Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices

Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.

Interaction of INPP5E with ARL13B is essential for its ciliary membrane retention but dispensable for its ciliary entry

Compositions of proteins and lipids within cilia and on the ciliary membrane are maintained to be distinct from those of the cytoplasm and plasma membrane, respectively, by the presence of the ciliary gate. INPP5E is a phosphoinositide 5-phosphatase that is localized on the ciliary membrane by anchorage via its C-terminal prenyl moiety. In addition, the ciliary membrane localization of INPP5E is determined by the small GTPase ARL13B. However, it remained unclear as to how ARL13B participates in the localization of INPP5E. We here show that wild-type INPP5E, INPP5E(WT), in ARL13B-knockout cells and an INPP5E mutant defective in ARL13B binding, INPP5E(ΔCTS), in control cells were unable to show steady-state localization on the ciliary membrane. However, not only INPP5E(WT) but also INPP5E(ΔCTS) was able to rescue the abnormal localization of ciliary proteins in INPP5E-knockout cells. Analysis using the chemically induced dimerization system demonstrated that INPP5E(WT) in ARL13B-knockout cells and INPP5E(ΔCTS) in control cells were able to enter cilia, but neither was retained on the ciliary membrane due to the lack of the INPP5E–ARL13B interaction. Thus, our data demonstrate that binding of INPP5E to ARL13B is essential for its steady-state localization on the ciliary membrane but is dispensable for its entry into cilia.

Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry and cell-based assays, including chemically induced dimerization and single-molecule pulldown, we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH domain–mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our work also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

Liquid condensation drives telomere clustering during ALT

Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liquid condensates, suggesting two potential mechanisms to promote telomere elongation: condensation to enrich DNA repair factors for telomere synthesis and coalescence to cluster telomeres to provide repair templates. Using chemically-induced dimerization, we show that telomere sumoylation nucleates APB condensation via SUMO-SIM (SUMO interaction motif) interactions and clusters telomeres. The induced APBs lack DNA repair factors, indicating that these factors are clients recruited to the APB scaffold rather than components that drive condensation. Telomere clustering, however, relies only on liquid properties of the condensate, as an alternative condensation chemistry also induces clustering. Our results demonstrate how the material properties and chemical composition of APBs independently contribute to ALT, suggesting a general framework for how liquid condensates promote cellular functions.