Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

Understanding Flavin-Dependent Halogenase Reactivity via Substrate Activity Profiling.

The activity of four native FDHs and four engineered FDH variants on 93 low molecular weight arenes was used to generate FDH substrate activity profiles. These profiles provided insights into how substrate class, functional group substitution, electronic activation, and binding impact FDH activity and selectivity. The enzymes studied could halogenate a far greater range of substrates than previously recognized, but significant differences in their substrate specificity and selectivity were observed. Trends between the electronic activation of each site on a substrate and halogenation conversion at that site were established, and these data, combined with docking simulations, suggest that substrate binding can override electronic activation even on compounds differing appreciably from native substrates. These findings provide a useful framework for understanding and exploiting FDH reactivity for organic synthesis.

Sodium channel beta4, a new disulfide-linked auxiliary subunit with similarity to beta2.

The principal alpha subunit of voltage-gated sodium channels is associated with auxiliary beta subunits that modify channel function and mediate protein-protein interactions. We have identified a new beta subunit termed beta4. Like the beta1-beta3 subunits, beta4 contains a cleaved signal sequence, an extracellular Ig-like fold, a transmembrane segment, and a short intracellular C-terminal tail. Using TaqMan reverse transcription-PCR analysis, in situ hybridization, and immunocytochemistry, we show that beta4 is widely distributed in neurons in the brain, spinal cord, and some sensory neurons.beta4 is most similar to the beta2 subunit (35% identity), and, like the beta2 subunit, the Ig-like fold of beta4 contains an unpaired cysteine that may interact with the alpha subunit. Under nonreducing conditions, beta4 has a molecular mass exceeding 250 kDa because of its covalent linkage to Nav1.2a, whereas on reduction, it migrates with a molecular mass of 38 kDa, similar to the mature glycosylated forms of the other beta subunits. Coexpression of beta4 with brain Nav1.2a and skeletal muscle Nav1.4 alpha subunits in tsA-201 cells resulted in a negative shift in the voltage dependence of channel activation, which overrode the opposite effects of beta1 and beta3 subunits when they were present. This novel, disulfide-linked beta subunit is likely to affect both protein-protein interactions and physiological function of multiple sodium channel alpha subunits.

TRPV3 is a calcium-permeable temperature-sensitive cation channel.

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.