Biology subjects

Englander, S. W.

Publications and source records attributed to Englander, S. W..

4 recordsLinked to original sources

bioRxiv2021

ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes

SUMMARYWNTs play key roles in development and disease, by binding both Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors that include the ROR and RYK receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2). RORs bind WNTs though a FZD-related cysteine-rich domain (CRD), and RYKs through a WNT-inhibitory factor (WIF) domain. Our structures suggest that neither the Nrk CRD nor the Drl-2 WIF domain can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable with a WNT acyl chain. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also show that DWnt-5, which regulates Drosophila ROR and RYK orthologs, lacks an acyl chain. Together with analysis of WNT/receptor interaction sites, these structures provide new insight into how WNTs recruit their RTK co-receptors into signaling complexes.

biochemistry↗

bioRxiv2020

ApoC-III helical structure determines its ability to bind plasma lipoproteins and inhibit Lipoprotein Lipase-mediated triglyceride lipolysis.

In humans, apolipoprotein C-III (apoC-III) plasma levels have been associated with increased risk of cardiovascular disease. This association is in part explained by the effects of apoC-III on triglyceride (TG) metabolism; apoC-III raises plasma TG by increasing very low density lipoprotein (VLDL) secretion, inhibiting lipoprotein lipase (LPL)-mediated TG lipolysis, and impairing the removal of triglyceride-rich lipoprotein (TRL) remnants from the circulation. In this study, we explored the structure-function relationship the interaction of apoC-III with plasma lipoproteins and its ultimate impact on LPL activity. The structural and functional properties of wild-type (WT) apoC-III were compared with two missense variants previously associated with lower (A23T) and higher (Q38K) plasma TG. ApoC-III in the lipid-free state is unstructured but its helix content and stability increases when bound to lipid. Lipid-bound apoC-III contains two alpha helices spanning residues amino acids 11 - 38 (helix 1) and 44 – 64 (helix 2). Investigation of the structural and functional consequences of the A23T and Q38K variants showed that these amino acid substitutions within helix 1 do not significantly alter the stability of the helical structure but affect its hydrophilic-lipophilic properties. The A23T substitution impairs lipoprotein binding capacity, reduces LPL inhibition, and ultimately leads to lower plasma TG levels. Conversely, the Q to K substitution at position 38 enhances the lipid affinity of helix 1, increases TRL binding capacity and LPL inhibition, and is associated with hypertriglyceridemia. This study indicates that structural modifications that perturb the hydrophilic/lipophilic properties of the alpha helices can modulate the hypertriglyceridemic effects of apoC-III.Competing Interest StatementDJR is a a co-founder of Staten Biotechnology, he is a member of the Scientific Advisory Boards of Alnylam, Novartis, and Pfizer, and he was previously a consultant for Akcea. The other authors declare no competing financial interests.View Full Text

biochemistry↗

bioRxiv2020

Characterization of small molecule induced changes in Parkinson's-related trafficking via the Nedd4 ubiquitin signaling cascade

The benzdiimidazole NAB2 rescues -synuclein-associated trafficking defects associated with early onset Parkinsons disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (KDapp = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro. Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its colocalization. Ubiquitin-enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking and transport associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism.

biochemistry↗

bioRxiv2018

Hydrogen-deuterium exchange coupled to top- and middle-down mass spectrometry enables high-resolution measurements of histone tail dynamics before and after nucleosome assembly

Until recently, a major limitation of hydrogen deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization information was limited to the length of the peptide generated during proteolysis. Recently, however, it has been demonstrated that electron transfer dissociation (ETD) allows for preservation of deuterium label in the gas phase and therefore can be used to obtain more resolved information. To date, this technology has remained mostly limited to single, small, already well-characterized model proteins. Here, we optimize, expand, and adapt HDX-MS/MS capabilities to accommodate histone and nucleosomal complexes on top-down (TD) HDX-MS/MS and middle-down (MD) HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded rigorous analysis by traditional structural biology techniques for decades, revealing important novel insights into chromatin biology. This work represents the first heterogeneous protein complex and protein-DNA complex to be analyzed by TD- and MD-HDX-MS/MS, respectively. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.

biochemistry↗