Biology subjects

Arold, S. T.

Publications and source records attributed to Arold, S. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Host specificity in cereal rust fungi is mediated by a conserved glycoside hydrolase family

Non-host resistance refers to the immunity of plant species to virtually all isolates of a potential pathogen and represents an underexplored avenue for breeding and engineering disease resistance. In domesticated and wild barley, cell surface-localized lectin receptor kinases (LecRKs) contribute to determining the host status to leaf rust fungi, which pose a major threat to global cereal production. Here, we identify a conserved family of leaf rust glycoside hydrolases as ligands for these barley LecRKs and show that direct ligand-receptor binding triggers immune responses. This mechanism of pathogen perception is conserved across multiple cereal species and can be functionally transferred between them. We also uncover previously uncharacterized recognition specificities among distinct LecRK variants, expanding the repertoire of LecRK-mediated rust pathogen detection. Our findings define a molecular mechanism underlying non-host resistance in cereals and provide a basis for harnessing non-host rust resistance across diverse crop-pathogen systems.

plant biology↗

bioRxiv2026

In Situ Landscape of Focal Adhesions and Cytoskeletal Integration Revealed by Cryo-Electron Tomography

Focal adhesions (FAs) are dynamic hubs for mechanotransduction, linking the extracellular matrix to actin fibers, intermediate filaments, and microtubules. Using cryo-electron tomography combined with 3-dimensional segmentation and subtomogram averaging, we visualize in situ the structural architecture of the FA environment at the leading edge of human fibroblasts. Our analysis reveals a rich architectural diversity within the FA landscape, where the spatial organisation and interplay of FA protein clusters, actin, vimentin, and microtubules change from the actin bundle core to its tip and across adjacent regions. Notably, we reveal diverse arrangements and connections of vimentin filaments, supporting their multifaceted role in the control and mechanics of adhesions. Together, these findings establish a structural framework for FA maturation and cytoskeletal integration, extending classical lamellipodial adhesion models and providing mechanistic insight into how FAs coordinate force transmission during cell migration.

cell biology↗

bioRxiv2025

Molecular Basis and Cellular Effects of Janus-Class-Driven Cytoplasmic PYK2 Coacervates

Kinase activity is increasingly associated with biomolecular phase separation. Focal adhesion kinase (FAK) forms membrane-associated condensates with paxillin to promote adhesion. Here we show that its paralogue, proline-rich tyrosine kinase 2 (PYK2), undergoes phase separation via a distinct mechanism. PYK2 forms cytoplasmic condensates primarily driven by its kinase- FAT linker (KFL) region. Overexpression of PYK2 induces condensates enriched in its autophosphorylated form, which sequester paxillin from focal adhesions and impair cell adhesion. We uncover an autoregulatory mechanism involving the KFL, linking self-association, autophosphorylation, and condensation. Uncommon among known phase separation drivers, KFL condensation is phosphorylation-independent and its sequence belongs to the "Janus" class. Using a transformer-based protein language model, we identified non-homologous sequences with similar features, many from adhesion and cytoskeletal regulators. We validated the phase-separating potential of several of these sequences in cells. These findings reveal a novel mechanism linking phase separation with kinase activation, and demonstrate distinct condensation behavior in close homologues. Our results also highlight how protein concentration modulates condensate function, with implications for disease, and expand the landscape of phase separation drivers.

cell biology↗

bioRxiv2025

Conformation-specific Design: a New Benchmark and Algorithm with Application to Engineer a Constitutively Active MAP Kinase

AO_SCPLOWBSTRACTC_SCPLOWA general method for designing proteins with high conformational specificity is desirable for a variety of applications, including enzyme design and drug target redesign. To assess the ability of algorithms to design for conformational specificity, we introduce MotifDiv, a benchmark dataset of 200 conformational specificity design challenges. We also introduce CSDesign, an algorithm for designing proteins with high preference for a target conformation over an alternate conformation. On the MotifDiv benchmark, CSDesign designs protein sequences that are predicted to prefer the target conformation. We apply this method in vitro to redesign human MAP kinase ERK2, an enzyme with active and inactive conformations. Out of two designs for the active conformation, one increased activity sufficiently to retain activity in the absence of activating phosphorylations, a property not present in the wild type protein.

synthetic biology↗

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

Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

SummaryThrough the Astrocyte Neuron Lactate Shuttle, astrocyte-derived lactate fuels the high-energy demands of neurons and acts as a signaling molecule, promoting synaptic plasticity and memory consolidation. Lactate regulates neuronal excitability and modulates the expression of genes related to synaptic plasticity and neuroprotection, but the molecular mode for these signaling actions is uncertain. Using patch-clamp recordings in cultured cortical neurons, we found that lactate enhances both the amplitude and the inactivation time constant of NMDA receptor currents (INMDAR) evoked by brief applications of glutamate and glycine. Not reproduced by HCAR1 agonists, this modulation depends on monocarboxylate transporters and lactate dehydrogenase, indicating the requirement for lactate entry and metabolic conversion into pyruvate and NADH formation within neurons. Disruption of intracellular calcium dynamics or inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a NMDAR-associated kinase linking Ca2+ signal to long-term potentiation (LTP), significantly diminishes the effects of lactate on INMDAR. We identified two redox-sensitive cysteine- containing sequences in the intrinsically disordered intracellular C-terminal domain of the GluN2B subunit that play a role in the potentiation of NMDAR by lactate. In a compelling set of experiments using HEK cells, we observed that the presence of functional CaMKII and GluN2B-containing NMDARs is necessary for the lactate-enhancing effects. Mutations in GluN2B that prevent CaMKII binding or redox regulation via cysteines abrogate the modulatory action of lactate. Immunoprecipitation experiments in neurons attest that lactate increases the association between CaMKII and GluN2B. This interaction is crucial for the potentiation of INMDAR amplitude by lactate. Proximity ligation assays between GluN2B and the postsynaptic density marker PSD-95 revealed that lactate induced an accumulation of GluN2B in dendritic spines, an effect that was prevented by a CaMKII peptide inhibitor. These results highlight a mechanistic pathway whereby lactate boosts NMDAR function through intracellular metabolic conversion and redox-sensitive interactions requiring CaMKII, establishing a link between astrocyte metabolism and synaptic modulation in neurons.

neuroscience↗

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↗

bioRxiv2024

A wheat tandem kinase sensor activates an NLR helper to trigger immunity

Most plant resistance genes encode membrane-anchored receptor-like proteins or intracellular nucleotide-binding and leucine-rich repeat (NLR) receptors. In wheat and barley, tandem kinases (TKs) have emerged as a new class of resistance determinants. To understand the modus operandi of the wheat stem rust resistance protein Sr62TK, we identified two genetic interactors-- a host gene required for Sr62TK function and the corresponding fungal AvrSr62 effector. We discovered that the SR62 locus consists of a digenic module encoding Sr62TK and an NLR (Sr62NLR). AvrSr62 binds to the N-terminal kinase of Sr62TK. This triggers displacement of the C-terminal kinase allowing it to recruit Sr62NLR for activation of immune responses. Understanding the mechanism of this two-component resistance complex will help engineering and breeding for durable resistance.

plant biology↗

bioRxiv2024

DWARF14 and KARRIKIN INSENSITIVE2 mediate signaling of the apocarotenoid zaxinone in Arabidopsis

The natural growth regulator zaxinone increases the levels of the phytohormones strigolactone (SL) and abscisic acid in Arabidopsis (Arabidopsis thaliana) via unknown mechanisms. We demonstrate that parts of the effects of zaxinone in Arabidopsis depend on the SL receptor DWARF14 (AtD14), the karrikin receptor KARRIKIN INSENSITIVE2 (AtKAI2), and the F-Box protein MORE AXILLARY BRANCHING2 (AtMAX2) that mediates the signaling of SLs and karrikins. Binding assays and co-crystallization revealed zaxinone as an additional ligand of AtD14 and an SL antagonist that interrupts the interaction of AtD14 with AtMAX2. Zaxinone also bound to AtKAI2. These findings unveil a perception mechanism for zaxinone in Arabidopsis and demonstrate the capability of AtD14 and AtKAI2 to bind signaling molecules, other than strigolactones or karrikins, and mediate their transduction.

plant biology↗

bioRxiv2024

Functional selection in SH3-mediated activation of the PI3 kinase

The phosphoinositide-3 kinase (PI3K), a heterodimeric enzyme, plays a pivotal role in cellular metabolism and survival. Its deregulation is associated with major human diseases, particularly cancer. The p85 regulatory subunit of PI3K binds to the catalytic p110 subunit via its C-terminal domains, stabilising it in an inhibited state. Certain Src homology 3 (SH3) domains can activate p110 by binding to the proline-rich (PR) 1 motif located at the N-terminus of p85. However, the mechanism by which this N-terminal interaction activates the C-terminally bound p110 remains elusive. Moreover, the intrinsically poor ligand selectivity of SH3 domains raises the question of how they can control PI3K. Combining structural, biophysical, and functional methods, we demonstrate that the answers to both these unknown issues are linked: PI3K-activating SH3 domains engage in additional "tertiary" interactions with the C-terminal domains of p85, thereby relieving their inhibition of p110. SH3 domains lacking these tertiary interactions may still bind to p85 but cannot activate PI3K. Thus, p85 uses a functional selection mechanism that precludes nonspecific activation rather than nonspecific binding. This separation of binding and activation may provide a general mechanism for how biological activities can be controlled by promiscuous protein-protein interaction domains.

biochemistry↗

bioRxiv2024

Regulation of Arabidopsis polyamine acetylation by NATA1 and NATA2

Polyamines have vital functions in organisms, including bacteria, plants, and animals, with key roles in growth, development, and stress responses. Spermine/spermidine N1-acetyl transferases (SSATs) regulate polyamine abundance by catalysing their N-acetylation, thereby reducing the pool of polyamines and producing other bioactive components. The regulatory mechanisms controlling SSAT enzymes are incompletely understood. Here, we investigate the biological role and regulation of the two SSAT isoforms present in Arabidopsis thaliana, N-ACETYLTRANSFERASE ACTIVITY (NATA) 1 and 2. We show that NATA2 is a heat-stable isoform, induced in response to heat. Intriguingly, a nata2 knockout mutation proved beneficial for growth and pathogen defence under heat stress in Arabidopsis, aligning with the stress-mitigating effect of polyamines. In contrast, the double knockout of nata1 and nata2 was lethal, highlighting the essential role of basal SSAT activity. Our numerous crystal structures of both NATAs, supported by functional assays, revealed that stress-produced acidic metabolites can selectively inhibit polyamine acetylation by occupying the NATA substrate-binding pocket. This environment-responsive regulation mechanism may allow Arabidopsis to adjust the deleterious action of NATAs under stress conditions, without eliminating the enzyme. More generally, metabolite-ensemble inhibition may be a novel paradigm for non-genetic feedback regulation of plant enzymes.

plant biology↗

bioRxiv2024

AlphaCRV: A Pipeline for Identifying Accurate Binder Topologies in Mass-Modeling with AlphaFold

The speed and accuracy of deep learning-based structure prediction algorithms makes it now possible to perform in silico "pull-downs" to identify protein-protein interactions at a proteome-wide scale. However, existing scoring algorithms struggle to accurately identify correct interactions at such a large scale, resulting in an excessive number of false positives. Here, we introduce AlphaCRV, a Python package that helps identify correct interactors in a one-against-many AlphaFold screen by clustering, ranking, and visualizing conserved binding topologies, based on protein sequence and fold.

bioinformatics↗

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

DeepGO-SE: Protein function prediction as Approximate Semantic Entailment

The Gene Ontology (GO) is one of the most successful ontologies in the biological domain. GO is a formal theory with over 100,000 axioms that describe the molecular functions, biological processes, and cellular locations of proteins in three sub-ontologies. Many methods have been developed to automatically predict protein functions. However, only few of them use the background knowledge provided in the axioms of GO for knowledge-enhanced machine learning, or adjust and evaluate the model for the differences between the sub-ontologies. We have developed DeepGO-SE, a novel method which predicts GO functions from protein sequences using a pretrained large language model combined with a neuro-symbolic model that exploits GO axioms and performs protein function prediction as a form of approximate semantic entailment. We specifically evaluate DeepGO-SE on proteins that have no significant similarity with training proteins and demonstrate that DeepGO-SE can improve function prediction for those proteins.

bioinformatics↗

bioRxiv2022

Genetic architecture of behavioural resilience to ocean acidification

Genetic variation is essential for adaptation to rapid environmental changes. Identifying genetic variation associated with climate-change related phenotypes is therefore the necessary first step towards predictive models of genomic vulnerability. Here we used a whole-genome scan to identify candidate genetic variants associated with differences in behavioural resilience to ocean acidification in a coral reef fish. We identified three genomic regions that differ between individuals that are behaviourally tolerant compared with behaviourally sensitive to elevated CO2. These include a dopamine receptor (drd4rs), cadherin related family member 5-like (cdhr5l), Synapse-associated protein 1 (syap1), and GRB2 Associated Regulator of MAPK1 Subtype 2 (garem2), which have previously been found to modify behaviour related to boldness, novelty seeking, and learning in other species, and differ between behaviourally tolerant and sensitive individuals. Consequently, the identified genes are promising candidates in the search of the genetic underpinnings and adaptive potential of behavioural resilience to ocean acidification in fishes.

ecology↗

bioRxiv2022

The purified recombinant FAT domain of the Focal Adhesion Kinase does not bind directly to recombinant Talin or MBD2 in vitro

Controlled localization and activation of the focal adhesion kinase (FAK) functionally links adhesion, migration and survival of the cell. The C-terminal focal adhesion targeting (FAT) domain of FAK is an important regulator of the localization, activation and molecular associations of FAK. Here, we aimed to investigate the structural basis for how FAK FAT binds to Talin and MBD2, which were previously reported to be cytoplasmic and nuclear ligands, respectively. Using several biophysical methods with purified recombinantly expressed protein constructs, we failed to observe measurable interactions between FAT and either the Talin FERM domain or MBD2. We conclude that the association of FAT with these proteins requires additional factors or post-translational modifications not present in bacterially produced purified proteins.

biophysics↗

bioRxiv2021

Efficient multi-gene expression in cell-free droplet microreactors

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.

bioengineering↗

bioRxiv2020

Synergy and allostery in ligand binding by HIV-1 Nef

The Nef protein of human and simian immunodeficiency viruses (HIV and SIV, respectively) boosts viral pathogenicity through its interactions with host cell proteins. Nef has a folded core domain and large flexible regions, each carrying several protein interaction sites. By combining the polyvalency intrinsic to unstructured regions with the binding selectivity and strength of a 3D folded domain, Nef can bind to many different host cell proteins, perturbing their cellular functions. For example, the combination of a linear proline-rich motif and a hydrophobic core domain surface allows Nef to increase affinity and selectivity for particular Src family SH3 domains. Here we investigated whether the interplay between Nefs flexible regions and its core domain can allosterically influence ligand selection. We found that the flexible regions can bind back to the core domain in different ways, producing distinct conformational states that alter the SH3 domain selectivity and availability of Nefs functional motifs. The resulting cross-talk might help synergising certain subsets of ligands while excluding others, promoting functionally coherent Nef-bound protein ensembles. Further, we combined proteomic and bioinformatic analyses to identify human proteins that select SH3 domains in the same way as does Nef. We found that only 2-3% of clones from a whole human fetal library displayed a Nef-like SH3 selectivity. However, in most cases this selectivity appears to be achieved by a canonical linear interaction rather than a Nef-like tertiary interaction. This analysis suggests that Nefs SH3 recognition surface has no (or marginally few) cellular counterparts, validating the Nef tertiary binding surface as a promising unique drug target.

biophysics↗