Biology subjects

Fung, H. Y. J.

Publications and source records attributed to Fung, H. Y. J..

9 recordsLinked to original sources

bioRxiv2025

A genome-scale drug discovery pipeline uncovers new therapeutic targets and a unique p97 allosteric binding site in Schistosoma mansoni

Schistosomes are parasitic flatworms that infect more than 200 million people globally. However, there is a shortage of molecular tools that enable the discovery of potential drug targets within schistosomes. Thus, praziquantel has remained the frontline treatment for schistosomiasis despite known liabilities. Here, we have conducted a genome-wide study in S. mansoni using the human druggable genome as a bioinformatic template to identify essential genes within schistosomes bearing similarity to catalogued drug targets. Then, we assessed these candidate targets in silico using a set of unbiased criteria to determine which possess ideal characteristics for a ready-made drug discovery campaign. Following this prioritization, we pursued a parasite p97 ortholog as a bona-fide drug target for the development of therapeutics to treat schistosomiasis. From this effort, we identified a covalent inhibitor series that kills schistosomes through an on-target killing mechanism by disrupting the ubiquitin proteasome system. Fascinatingly, these inhibitors induce a conformational change in the conserved D2 domain P-loop of schistosome p97 upon modification of Cys519. This conformational change reveals an allosteric binding site adjacent to the D2 domain active site reminiscent of the DFG flip in protein kinases. This allosteric binding site can potentially be utilized to generate new classes of species-selective p97 inhibitors. Furthermore, these studies provide a resource for the development of alternative therapeutics for schistosomiasis and a workflow to identify potential drug targets in similar systems with few available molecular tools. Significance StatementSchistosomes cause widespread infections in humans, leading to severe chronic illnesses in endemic regions. There is no vaccine for schistosomiasis, and there has been limited success using the current standard-of-care treatment, praziquantel. Therefore, it is essential to identify drug targets within these parasites. Here, we identify potential drug targets in schistosomes bearing similarity to established human therapeutic targets, evaluate their essentiality for parasite survival, then prioritize them using an unbiased set of criteria to uncover high-value targets for the treatment of schistosomiasis. We investigated one candidate as a proof-of concept, a p97 ortholog, to discover newly characterized inhibitors of the parasite enzyme. This study demonstrates that this workflow can lead to the identification of small molecules that kill schistosomes.

microbiology↗

bioRxiv2024

Allosteric degraders induce CRL5ASB8 mediated degradation of XPO1

SummaryThe nuclear export receptor exportin 1 (XPO1/CRM1) is often overexpressed in cancer cells, leading to the mislocalization of numerous cancer-related protein cargoes1,2. Selinexor, a covalent XPO1 inhibitor, and other Selective Inhibitor of Nuclear Export (SINEs) restore proper nuclear localization by blocking XPO1-cargo binding2-7. SINEs also induce XPO1 degradation via the Cullin-RING E3 ubiquitin ligase (CRL) substrate receptor ASB87. Here we elucidate the mechanism underlying the high-affinity engagement of CRL5ASB8 with SINE-conjugated XPO1. Cryogenic electron microscopy (cryoEM) structures reveal that ASB8 binds to a cryptic site on XPO1, which becomes accessible only upon SINE conjugation. While molecular glue degraders typically interact with both CRL and the substrate8-10, SINEs bind to XPO1 without requiring interaction with ASB8 for efficient XPO1 degradation. Instead, an allosteric mechanism facilitates high affinity XPO1-ASB8 interaction, leading to XPO1 ubiquitination and degradation. ASB8-mediated degradation is also observed upon treatment of the endogenous itaconate derivate 4-octyl itaconate, which suggests a native mechanism that is inadvertently exploited by synthesized XPO1 inhibitors. This allosteric XPO1 degradation mechanism of SINE compounds expands the known modes of targeted protein degradation beyond the well-characterized molecular glue degraders and proteolysis targeting chimeras of CRL4.

cancer biology↗

bioRxiv2024

Phosphate-dependent nuclear export via a novel NES class recognized by exportin Msn5

Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the phosphate-sensing transcription factor Pho4, which requires phosphorylation for nuclear export by the yeast exportin Msn5. Unlike the traditional hydrophobic nuclear export signal (NES) utilized by the Exportin-1/XPO1 system, cryogenic-electron microscopy structures reveal that Pho4 presents a novel, phosphorylated 35-residue NES that interacts with the concave surface of Msn5 through two Pho4 phospho-serines that align with two Msn5 basic patches, unveiling a previously unknown mechanism of phosphate-specific recognition. Furthermore, the discovery that unliganded Msn5 is autoinhibited explains the positive cooperativity of Pho4/Ran-binding and proposes a mechanism for Pho4s release in the cytoplasm. These findings advance our understanding of the diversity of signals that drive nuclear export and how cargo phosphorylation is crucial in regulating nuclear transport and controlling cellular signaling pathways.

biophysics↗

bioRxiv2024

Structural basis for Retriever-SNX17 assembly and endosomal sorting

During endosomal recycling, Sorting Nexin 17 (SNX17) facilitates the transport of numerous membrane cargo proteins by tethering them to the Retriever complex. Despite its importance, the mechanisms underlying this interaction have remained elusive. Here, we report the structure of the Retriever-SNX17 complex determined using cryogenic electron microscopy (cryo-EM). Our structure reveals that the C-terminal tail of SNX17 engages with a highly conserved interface between the VPS35L and VPS26C subunits of Retriever. Through comprehensive biochemical, cellular, and proteomic analyses, we demonstrate that disrupting this interface impairs the Retriever-SNX17 interaction, subsequently affecting the recycling of SNX17-dependent cargos and altering the composition of the plasma membrane proteome. Intriguingly, we find that the SNX17-binding pocket on Retriever can be utilized by other ligands that share a consensus acidic C-terminal tail motif. By showing how SNX17 is linked to Retriever, our findings uncover a fundamental mechanism underlying endosomal trafficking of critical cargo proteins and reveal a mechanism by which Retriever can engage with other regulatory factors.

biochemistry↗

bioRxiv2023

Structural mechanism of co-import of H2A-H2B and its chaperone Nap1 by nuclear import receptor Kap114

Core histones are synthesized and processed in the cytoplasm before transport into the nucleus for assembly into nucleosomes; however, they must also be chaperoned as free histones are toxic. The importin Kap114 binds and transports histone H2A-H2B into the yeast nucleus, where RanGTP facilitates H2A-H2B release. Kap114 and H2A-H2B also bind the Nap1 histone chaperone, which is found in both the cytoplasm and the nucleus, but how Nap1 and Kap114 cooperate in H2A-H2B processing and nucleosome assembly has been unclear. To understand these mechanisms, we used biochemical and structural analyses to reveal how Nap1, Kap114, H2A-H2B and RanGTP interact. We show that Kap114, H2A-H2B and a Nap1 dimer (Nap12) assemble into a 1:1:1 ternary complex. Cryogenic electron microscopy revealed two distinct Kap114/Nap12/H2A-H2B structures: one of H2A-H2B sandwiched between Nap12 and Kap114, and another in which Nap12 bound to the Kap114*H2A-H2B complex without contacting H2A-H2B. Another Nap12*H2A-H2B*Kap114*RanGTP structure reveals the nuclear complex. Mutagenesis revealed shared critical interfaces in all three structures. Consistent with structural findings, DNA competition experiments demonstrated that Kap114 and Nap12 together chaperone H2A-H2B better than either protein alone. When RanGTP is present, Kap114s chaperoning activity diminishes. However, the presence of Nap12 within the Nap12*H2A-H2B*Kap114*RanGTP quaternary complex restores its ability to chaperone H2A-H2B. This complex effectively deposits H2A-H2B into nucleosomes. Together, these findings suggest that Kap114 and Nap12 provide a sheltered path from cytoplasm to nucleus, facilitating the transfer of H2A-H2B from Kap114 to Nap12, ultimately directing its specific deposition into nucleosomes. Significance StatementFree histones are toxic and must be sequestered by other macromolecules in the cell. Nuclear import receptor Kap114 imports H2A-H2B into the nucleus while also chaperoning it. The histone chaperone Nap1 also chaperones H2A-H2B, but it is unclear how Nap1 and Kap114 cooperate to process H2A-H2B. We present biochemical and structural results that explain how Kap114, Nap1 and H2A-H2B assemble in the absence and presence of RanGTP, how Nap1 and Kap114 co-chaperone H2A-H2B, and how RanGTP and Nap1 coordinate the transfer of H2A-H2B from Kap114 to assembling nucleosomes in the nucleus.

biophysics↗

bioRxiv2023

Molecular basis of RanGTP-activated nucleosome assembly with Histones H2A-H2B bound to Importin-9

Padavannil et al. 2019 show that Importin-9 (Imp9) transports Histones H2A-H2B from the cytoplasm to the nucleus using a non-canonical mechanism whereby binding of a GTP-bound Ran GTPase (RanGTP) fails to evict the H2A-H2B cargo. Instead, a stable complex forms, comprised of equimolar RanGTP, Imp9, and H2A-H2B. Unlike the binary Imp9*H2A-H2B complex, this RanGTP*Imp9*H2A-H2B ternary complex can release H2A-H2B to an assembling nucleosome. Here, we define the molecular basis for this RanGTP-activated nucleosome assembly by Imp9. We use hydrogen-deuterium exchange coupled with mass spectrometry and compare the dynamics and interfaces of the RanGTP*Imp9*H2A-H2B ternary complex to those in the Imp9*H2A-H2B or Imp9*RanGTP binary complexes. Our data are consistent with the Imp9*H2A-H2B structure by Padavannil et al. 2019 showing that Imp9 HEAT repeats 4-5 and 18-19 contact H2A-H2B, as well as many homologous importin*RanGTP structures showing that importin HEAT repeats 1 and 3, and the h8 loop, contact RanGTP. We show that Imp9 stabilizes H2A-H2B beyond the direct binding site, similar to other histone chaperones. Importantly, we reveal that binding of RanGTP releases H2A-H2B interaction at Imp9 HEAT repeats 4-5, but not 18-19. This exposes DNA- and histone-binding surfaces of H2A-H2B, thereby facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. This may ensure that H2A-H2B is only released in high RanGTP concentrations near chromatin. We delineate the molecular link between the nuclear import of H2A-H2B and its deposition into chromatin by Imp9. SignificanceImp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP alone is insufficient to release H2A-H2B. The resulting stable RanGTP*Imp9*H2A-H2B complex gains nucleosome assembly activity as H2A-H2B can be deposited onto an assembling nucleosome. We show that H2A-H2B is allosterically stabilized via interactions with both N- and C-terminal portions of Imp9, reinforcing its chaperone-like behavior. RanGTP binding causes H2A-H2B release from the N-terminal portion of Imp9 only. The newly-exposed H2A-H2B surfaces can interact with DNA or H3-H4 in nucleosome assembly. Imp9 thus plays a multi-faceted role in histone import, storage, and deposition regulated by RanGTP, controlling histone supply in the nucleus and to chromatin.

biophysics↗

bioRxiv2022

Mechanism of RanGTP priming the release of H2A-H2B from Kap114 and Importin-9

Previously we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9*H2A-H2B complex and formation of the ternary RanGTP*Importin-9*H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for nucleosome assembly. Here we show cryo-EM structures of Importin-9*RanGTP and of its yeast homolog Kap114, including Kap114*RanGTP, Kap114*H2A-H2B, and RanGTP*Kap114*H2A-H2B to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9*RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9*H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP*Kap114*H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome. Significance StatementHistones and their chaperone networks are typically conserved in eukaryotes. The yeast importin Kap114 and its human homolog Importin-9 share low sequence identity, but both are primary nuclear import receptors for the core histone heterodimer H2A-H2B. Cryo-EM structures of Kap114*H2A-H2B, Kap114*RanGTP and Importin-9*RanGTP complexes show homologous structure and function for Kap114 and Importin-9. In the nucleus, RanGTP binding to Kap114/Imp9*H2A-H2B does not release H2A-H2B, but RanGTP binds to form an atypical ternary complex. Structure of the ternary RanGTP*Kap114*H2A-H2B complex explains how the GTPase and cargo bind simultaneously to Kap114 and how the presence of Ran in the complex primes H2A-H2B transfer to assembling nucleosomes.

biochemistry↗

bioRxiv2022

Structure of Importin-4 bound to the H3-H4·ASF1 histone·histone chaperone complex

Importin-4 is the primary nuclear import receptor of core histones H3 and H4. Importin-4 binds the H3-H4 dimer and histone-chaperone ASF1 prior to nuclear import, but available structures of Importin-4{middle dot}histone tail complexes do not explain how Importin-4 recognizes the biologically relevant heterotrimeric H3-H4{middle dot}ASF1 cargo. Our 3.5 [A] Importin-4{middle dot}H3-H4{middle dot}ASF1 cryo-electron microscopy structure revealed interactions with H3-H4{middle dot}ASF1 different those suggested by previous Importin-H3 tail peptide structures. The N-terminal half of Importin-4 clamps the globular histone domain and the H3 N helix while its C-terminal half binds the H3 N-terminal tail weakly, with negligible tail contribution to binding energy; ASF1 binds H3-H4 without contacting Importin-4. Together, ASF1 and Importin-4 shield nucleosomal interfaces of H3-H4 to chaperone and import it into the nucleus, where Importin-4 undergoes large conformational changes as RanGTP binds to release H3-H4{middle dot}ASF1. This work explains the mechanisms of nuclear import of full-length H3-H4.

molecular biology↗

bioRxiv2019

Structural characterization of tau in fuzzy tau:tubulin complexes

Tau is a neuronal microtubule (MT) associated protein of significant interest due to its association with several neurodegenerative disorders. Taus intrinsic disorder and the dynamic nature of its interactions with tubulin and MTs make its structural characterization challenging. Here we use an environmentally sensitive fluorophore as a site-specific probe of tau bound to soluble tubulin. Comparison of our results with recently published tau:MT cryo-EM model reveals structural similarities between tubulin- and MT-bound tau. Analysis of residues across the repeat regions reveal a hierarchy in tubulin occupancy, which may be relevant to taus ability to differentiate between tubulin and MTs. As binding to soluble tubulin is a critical first step in MT polymerization, our characterization of the structural features of tau in dynamic, fuzzy tau:tubulin assemblies advances our understanding of how tau functions in the cell and how function may be disrupted in disease.

biophysics↗