Biology subjects

Liew, C. W.

Publications and source records attributed to Liew, C. W..

6 recordsLinked to original sources

bioRxiv2025

Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases

Peptide asparaginyl ligases (PALs) are powerful tools for protein engineering but are limited by natural rarity and poor expression. We mined 23 cyclotide-rich Viola species, uncovering 29 new PALs that expanded the known repertoire to 47. A dual-objective screen identified VdiPAL1 as the best-performed natural PAL, with twice efficiency of wt-VyPAL2 and 12 mg L-1 soluble expression in E. coli. A broad P2 specificity including Trp/Ile/Leu/Phe/Tyr/Met was discovered across diverse PALs, which enables sequential click-compatible liposome dual-functionalization. 1.8L[A] crystal structure of VdiPAL1 reveals a pre-organized near-attack conformation (NAC), supported by constant-pH MD simulations linking pH-dependent reactivity to NAC geometry. Our homology- and structure-based design yielded VyOpt1, a quintuple mutant of VyPAL2 with over 24-fold improved expression via enhanced cap-domain foldability in a single design-test cycle. This work expands the PAL family and demonstrates a transferable cap-domain-based engineering strategy, highlighting natural diversity as a powerful driver of enzyme discovery and optimization. TOC summaryMining of cyclotide-rich Viola genus expanded the total number of natural peptide asparaginyl ligases (PALs) to 47, including the discovery of highly expressible VdiPAL1. Its high-resolution structure provided new insights for mechanism, and its sequence guided us to a generalized PAL engineering strategy, leading to 24-fold increment in VyPAL2 expression.

biochemistry↗

bioRxiv2025

Medial preoptic area FoxO1 controls metabolic adaptation in a sexually dimorphic manner

The medial preoptic area (MPOA) of the hypothalamus is essential for metabolic adaptation to environmental challenges, though the molecular mechanisms underlying this process remain poorly understood. Here, we investigate the role of Forkhead transcription factor O1 (FoxO1), a key mediator of stress adaptation, in MPOA-dependent metabolic responses to temperature and nutritional changes. Our findings reveal sex-specific responses to both nutritional and temperature challenges. In female mice, but not males, a high-fat diet (HFD) challenge decreased FoxO1 expression in the MPOA. Specific deletion of FoxO1 in MPOA neurons (FoxO1-KOMPOA) had no effect on body weight under normal chow-fed conditions but protected females from HFD-induced obesity (DIO). These protected females exhibited increased lean mass, decreased fat mass, enhanced thermogenesis, increased energy expenditure, and reduced food intake under HFD conditions. They also showed enhanced cold-induced heat production at 6{degrees}C, though this effect vanished at thermoneutrality (30{degrees}C). The protection against DIO was abolished by ovariectomy (OVX) and was not restored by 17{beta}-estradiol supplementation, suggesting an estrogen-independent mechanism. Conversely, constitutive activation of FoxO1 in MPOA neurons (FoxO1-CAMPOA) increased DIO susceptibility in both sexes. Together, these findings demonstrate that FoxO1MPOA plays a crucial role in coordinating metabolic adaptation to nutritional and temperature challenges specifically in female mice.

physiology↗

bioRxiv2022

Crystal structure of the Rubella virus protease reveals a unique papain-like protease fold

Rubella is well-controlled due to an effective vaccine, but outbreaks are still occurring without any available antiviral treatments. There is still much to learn about the rubella virus (RUBV) papain-like protease (RubPro) that could be a potential drug target. This protease is crucial to RUBV replication, cleaving the non-structural polyprotein p200 into 2 multi-functional proteins, p150 and p90. Here we report a novel crystal structure of RubPro at 1.64 [A] resolution. It has a similar catalytic core structure to that of SARS-CoV-2 and foot-mouth-disease virus (FMDV) proteases. RubPro has well-conserved sequence motifs that are also found in its newly discovered Rubivirus relatives. The RubPro construct was shown to have protease activity in trans against a construct of RUBV protease-helicase and fluorogenic peptide. A protease-helicase construct was also cleaved in E. coli expression. RubPro was demonstrated to possess deubiquitylation activity, suggesting a potential role of RubPro in modulating the hosts innate immune responses. The structural and functional insights of the RubPro will advance our current understanding of its function and point to more structure-based research into the RUBV replication machinery, in hopes of developing antiviral therapeutics in the future.

microbiology↗

bioRxiv2020

Crystal structures of full length DENV4 NS2B-NS3 reveal the dynamic interaction between NS2B and NS3 upon binding to protease inhibitors

Flavivirus is a genus of emerging and re-emerging arboviruses which include many significant human pathogens. Non-structural protein 3 (NS3), a multifunctional protein with N-terminal protease and C-terminal helicase, is essential in viral replication. The NS3 protease together with NS2B cofactor is an attractive antiviral target. A construct with an artificial glycine linker connecting the NS2B cofactor and NS3 protease has been used for structural, biochemical and drug-screening studies. The effect of this linker on dynamics and enzymatic activity of the protease was studied by several biochemical and NMR methods but the findings remained inconclusive. Here, we designed constructs of NS2B cofactor joined to full length DENV4 NS3 in three different manners, namely bNS2B47NS3 (bivalent), eNS2B47NS3(enzymatically cleavable) and gNS2B47NS3 (glycine-rich G4SG4 linker). We report the first crystal structures of linked and unlinked full-length NS2B-NS3 enzyme in its free state and also in complex with Bovine Pancreatic Trypsin Inhibitor (BPTI). These structures demonstrate that the NS2B-NS3 protease mainly adopts a closed conformation. BPTI binding is not essential to but favors the closed conformation by interacting with both NS2B and NS3. The artificial linker between NS2B and NS3 tends to induce the open conformation and interfere with the protease activity. This negative impact on the enzyme structure and function is restricted to the protease domain as the ATPase activities of these constructs are not affected.

biochemistry↗

bioRxiv2020

Human AKTIP interacts with ESCRT proteins and functions at the midbody in cytokinesis

To complete mitosis, the intercellular bridge that links daughter cells needs to be cleaved. This abscission step is carried out by the sequential recruitment of ESCRT proteins at the midbody. We report here that a new factor, named AKTIP, works in association with ESCRTs. We find that AKTIP binds to the ESCRT I subunit VPS28, and show by high resolution microscopy that AKTIP forms a ring in the dark zone of the intercellular bridge. This ring is positioned in between the circular structures formed by ESCRTs type III. Functionally, we observe that the reduction of AKTIP impinges on the recruitment of the ESCRT III member IST1 at the midbody and causes abscission defects. Taken together, these data indicate that AKTIP is a new factor that contributes to the formation of the ESCRT complex at the midbody and is implicated in the performance of the ESCRT machinery during cytokinetic abscission.

cell biology↗

bioRxiv2019

The Human Telomeric Nucleosome Displays Distinct Structural and Dynamic Properties

Telomeres protect the ends of our chromosomes and are key to maintaining genomic integrity during cell division and differentiation. However, our knowledge of telomeric chromatin and nucleosome structure at the molecular level is limited. Here, we aimed to define the structure, dynamics as well as properties in solution of the human telomeric nucleosome. We first determined the 2.2 [A] crystal structure of a human telomeric nucleosome core particle (NCP) containing 145 bp DNA, which revealed the same helical path for the DNA as well as symmetric stretching in both halves of the NCP as that of the 145 bp 601 NCP. In solution, the telomeric nucleosome exhibited a less stable and a markedly more dynamic structure compared to NCPs containing DNA positioning sequences. These observations provide molecular insights into how telomeric DNA forms nucleosomes and chromatin and advance our understanding of the unique biological role of telomeres.

biochemistry↗