Biology subjects

Sopipong, W.

Publications and source records attributed to Sopipong, W..

3 recordsLinked to original sources

bioRxiv2026

Dissecting KEAP1-NRF2 interactions: Structural and functional impact of cancer-associated KEAP1 mutants

Kelch-like ECH-associated protein 1 (KEAP1) is a cysteine-rich substrate adaptor for the CUL3-RBX1 E3 ubiquitin ligase complex, primarily regulating the transcription factor NRF2. By promoting NRF2 degradation under basal conditions, KEAP1 maintains redox homeostasis, while oxidative or electrophilic stress disrupts this regulation, enabling NRF2 activation of cytoprotective genes. Dysregulation of the KEAP1-NRF2 pathway, often through KEAP1 mutations, is implicated in cancer, where constitutive NRF2 activation promotes tumour growth, chemoresistance, and metabolic reprogramming. In this study, we investigated structural and functional consequences of three representative KEAP1 mutants R470C (superbinder), G480W (hypomorphic), and R554Q (null allele) using thermal shift assays (TSA), microscale thermophoresis (MST), isothermal calorimetry (ITC), and X-ray crystallography, complemented by AlphaFold3 modelling. TSA revealed that while inhibitor-induced thermal stability shifts followed similar pattern across mutants, baseline stability varied significantly, with R554Q showing the largest destabilisation. MST and ITC confirmed mutant-specific alterations in NRF2 ETGE peptide binding affinity, consistent with their known functional classifications. Structures of mutant-inhibitor and mutant-peptide complexes showed remarkable structural similarity to native KEAP1, with preserved inhibitor and peptide binding orientations despite residue substitutions. Spatial mapping of reported KEAP1 mutations revealed clustering of superbinder mutations near the IVR interface, while most loss-of-function variants localised within the KELCH domain. Our findings provide a detailed structural and biophysical characterization of clinically relevant KEAP1 mutations, offering mechanistic insights into their functional diversity. This work advances the understanding of KEAP1 mutation effects, with implications for therapeutic targeting of the KEAP1-NRF2 pathway in cancer.

cancer biology↗

bioRxiv2024

Structural extension of the human exocyst is enabled by a minimal interface

In multicellular organisms, the machinery responsible for polarized trafficking directs constitutive cargo secretion at distinct sites of the plasma membrane, cilia, and junctional structures. Central to this machinery is the exocyst complex, which tethers cargo vesicles to their destination membrane, alongside other intracellular membrane tethering roles. Precisely how the exocyst spatially integrates membranes and membrane resident binding partners is unclear. Here, we address the structural morphology and formation of the human exocyst complex. Through structural approaches coupled to predictive models, we determined that the exocyst and its subcomplexes have extended arm-like structures that help maximize its reach. Moreover, we demonstrate minimal intersubunit interaction, in contrast to prior models. Nucleation of the holocomplex occurs through a single site, explaining its spatial extension. Our results provide the biochemical basis for exocyst complex assembly, suggesting an ornate extended architecture. SignificanceCargo transport to the eukaryotic cell plasma membrane predominantly relies on the exocyst as a central, signal-integrating polarized trafficking complex. How this single cargo vesicle tethering complex can target diverse vesicles to destination membrane is poorly understood, partly due to inconsistent structural models. Here, we show an extended architecture of the human exocyst complex, revealing a minimal nucleation interface between its two subcomplexes. The distinct morphology of human exocyst, compared to yeast models, suggests a novel mechanism that supports its versatile role in membrane trafficking.

biochemistry↗

bioRxiv2022

Emerging variants of SARS-CoV-2 NSP10 highlight strong functional conservation of its binding to two non-structural proteins, NSP14 and NSP16

The coronavirus SARS-CoV-2 protects its RNA from being recognized by host immune responses by methylation of its 5 end, also known as capping. This process is carried out by two enzymes, non-structural protein 16 (NSP16) containing 2-O-methyltransferase and NSP14 through its N7 methyltransferase activity, which are essential for the replication of the viral genome as well as evading the hosts innate immunity. NSP10 acts as a crucial cofactor and stimulator of NSP14 and NSP16. To further understand the role of NSP10, we carried out a comprehensive analysis of >13 million globally collected whole-genome sequences (WGS) of SARS-CoV-2 obtained from the Global Initiative Sharing All Influenza Data (GISAID) and compared it with the reference genome Wuhan/WIV04/2019 to identify all currently known variants in NSP10. T12I, T102I, and A104V in NSP10 have been identified as the three most frequent variants and characterized using X-ray crystallography, biophysical assays and enhanced sampling simulations. In contrast to other proteins such as spike and NSP6, NSP10 is significantly less prone to mutation due to its crucial role in replication. The functional effects of the variants were examined for their impact on the binding affinity and stability of both NSP14-NSP10 and NSP16-NSP10 complexes. These results highlight the limited changes induced by variant evolution in NSP10 and reflect on the critical roles NSP10 plays during the SARS-CoV-2 life cycle. These results also indicate that there is limited capacity for the virus to overcome inhibitors targeting NSP10 via the generation of variants in inhibitor binding pockets. Significance StatementThe SARS-CoV-2 proteins have constantly been evolving. These variants assist the virus to survive, adapt and evade the host immune responses. While the main focus has been on structural proteins like Spike, there is very limited structural and functional information on the effects of emerging mutations on other essential non-structural viral proteins. One such protein is NSP10, an essential cofactor for NSP14 and NSP16. This study demonstrates that NSP10 is more resistant to genetic variations than other SARS-CoV-2 non-structural proteins and that the presence of mutations conserve structural and dynamic changes in NSP10. The effects of naturally occurring mutations reflect the evolutionary relationship between structurally conserved essential cofactors, their function and the role they play in the survival of the virus.

biophysics↗