RAB3GAP1 is GTPase activating protein localized to the ER and Golgi compartments. In humans, mutations in RAB3GAP1 are the most common cause of Warburg Micro syndrome, a neurodevelopmental disorder associated with intellectual disability, microcephaly, and agenesis of the corpus callosum. We found that downregulation of RAB3GAP1 leads to a reduction in neurite outgrowth and complexity in human stem cell derived neurons. To further define the cellular function of RAB3GAP1, we sought to identify novel interacting proteins. We used a combination of mass spectrometry, co-immunoprecipitation and colocalization analysis and identified two novel interactors of RAB3GAP1: the axon elongation factor Dedicator of cytokinesis 7 (DOCK7) and the TATA modulatory factor 1 (TMF1) a modulator of Endoplasmic Reticulum (ER) to Golgi trafficking. To define the relationship between RAB3GAP1 and its two novel interactors, we analyzed their localization to different subcellular compartments in neuronal and non-neuronal cells with loss of RAB3GAP1. We find that RAB3GAP1 is important for the sub-cellular localization of TMF1 and DOCK7 across different compartments of the Golgi and endoplasmic reticulum. In addition, we find that loss of function mutations in RAB3GAP1 lead to dysregulation of pathways that are activated in response to the cellular stress like ATF6, MAPK, and PI3-AKT signaling. In summary, our findings suggest a novel role for RAB3GAP1 in neurite outgrowth that could encompass the regulation of proteins that control axon elongation, ER-Golgi trafficking, as well as pathways implicated in response to cellular stress.

Allosteric transcription factors (aTFs) are used in a myriad of processes throughout biology and biotechnology. aTFs have served as the workhorses for developments in synthetic biology, fundamental research, and protein manufacturing. One of the most utilized TFs is the lactose repressor (LacI). In addition to being an exceptional tool for gene regulation, LacI has also served as an outstanding model system for understanding allosteric communication. In this perspective, we will use the LacI TF as the principal exemplar for engineering alternate functions related to allostery-i.e., alternate protein DNA interactions, alternate protein-ligand interactions, and alternate phenotypic mechanisms. In addition, we will summarize the design rules and heuristics for each design goal and demonstrate how the resulting design rules and heuristics can be extrapolated to engineer other aTFs with a similar topology-i.e., from the broader LacI/GalR family of TFs.

Protein allostery is a vitally important protein function that has proven to be a vexing problem to understand at the molecular level. Allosteric communication is a hallmark of many protein functions. However, despite more than four decades of study the details regarding allosteric communication in protein systems are still being developed. Engineering of LacI and related homologues to confer alternate allosteric communication has shed light on the pre-requisites for the de novo design of allosteric communication. While the de novo design of an allosteric pathway and complementary functional surfaces has not been realized, this review highlights recent advances that set the stage for true predictive design for a given protein topology.