CTD at its three cdk1 sites is required for proper mitotic progression, for dynein loading onto prometaphase kinetochores, and for spindle assembly checkpoint inactivation in human cells. Mitotic LIC1-CTD phosphorylation also engages the prolyl isomerase Pin1 predominantly to Hook2-dynein-Nde1-Lis1 complexes, but not to dynein-spindly-dynactin complexes. LIC1-CTD dephosphorylation abrogates dynein-Pin1 binding, promotes prophase centrosome-nuclear envelope detachment, and impairs metaphase chromosome congression and mitotic Golgi fragmentation, without affecting interphase membrane transport. Phosphomutation of a conserved LIC1-CTD SP site in zebrafish leads to early developmental defects. Our work reveals that LIC1-CTD phosphorylation differentially regulates distinct mitotic dynein pools and suggests the evolutionary conservation of this phosphoregulation.

Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes.

Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing-issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a chemogenetic system that brings Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome. Despite the large size of Cas9, we were able to demonstrate efficient degradation in cells from multiple species. Furthermore, by controlling the Cas9 lifetime, we were able to bias the DNA repair pathways and the genotypic outcome for both templated and nontemplated genome editing. Finally, we were able to dosably control the Cas9 activity and specificity to ameliorate the off-target effects. The ability of this system to change the Cas9 lifetime and, therefore, bias repair pathways and specificity in the desired direction allows precision control of the genome editing outcome.

The exocyst complex and Rab5 are required for abscission by localizing ESCRT III subunits to the cytokinetic bridge.

Cytokinesis is the final step of cell division following chromosome segregation that generates two daughter cells. The conserved exocyst complex is required for scission of the intercellular cytokinetic bridge, although the molecular mechanisms it employs in this process are unclear. We identify and validate the early endocytic GTPase Rab5 as interacting with the exocyst complex in mammalian cells. Rab5 localizes in the cytokinetic bridge and on the midbody ring in a manner similar to the exocyst complex. Depletion of Rab5 led to delayed abscission. Caenorhabditis elegans orthologs of both exocyst complex subunits and Rab5 localize along the cleavage furrow and are required for cytokinesis in early embryos. Cytokinetic cells depleted of either Rab5 or the exocyst subunits Exoc3 and Exoc4 showed impaired deposition of the endosomal sorting complexes required for transport (ESCRT) III subunits CHMP2B and/or CHMP4B near the midbody ring. The study reveals an evolutionarily conserved role for the early endocytic marker Rab5 in cytokinetic abscission. In addition, it uncovers a key requirement of the exocyst and Rab5 for the delivery of components of the membrane-severing ESCRT III machinery to complete cytokinesis.

The chaperone ERp29 is required for tunneling nanotube formation by stabilizing MSec.

Tunneling nanotubes (TNTs) are membrane conduits that mediate long-distance intercellular cross-talk in several organisms and play vital roles during development, pathogenic transmission, and cancer metastasis. However, the molecular mechanisms of TNT formation and function remain poorly understood. The protein MSec (also known as TNFα-induced protein 2 (TNFAIP2) and B94) is essential for TNT formation in multiple cell types. Here, using affinity protein purification, mass spectrometric identification, and confocal immunofluorescence microscopy assays, we found that MSec interacts with the endoplasmic reticulum (ER) chaperone ERp29. siRNA-mediated ERp29 depletion in mammalian cells significantly reduces TNT formation, whereas its overexpression induces TNT formation, but in a strictly MSec-dependent manner. ERp29 stabilized MSec protein levels, but not its mRNA levels, and the chaperone activity of ERp29 was required for maintaining MSec protein stability. Subcellular ER fractionation and subsequent limited proteolytic treatment suggested that MSec is associated with the outer surface of the ER. The ERp29-MSec interaction appeared to require the presence of other bridging protein(s), perhaps triggered by post-translational modification of ERp29. Our study implicates MSec as a target of ERp29 and reveals an indispensable role for the ER in TNT formation, suggesting new modalities for regulating TNT numbers in cells and tissues.