Biology subjects

Goldfarb, D.

Publications and source records attributed to Goldfarb, D..

4 recordsLinked to original sources

bioRxiv2020

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including double electron-electron resonance (DEER), on reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.

biochemistry↗

bioRxiv2020

TRIM67 Regulates Exocytic Mode and Neuronal Morphogenesis via SNAP47

Neuronal morphogenesis involves dramatic plasma membrane expansion, likely fueled by SNARE-mediated exocytosis. Distinct fusion modes described at neuronal synapses include full-vesicle-fusion (FVF) and kiss-and-run fusion (KNR). During FVF, lumenal cargo is secreted and vesicle membrane incorporates into the plasma membrane. During KNR a transient fusion pore secretes cargo, but closes without membrane addition. In contrast, fusion modes are not described in developing neurons where plasma membrane expansion is significant. Here, we resolve individual exocytic events in developing murine cortical neurons and use new classification tools to identify four distinguishable fusion modes: two FVF-like modes that insert membrane material and two KNR-like modes that do not. Discrete fluorescence profiles suggest distinct behavior of the fusion pore with each mode. Simulations and experiments agree that FVF-like exocytosis provides sufficient membrane material for morphogenesis. We find the E3 ubiquitin ligase TRIM67 promotes FVF-like exocytosis. Our data suggest this is accomplished in part by limiting incorporation of the Qb/Qc SNARE SNAP47 into SNARE complexes and thus, SNAP47 involvement in exocytosis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis, and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector {beta}-catenin. The multiprotein destruction complex binds and phosphorylates {beta}-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase, for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing {beta}-catenin. Recent work clarified underlying mechanisms, but important questions remain. We explore {beta}-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axins RGS domain plays an important role. We find that elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. SIM imaging in mammalian cells suggests that this may result by promoting Dishevelled: Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high.

cell biology↗

bioRxiv2019

Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor

NFE2L2/NRF2 is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, and conversely NRF2 protein levels as well as activity is diminished with age and in neurodegenerative disorders. Though NRF2 activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here we used a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the understudied protein kinase Brain Specific Kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives AMPK activation and suppresses mTOR signaling. As a result, BRSK2 kinase activation suppressed ribosome-RNA complexes, global protein synthesis, and NRF2 protein levels. Collectively, our data establish the catalytically active BRSK2 kinase as a negative regulator of NRF2 via the AMPK/mTOR signaling. This signaling axis may prove useful for therapeutically targeting NRF2 in human diseases.\n\nSummary StatementBRSK2 suppresses NRF2 signaling by inhibiting protein synthesis through mTOR downregulation.

cell biology↗