Biology subjects

Goldfarb, D.

Publications and source records attributed to Goldfarb, D..

24 records · Page 2Linked to original sources

bioRxiv2020

Nanotrap(R) particles improve detection of SARS-CoV-2 for pooled sample methods, extraction-free saliva methods, and extraction-free medium methods

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from transport medium and saliva using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus prior to RNA extraction, thus significantly improving detection of the virus using a real-time RT-PCR assay across a range of viral titers, from 100 to 1,000,000 viral copies/mL; in particular, detection of virus in low viral load samples is enhanced when using the method coupled with the IDT 2019-nCoV CDC EUA Kit. This method is compatible with commercially available nucleic acid extraction kits, as well with a simple heat and detergent method. Using transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2 using either the Abbott RealTime SARS-CoV-2 EUA Test (n=14) or the Cepheid Xpert Xpress SARS-CoV-2 EUA Test (n=35), we demonstrate that our method not only correctly identifies all positive samples (n = 17) but also significantly improves detection of the virus in low viral load samples. The average improvement in cycle threshold (Ct) value as measured with the IDT 2019-nCoV CDC EUA Kit was 3.1; n = 10. Finally, to demonstrate that the method could potentially be used to enable pooled testing, we spiked infectious virus or a confirmed positive diagnostic remnant sample into 5 mL and 10 mL of negative transport medium and observed significant improvement in the detection of the virus from those larger sample volumes.

microbiology↗

bioRxiv2020

In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

The complexity of the cellular medium can affect proteins properties and therefore in-cell characterization of proteins is essential. We explored the stability and conformation of BIR1, the first baculoviral IAP repeat domain of X-chromosome-linked inhibitor of apoptosis (XIAP), as a model for a homo-dimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three protein residues, C12 (flexible region), E22C and N28C (part of helical residues 26-31) in the N-terminal region. In contrast to predictions by excluded volume crowding theory, the dimer-monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was recapitulated under conditions of high salt concentrations given that a conserved salt bridge at the dimer interface is critically required for association. Unexpectedly, however, also the addition of a crowding agent such as Ficoll destabilized the dimer, suggesting that Ficoll forms specific interactions with the monomeric protein. Changes in DEER distance distributions were observed for the E22C site, which displayed reduced conformational freedom in cells. Although overall DEER behaviors at E22C and N28C were compatible with a predicted compaction of disordered protein regions by excluded volume effects, we were unable to reproduce E22C properties in artificially crowded solutions. These results highlight the importance of in-cell DEER measurements to appreciate the complexities of cellular in vivo effects on protein structures and functions.

biophysics↗

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↗