Biology subjects

Huntington, B.

Publications and source records attributed to Huntington, B..

5 recordsLinked to original sources

bioRxiv2025

Plant-specific adaptations of the CDC48 unfoldase

Targeted protein degradation through the CDC48 unfoldase enables the maintenance and rapid adaptation of proteomes across eukaryotes. However, the profound differences between animals, fungi, and plants are expected to have led to a significant adaptation of the CDC48-mediated degradation. While animal and fungal CDC48 systems have shown structural and functional preservation, such analysis is lacking for plants. We determined the structural and functional characteristics of Arabidopsis thaliana CDC48A in various states and bound to the target-identifying cofactors UFD1 and NPL4. Our analysis reveals several features that distinguish AtCDC48 from its animal and yeast counterparts, despite an 80% sequence identity. Key features are that AtCDC48A displays distinct domain dynamics and interacts differently with AtNPL4. Moreover, AtNPL4 and AtUFD1 do not form an obligate heterodimer, but independently bind to AtCDC48A and mediate target degradation; however, their joint action is synergistic. An evolutionary analysis supports that these Arabidopsis features are conserved across plants and represent the ancestral state of eukaryotic CDC48 systems. Jointly, our findings support that plant CDC48 retains a greater modular and combinatorial cofactor usage, highlighting a specific adaptation of targeted protein degradation in plants.

plant biology↗

bioRxiv2024

Mechanism of cooperative strigolactone perception by the MAX2 ubiquitin ligase-receptor-substrate complex

Strigolactones (SLs) are a group of plant hormones that regulate various aspects of plant growth and development. Additionally, SLs exuded into the soil promote symbiotic relationships with arbuscular mycorrhizal fungi and stimulate the germination of parasitic plants such as Striga hermonthica. The binding and hydrolysis of SLs by their receptors (D14 in Arabidopsis and HTL in Striga) promote the ubiquitination of transcriptional repressors by Skp1-cullin-F-box (SCF)-type E3 ubiquitin ligases. The mechanistic link between SL perception by D14/HTL and substrate recognition by the E3 remains unclear. We identified an E3-HTL-substrate complex that is sufficiently stable for cryogenic electron microscopy. This complex, composed of SKP1 (ASK1) and substrate (SMAX1) from Arabidopsis, and Striga F-box (MAX2) and SL receptor (HTL7), reveals that the substrate engages in a bidentate association through its N and D2 domains. This interaction, which is both conformationally and compositionally dynamic, directly and allosterically stabilises the MAX2-(SL)HTL7 complex and affects the positioning of ASK1 relative to MAX2. This dynamic positioning influences the proximity between the substrate D2 domain and the ubiquitin-conjugated E2 enzyme. This work advances our understanding of how E3 ligases in plants translate hormone perception into genetic adaptations.

biophysics↗

bioRxiv2023

P-NADs: PUX-based NAnobody Degraders for Ubiquitin-Independent Degradation of Target Proteins

Targeted protein degradation (TPD) allows cells to maintain a functional proteome and to rapidly adapt to changing conditions. Methods that repurpose TPD for the deactivation of specific proteins have demonstrated significant potential in therapeutic and research applications. Most of these methods are based on proteolysis targeting chimaera (PROTAC) which link the protein target to an E3 ubiquitin ligase binding moiety, resulting in the ubiquitin-based degradation of the target protein. In this study, we introduce a method for ubiquitin-independent TPD based on nanobody-conjugated plant ubiquitin regulatory X domain-containing (PUX) adaptor proteins. We show that the PUX-based NAnobody Degraders (P-NADs) can unfold a target protein through the Arabidopsis and human orthologues of the CDC48 unfoldase without the need for ubiquitination or initiating motifs. Despite originating from plants, P-NAD plasmids can be transfected into a human cell line, where produced proteins use the endogenous CDC48 machinery for ubiquitin-independent TPD. Thus, P-NADs pave the road for ubiquitin-independent therapeutic TPD approaches. The P-NAD design combined with established in vitro and cellular assays make this system also a versatile platform for elucidating functional aspects of CDC48-based TPD in plants and animals.

synthetic biology↗

bioRxiv2023

AlphaFold2 modeling and molecular dynamics simulations of an intrinsically disordered protein

We use AlphaFold2 (AF2) to model the monomer and dimer structures of an intrinsically disordered protein (IDP), Nvjp-1, assisted by molecular dynamics (MD) simulations. We observe relatively rigid dimeric structures of Nvjp-1 when compared with the monomer structures. We suggest that protein conformations from multiple AF2 models and those from MD trajectories exhibit a coherent trend: the conformations of an IDP are deviated from each other and the conformations of a well-folded protein are consistent with each other. We use a residue-residue interaction network (RIN) derived from the contact map which show that the residue-residue interactions in Nvjp-1 are mainly transient; however, those in a well-folded protein are mainly persistent. Despite the variation in 3D shapes, we show that the AF2 models of both disordered and ordered proteins exhibit highly consistent profiles of the pLDDT (predicted local distance difference test) scores. These results indicate a potential protocol to justify the IDPs based on multiple AF2 models and MD simulations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/551023v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@65c7f1org.highwire.dtl.DTLVardef@29cb87org.highwire.dtl.DTLVardef@1f126d2org.highwire.dtl.DTLVardef@12bb76d_HPS_FORMAT_FIGEXP M_FIG The TOC figure: C_FIG

biophysics↗

bioRxiv2018

Systematic evaluation of isoform function in literature reports of alternative splicing

Although most mammalian genes have multiple isoforms, an ongoing debate is whether these isoforms are all functional as well as the extent to which they increase the genomes functional repertoire. To ground this debate in data, we established a curation framework for evaluating experimental evidence of functionally distinct splice isoforms (FDSIs) and analyzed splice isoform function for over 700 human and mouse genes. Despite our bias towards prominently studied genes, we found experimental evidence meeting the classical definition for functionally distinct isoforms for only ~5% of the curated genes. If we relax our criteria, the fraction of genes with support for FDSIs remains low (~13%). We provide evidence that this picture will not change substantially with further curation. Furthermore, many FDSIs did not trace to a specific isoform in Ensembl. Our work has implications for computational analyses of alternative splicing and should help shape research around the role of splicing on gene function from presuming large general effects to acknowledging the need for stronger experimental evidence.

molecular biology↗