Biology subjects

Blacklow, S. C.

Publications and source records attributed to Blacklow, S. C..

22 records · Page 2Linked to original sources

bioRxiv2019

Molecular basis for feedback inhibition of Notch signaling by the Notch regulated ankyrin repeat protein NRARP

The Notch regulated ankyrin repeat protein (NRARP) is a negative feedback regulator of Notch signaling in higher organisms. The molecular basis for NRARP function, however, has remained elusive. Mass spectrometry-based proteomic studies show that human NRARP associates with the core Notch transcriptional activation complex (NTC), containing the RBPJ transcription factor, the Notch intracellular domain (NICD), and a Mastermind-like co-activator. Binding of NRARP is direct, requires both RBPJ and NICD, and is independent of Mastermind-like proteins or DNA. The X-ray structure of an NRARP/RBPJ/NOTCH1/DNA complex, determined to 3.75 [A] resolution, reveals a non-canonical mode of binding in which NRARP extends the NOTCH1 ankyrin-repeat stack by three additional repeats. Mutations of NRARP residues at the binding interface disrupt entry into complexes and suppress feedback inhibition in signaling assays. These studies establish the structural basis for NTC engagement by NRARP and provide insight into its mechanism of feedback inhibition of Notch signaling.

biochemistry↗

bioRxiv2019

Development of a Covalent Inhibitor of Gut Bacterial Bile Salt Hydrolases

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria in order to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSH. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. Strikingly, this inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.

microbiology↗

bioRxiv2019

Time resolved quantitative phosphoproteomics reveals distinct patterns of SHP2 dependence in EGFR signaling

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines; it is frequently mutated in childhood leukemias and other cancers. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by the small molecule SHP099. Data on phosphotyrosine abundance at more than 400 tyrosine residues reveals six distinct response signatures following SHP099 treatment and washout. These include putative substrate sites with increased phosphotyrosine abundance at early or late timepoints, and another class of sites that shows reduced phosphotyrosine abundance when SHP099 is present. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight how analysis of phosphoproteomic dynamics can provide insight into transmembrane signaling responses.

cell biology↗

bioRxiv2019

A flow extension tethered particle motion assay for single-molecule proteolysis

Regulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads are not restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow extension (FLEX) strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allows assignment of tethered substrates into singly-captured and multiply-tethered bins, with the fraction of fully mobile, single-tethered substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiply-tethered beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.

biophysics↗