Biology subjects

Lim, L.

Publications and source records attributed to Lim, L..

25 records · Page 2Linked to original sources

bioRxiv2020

Influenza C and D viral load in cattle correlates with bovine respiratory disease (BRD): Emerging role of orthomyxoviruses in the pathogenesis of BRD

Bovine respiratory disease (BRD) is the costliest disease affecting the cattle industry globally. Despite decades of research, the pathophysiology of BRD is not yet fully understood. It is widely believed that viruses predispose cattle to bacterial infection by causing direct damage to the respiratory tract and interfering with the immune system, leading to bacterial pneumonia. BRD remains a major challenge despite extensive vaccination against all major viral pathogens associated with the disease. Orthomyxoviruses (Influenza C & D viruses), have recently been found to infect cattle throughout the United States and are implicated to play a role in BRD. Here, we use the largest cohort study to date to investigate the association of influenza viruses in cattle with BRD. Cattle (n=599) from 3 locations were individually observed and scored for respiratory symptoms using the McGuirk scoring system. Deep pharyngeal and mid-nasal swabs were collected from each animal and were tested quantitatively for bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus, bovine coronavirus, influenza C virus (ICV) and influenza D virus (IDV) by real-time PCR. Cattle that have higher viral loads of IDV and ICV also have greater numbers of co-infecting viruses than controls. More strikingly, in BRD-symptomatic cattle, the geometric mean of detectable IDV viral RNA was nearly 2 logs higher in co-infected animals (1.30x104) than those singly infected with IDV (2.19x102). This is strong evidence that viral coinfections can lead to higher replication of IDV. Our results strongly suggest that orthomyxoviruses may be significant contributors to BRD.

microbiology↗

bioRxiv2019

ATP emerged to induce protein folding, inhibit aggregation and increase stability

By NMR characterization of effects of ATP and related molecules on the folding and dynamics of the ALS-causing C71G-PFN1 and nascent hSOD1, we reveal for the first time that ATP has a general capacity in inducing protein folding with the highest efficiency known so far. This capacity was further identified to result from triphosphate, a key intermediate in prebiotic chemistry, which, however, can severely trigger protein aggregation. Remarkably, by joining adenosine and triphosphate together, ATP integrates three abilities to simultaneously induce protein folding, inhibit aggregation and increase thermodynamic stability. Our study implies that the emergence of ATP might represent an irreplaceable step essential for the Origin of Life, and decrypts a principle for engineering small molecules with three functions to treat aggregation-associated ageing and diseases. One sentence summaryBy joining adenosine and triphosphate, ATP integrates three abilities to control protein homeostasis for the Origin of Life.

biochemistry↗

bioRxiv2019

Spatial control of oxygen delivery to 3D cultures alters cancer cell growth and gene expression

Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional bioreactor system where cancer cells are grown in Matrigel in modified 6-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3% to 0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional 2D monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.

bioengineering↗

bioRxiv2018

TMEM106B, a risk factor for FTLD and aging, has an intrinsically disordered cytoplasmic domain

TMEM106B was initially identified as a risk factor for FTLD, but recent studies highlighted its general role in neurodegenerative diseases. Very recently TMEM106B has also been characterized to regulate aging phenotypes. TMEM106B is a 274-residue lysosomal protein whose cytoplasmic domain functions in the endosomal/autophagy pathway by dynamically and transiently interacting with diverse categories of proteins but the underlying structural basis remains completely unknown. Here we conducted bioinformatics analysis and biophysical characterization by CD and NMR spectroscopy, and obtained results reveal that the TMEM106B cytoplasmic domain is intrinsically disordered with no well-defined three-dimensional structure. Nevertheless, detailed analysis of various multi-dimensional NMR spectra allowed defining residue-specific conformations and dynamics. Overall, the TMEM106B cytoplasmic domain is lacking of any tight tertiary packing and relatively flexible. However, several segments are populated with dynamic/nascent secondary structures and have relatively restricted backbone motions. In particular, the fragment Ser12-Met36 is highly populated with - helix conformation. Our study thus decodes that being intrinsically disordered allows the TMEM106B cytoplasmic domain to dynamically and transiently interact with a variety of distinct partners.

biophysics↗

bioRxiv2018

Misfolded proteins share a common capacity in disrupting LLPS organizing membrane-less organelles

Profilin-1 mutants cause ALS by gain of toxicity but the underlying mechanism remains unknown. Here we showed that three PFN1 mutants have differential capacity in disrupting dynamics of FUS liquid droplets underlying the formation of stress granules (SGs). Subsequently we extensively characterized conformations, dynamics and hydrodynamic properties of C71G-PFN1, FUS droplets and their interaction by NMR spectroscopy. C71G-PFN1 co-exists between the folded (55.2%) and unfolded (44.8%) states undergoing exchanges at 11.7 Hz, while its unfolded state non-specifically interacts with FUS droplets. Results together lead to a model for dynamic droplets to recruit misfolded proteins, which functions seemingly at great cost: simple accumulation of misfolded proteins within liquid droplets is sufficient to reduce their dynamics. Further aggregation of misfolded proteins within droplets might irreversibly disrupt/destroy structures and dynamics of droplets, as increasingly observed on SGs, an emerging target for various neurodegenerative diseases. Therefore, our study implies that other misfolded proteins might also share the capacity in disrupting LLPS.

biophysics↗

bioRxiv2017

NMR studies reveal that protein dynamics critically mediate aggregation of the well-folded and very soluble E. coli S1 ribosomal protein

Unlike mammalian aging associated with many hallmarks, E. coli aging is only significantly characterized by protein aggregation, thus offering an excellent model for addressing the relationship between protein aggregation and aging. Here we characterized conformations, unfolding and dynamics of ribosomal protein S1 and its D3/D5 domains using NMR, CD and fluorescence spectroscopy. S1 is a 557-residue modular protein containing six S1 motifs. Paradoxically, while S1 is well-folded and very soluble in vitro, it was found in various lists of aggregated E. coli proteins. Our results decipher: 1) S1 has dynamic inter-domain interactions. Strikingly, S1 and its D3/D5 domains have significantly exposed hydrophobic patches characterized by irreversible unfolding. 2) Although D5 has significantly restricted backbone motion on ps-ns time scale, it has global s-ms conformational dynamics and particularly high \"global breathing\" motions. 3) D5 assumes the conserved {beta}-barrel fold but contains large hydrophobic patches at least dynamically accessible. Taken together, our study reveals that S1 could be prone to aggregation due to significant dynamics at two levels: inter-domain interactions and individual domains, which may even render buried hydrophobic patches/cores accessible for driving aggregation. This mechanism is most likely to operate in many proteins of E. coli and other organisms including human.

biophysics↗

bioRxiv2017

RRM domain of ALS/FTD-causing FUS interacts with membrane: an anchor of membraneless organelles to membranes?

526-residue FUS functions to self-assemble into reversible droplets/hydrogels, which could be further solidified into pathological fibrils. FUS is composed of N-terminal low-sequence complexity (LC); RNA-recognition motif (RRM) and C-terminal LC domains. FUS belongs to an emerging category of proteins which are capable of forming membraneless organelles in cells via phase separation. On the other hand, eukaryotic cells contain a large network of internal membrane systems. Therefore, it is of fundamental importance to address whether membraneless organelles can interact with membranes. Here we attempted to explore this by NMR HSQC titrations of three FUS domains with gradual addition of DMPC/DHPC bicelle, which mimics the bilayer membrane. We found that both N- and C-terminal LC domains showed no significant interaction with bicelle, but its well-folded RRM domain does dynamically interact with bicelle with an interface opposite to that for binding nucleic acids including RNA and ssDNA. If this in vitro observation also occurs in cells, to interact with membrane might represent a mechanism for dynamically organizing membraneless organelles to membranes to facilitate their physiological functions.

biophysics↗