Full metadata record
| DC Field | Value | Language |
|---|---|---|
| dc.contributor | Department of Applied Biology and Chemical Technology | en_US |
| dc.contributor.advisor | Zhao, Yanxiang (ABCT) | en_US |
| dc.creator | Zhang, Shuqi | - |
| dc.identifier.uri | https://theses.lib.polyu.edu.hk/handle/200/13998 | - |
| dc.language | English | en_US |
| dc.publisher | Hong Kong Polytechnic University | en_US |
| dc.rights | All rights reserved | en_US |
| dc.title | Dissecting the molecular machinery for tonicity-driven nuclear import of NFAT5 - role of KPNB1 and liquid-liquid phase separation | en_US |
| dcterms.abstract | Mammalian cells undergo osmoadaptation to maintain cell volume when exposed to an anisotonic environment. Nuclear factor of activated T-cells 5 (NFAT5) is the only transcription factor regulated by hypertonicity. Studies have shown that NFAT5 becomes significantly enriched in the nucleus under hypertonic conditions and activate transcription of target genes like osmolyte transporters to quickly restore intracellular osmolality. NFAT5 contains a leucine-rich canonical nuclear export sequence (NES) and a consensus bipartite nuclear localization signal (NLS) within its N-terminal intrinsically disordered region that is required for isotonicity-driven nucleocytoplasmic shuttling. Four phosphorylation sites, tyrosine 143 (Y143), threonine 135 (T135), serine 155 and 158 (S155 and S158), have been identified as crucial for NFAT5 activity regulation. However, the molecular machinery responsible for NFAT5 nuclear import and its regulation by tonicity are not well understood. | en_US |
| dcterms.abstract | Here we report molecular and structural studies to dissect the tonicity-regulated nucleocytoplasmic shuttling process of NFAT5. We first used siRNA screening to confirm that the nuclear import of NFAT5 under hypertonicity required only karyopherin β1 (KPNB1), but not karyopherin α. We then carried out mapping studies to show that a short segment (residues 199-216) termed canonical NLS (cNLS) is required for KPNB1 binding but insufficient for nuclear import of NFAT5. Instead, a longer segment (residues 171-253) termed the full NLS is required to recapitulate the tonicity-driven nuclear import of NFAT5. | en_US |
| dcterms.abstract | Given the disordered nature of the full NLS region in NFAT5, we reason it might undergo tonicity-mediated LLPS. We first did in vitro biochemical assays to confirm that the full NLS of NFAT5 indeed underwent liquid-liquid phase separation (LLPS) in salt-dependent manner. Under low salt conc entration of 100 mM NaCl, the full NLS of NFAT5 formed dynamic liquid droplets. However, these droplets readily disappeared when the salt concentration reached 200 mM. Intriguingly, organic solvents like sorbitol had no such effect. This LLPS phenomenon was also observed for a longer segment that included both the AED domain and the full NLS (residues 132-253). | en_US |
| dcterms.abstract | To validate our in vitro findings, we used immunofluorescence imaging studies to assess possible LLPS for NFAT5 in vivo. Our imaging studies of HeLa cells showed that endogenous NFAT5 formed distinct puncta in the cytosol under all tonicity conditions. To further confirm these puncta are related to LLPS, we generated a GFP-tagged NFAT5 construct (residues 1-543) and assessed its liquid-like property using FRAP experiments. Our data shows that the puncta formed by GFP-NFAT5-1-543 readily recovered their fluorescence signal after photobleaching, a defining feature of LLPS. Overall, our studies confirm that NFAT5 undergoes LLPS both in vitro and in vivo. | en_US |
| dcterms.abstract | As LLPS would sequester NFAT5 and renders it not available for nuclear import, we then investigated how KPNB1 would impact the LLPS of NFAT5. Interestingly, KPNB1 readily disrupted the LLPS of NFAT5 full NLS in vitro. Additionally, siRNA knockdown of KPNB1 in vivo increased the number of puncta for endogenous NFAT5. Furthermore, mutations that mimic the tonicity-driven phosphorylation events also disrupted LLPS of NFAT5. These results suggest that LLPS of NFAT5 is a key factor to regulate its nuclear import by KPNB1. Under hypo- or isotonic conditions, NFAT5 may undergo LLPS, which impedes its binding to KPNB1 and thus impairs its nuclear import. Under hypertonic conditions, the LLPS level for NFAT5 is reduced, which facilities binding to KPNB1 and promotes nuclear import. | en_US |
| dcterms.abstract | Lastly, we also determined the Cryo-EM structure of KPNB1 in complex with the full NLS of NFAT5 at 4.09 Å resolution. In this structure, KPNB1 adopts a conformation that largely resembles the structure of KPNB1-aIBB. Only small amount of density was visible for the cNLS of NFAT5 while the N- and C-terminal flanking segments were absent. Notably, binding site for cNLS of NFAT5 overlaps with that for RanGTP and IBB of KPNA1. Mutational studies confirm that residues in these two regions are critical for NFAT5 bindings. This overlap explains why the nuclear import of NFAT5 is KPNA-independent as its cNLS competes with the IBB of KPNA1 for KPNB1 binding. | en_US |
| dcterms.abstract | In summary, using a combination of biochemical, structural, and functional studies, my thesis work has delineated the molecular mechanism of tonicity-regulated nuclear import of NFAT5 with LLPS as a novel critical factor. This finding may have broad implications for the nuclear import process of LLPS-competent cargos. | en_US |
| dcterms.extent | xvii, 164 pages : color illustrations | en_US |
| dcterms.isPartOf | PolyU Electronic Theses | en_US |
| dcterms.issued | 2023 | en_US |
| dcterms.educationalLevel | Ph.D. | en_US |
| dcterms.educationalLevel | All Doctorate | en_US |
| dcterms.accessRights | open access | en_US |
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