Biology subjects

Yan, R.

Publications and source records attributed to Yan, R..

82 records · Page 5Linked to original sources

bioRxiv2020

Structure of dimeric full-length human ACE2 in complex with B0AT1

Angiotensin-converting enzyme 2 (ACE2) is the surface receptor for SARS coronavirus (SARS-CoV), directly interacting with the spike glycoprotein (S protein). ACE2 is also suggested to be the receptor for the new coronavirus (2019-nCoV), which is causing a serious epidemic in China manifested with severe respiratory syndrome. B0AT1 (SLC6A19) is a neutral amino acid transporter whose surface expression in intestinal cells requires ACE2. Here we present the 2.9 [A] resolution cryo-EM structure of full-length human ACE2 in complex with B0AT1. The complex, assembled as a dimer of ACE2-B0AT1 heterodimers, exhibits open and closed conformations due to the shifts of the peptidase domains (PDs) of ACE2. A newly resolved Collectrin-like domain (CLD) on ACE2 mediates homo-dimerization. Structural modelling suggests that the ACE2-B0AT1 complex can bind two S proteins simultaneously, providing important clues to the molecular basis for coronavirus recognition and infection.

biochemistry↗

bioRxiv2020

Structural basis for catalysis and substrate specificity of human ACAT1

Acyl-coenzyme A: cholesterol acyltransferases (ACATs) catalyze acyl transfer from acyl-coenzyme A (CoA) to cholesterol to generate cholesteryl ester, which is the primary form for cellular storage and plasma transport of cholesterol. Because of their close relationship with cholesterol metabolism, ACATs represent potential drug target for the treatment of atherosclerosis and other cholesterol-related disorders. Here we present the cryo-EM structure of human ACAT1 at 3.3 [A] resolution for dimer of dimers and 3.0 [A] for a dimer. Each protomer consists of nine transmembrane segments that enclose a cytosolic (C) and a transmembrane (T) tunnel. The tunnels, each accommodating an elongated density, converge at the predicted catalytic site. Structure-guided mutational analyses suggest the cytosolic and lateral entry for acyl-CoA and cholesterol, respectively. Our structural, biochemical, and mass spectrometric characterizations reveal the catalytic mechanism and substrate preference for unsaturated acyl chain by ACAT1.

biochemistry↗

bioRxiv2019

Measurement and models accounting for cell death capture hidden variation in compound response

Cancer cell sensitivity or resistance is almost universally quantified through a direct or surrogate measure of cell number. However, compound responses can occur through many distinct phenotypic outcomes including changes in cell growth, apoptosis, and non-apoptotic cell death. These outcomes have distinct effects on the tumor microenvironment, immune responses, and resistance mechanisms. Here, we show that quantifying cell viability alone is insufficient to distinguish between these compound responses. Using an alternative assay and drug response analysis amenable to high-throughput measurement, we find that compounds with identical viability outcomes can have very different effects on cell growth and death. Moreover, compound pairs with additive cell growth and death effects can appear synergistic when only assessed by viability. Overall, these results demonstrate an approach to incorporating measurements of cell death when characterizing a pharmacologic response. Summary PointsO_LIMeasurements of solely live cell numbers mask important differences in compound effects. C_LIO_LIAdditive effects on growth and death rates can appear synergistic when analyzed solely via live cell number. C_LIO_LIAutomated imaging can provide reasonable throughput to analyze cell response in terms of cell growth and death, and endpoint analysis is similarly informative. C_LI

cancer biology↗

bioRxiv2019

Increased Dynamics of α-Synuclein Fibrils by β-Synuclein Leads to Reduced Seeding and Cytotoxicity

Alpha-synuclein (S) fibrils are toxic to cells and contribute to the pathogenesis and progression of Parkinsons disease and other synucleinopathies. {beta}-Synuclein ({beta}S), which co-localizes with S, has been shown to provide a neuroprotective effect, but the molecular mechanism by which this occurs remains elusive. Here we show that S fibrils formed in the presence of {beta}S are less cytotoxic, exhibit reduced cell seeding capacity and are more resistant to fibril shedding compared to S fibrils alone. Using solid-state NMR, we found that the overall structure of the core of S fibrils when co-incubated with {beta}S is minimally perturbed, however, the dynamics of Lys and Thr residues, located primarily in the imperfect KTKEGV repeats of the S N-terminus, are increased. Our results suggest that amyloid fibril dynamics may play a key role in modulating toxicity and seeding. Thus, enhancing the dynamics of amyloid fibrils may be a strategy for future therapeutic targeting of neurodegenerative diseases.

biophysics↗

bioRxiv2019

Hypotonic stress induces fast, reversible degradation of the vimentin cytoskeleton via intracellular calcium release

The dynamic response of the cell to osmotic changes is critical to its physiology and has been widely exploited for cell manipulation. Using 3D-STORM super-resolution microscopy, here we examine the hypotonic stress-induced ultrastructural changes of the cytoskeleton of a common fibroblast cell type. Unexpectedly, we observe a fast, yet reversible dissolution of the vimentin intermediate filament system that precedes ultrastructural changes of the supposedly more dynamic actin and tubulin cytoskeletal systems, as well as changes in cell morphology. In combination with calcium imaging and biochemical analysis, we next show that the vimentin-specific fast cytoskeletal degradation under hypotonic stress is due to proteolysis by the calcium-dependent protease calpain. We find the process to be activated by the hypotonic stress-induced calcium release from intracellular stores, and so is efficiently suppressed by inhibiting any part of the IP3-Ca2+-calpain pathway we establish. Together, our findings highlight an unexpected, fast degradation mechanism for the vimentin cytoskeleton in response to the external stimuli, and point to the significant, yet previously overlooked physiological impacts of hypotonic stress-induced intracellular calcium release on cell ultrastructure and function.

cell biology↗

bioRxiv2019

Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

Intracellular diffusion underlies vital processes of the cell. However, it remains difficult to elucidate how an average-sized protein diffuses in the cell with good spatial resolution and sensitivity. Here we report single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through the local statistics of instantaneous displacements of freely diffusing single molecules. We thus show that diffusion in the cytoplasm and in the nucleus to both be spatially heterogeneous at the nanoscale, and such variations in local diffusivity correlate strongly with the ultrastructure of the actin cytoskeleton and the chromosome, respectively. Moreover, we identify the net charge of the diffuser as a key determinant of diffusion rate: intriguingly, the possession of positive, but not negative, net charges significantly impedes diffusion, and the exact degree of slowdown is determined by the specific subcellular environments.

biophysics↗

bioRxiv2018

Ex vivo dynamics of human glioblastoma cells in a microvasculature-on-a-chip system correlates with tumor heterogeneity and subtypes

The perivascular niche (PVN) plays an essential role in brain tumor stem-like cell (BTSC) fate control, tumor invasion, and therapeutic resistance. Herein we report on the use of a microvasculature-on-a-chip system as a PVN model to evaluate the dynamics of BTSCs ex vivo from 10 glioblastoma patients. We observed that BTSCs preferentially localize in the perivascular zone. Live cell tracking revealed that the cells residing in the vicinity of microvessels had the lowest motility, while a fraction of cells on the microvessels unexpectedly possessed the highest motility and migrated over the longest distance. These results indicate that the perivascular zone is a niche for BTSCs, while the microvascular tracks are also a path for long-distance tumor cell migration and invasion. Additionally, the degree of co-localization between tumor cells and microvessels varied significantly across patients. To validate the results from our microvasculature-on-a-chip system, we used single-cell transcriptome sequencing (10 patients and 21,750 single cells in total) to identify the subtype of each tumor cell. The co-localization coefficient was found to correlate positively with proneural (stem-like) or mesenchymal (invasive) but not classical (proliferative) tumor cells. Furthermore, we found that a gene signature profile including PDGFRA correlated strongly with the \"homing\" of brain tumor cells to the PVN. Our findings demonstrated that ex vivo dynamics of human brain tumor cells in a microvasculature-on-a-chip model can recapitulate in vivo tumor cell dynamics, heterogeneity, and subtypes, representing a new route to the study of human tumor cell biology and uncover patient-specific tumor cell functions.

bioengineering↗

bioRxiv2018

MBIR: A Cryo-electron Tomography 3D Reconstruction Method that Effectively Minimizes Missing Wedge Artifacts and Restores Missing Information

Cryo-Electron Tomography (cryo-ET) has become an essential technique in revealing cellular and macromolecular assembly structures in their native states. However, due to radiation damage and the limited tilt range, cryo-ET suffers from low contrast and missing wedge artifacts, which limits the tomograms to low resolution and hinders further biological interpretation. In this study, we applied the Model-Based Iterative Reconstruction (MBIR) method to obtain tomographic 3D reconstructions of experimental cryo-ET datasets and demonstrated the advantages of MBIR in contrast improvement, missing wedge artifacts reduction, and missing information restoration compared with other reconstruction approaches. Considering the outstanding reconstruction quality, MBIR has a great potential in the determination of high resolution biological structures with cryo-ET.

biophysics↗

bioRxiv2018

The structure of the Pro-domain of mouse proNGF in contact with the NGF domain

Nerve Growth Factor (NGF) is an important neurotrophic factor involved in the regulation of cell differentiation, maintenance, growth and survival of target neurons. Expressed as a proNGF precursor, NGF is then matured by furin-mediated protease cleavage. Increasing evidence suggests that NGF and proNGF have distinct cellular partners which account for different functional roles. While the structure of mature NGF is available, little is known about the structure of the pro-domain within the context of proNGF because the dynamical and structural features of the protein have so far prevented its structure determination. We have exploited a new hybrid strategy based on nuclear magnetic resonance and modelling validated by small angle X-ray scattering to gain novel insights on the pro-domain, both in isolation and in the context of proNGF. We show that the isolated pro-domain is intrinsically unstructured but has a clear tertiary structure propensity and forms transient tertiary intramolecular contacts. It is also able to interact, albeit weakly, with mature NGF and has per se the ability to induce growth cone collapse, indicating functional independence. Based on paramagnetic relaxation enhancement data and advanced molecular modelling, we have then reconstructed the overall properties of the pro-domain in the context of proNGF and showed that it has a compact structure. Our data represent an important step towards the structural and functional characterization of the properties of proNGF and its pro-domain.

biophysics↗

bioRxiv2017

Defocus and magnification dependent variation of TEM image astigmatism

Daily alignment of the microscope is a prerequisite to reaching optimal illumination lens and imaging lens conditions for high resolution imaging in cryo-EM. In contrast to the dramatic progress in automated image acquisition and post-image processing techniques, less attention has been paid to the improvement of microscope alignment before data collection. In this study, we have employed our recently published tool, s2stigmator, to study how image astigmatism varies with the imaging conditions (e.g. defocus, magnification). We have found that the large change of defocus/magnification between visual correction of astigmatism and subsequent data collection tasks, or even during data collection will inevitably result in undesirable residual astigmatism in the final images. Furthermore, the dependence of astigmatism on the imaging conditions varies significantly from time to time, so that it cannot be reliably compensated by pre-calibration of the microscope. These findings have essentially invalidated a basic assumption of current cryo-EM imaging strategies that assumes invariant astigmatism for different defocuses/magnifications used in the microscope alignment stage and the final data acquisition stage. Based on these findings, we recommend the same magnification and the median defocus of the intended defocus range for final data collection are used in the objective lens astigmatism correction task during microscope alignment and in the focus mode of the iterative low-dose imaging. It is also desirable to develop a fast, accurate method that can perform dynamic correction of the astigmatism for different intended defocuses during automated imaging.

biophysics↗