Biology subjects

Toth, R.

Publications and source records attributed to Toth, R..

At least 19 recordsLinked to original sources

bioRxiv2026

The Arabidopsis outward K+ channel GORK mediates flg22-induced K+ efflux and stomatal closure

Transient K+ efflux occurs rapidly following pathogen-associated molecular pattern (PAMP) perception by pattern recognition receptors (PRRs) as part of pattern-triggered immunity (PTI). However, the molecular identities of the K+ channels responsible for PTI-associated K+ efflux and the mechanisms linking immune signaling to K+ channel regulation remain largely unknown. Here, we show that the channel GUARD CELL OUTWARD RECTIFYING K+ (GORK) is required for K+ efflux and stomatal closure induced by flg22, a model PAMP derived from bacterial flagellin. BOTRYTIS-INDUCED KINASE 1 (BIK1), a receptor-like cytoplasmic kinase associated with immune receptor complexes that functions as a central regulator of PTI, interacts with GORK and phosphorylates the channel on the conserved T199 residue in vitro. Electrophysiological analyses indicate that BIK1 inhibits GORK activity in a kinase-dependent manner and that T199 plays an essential role for GORK function. Together, this study identifies GORK as the key K+ channel executing rapid PTI-associated K+ efflux and associated stomatal closure, thus shedding light on the mechanisms of early immune signaling.

plant biology↗

bioRxiv2026

The multifunctional scaffold protein Small ovary couples piRNA-guided transposon recognition to nuclear RNA decay and heterochromatin formation

The piRNA pathway maintains genome integrity by silencing transposons cotranscriptionally in the nucleus through recognition of nascent transposon RNAs and recruitment of endogenous transcriptional and chromatin-level repressive mechanisms to transposon loci. However, the molecular link between the SFiNX complex, which recognizes nascent transposon RNA, and downstream effector complexes has remained elusive. Here, we demonstrate that the Small ovary (Sov) protein mediates this connection. By mapping the functional activities of its structural elements, we reveal that Sov contributes to transposon silencing through two distinct molecular mechanisms. First, Sov specifically directs nascent transposon transcripts toward nuclear RNA exosome-mediated degradation by physically interacting with the RNA decay factor TEsup1. Second, Sov directly binds the heterochromatin protein HP1a via multiple conserved motifs and undergoes phase separation, facilitating heterochromatin formation and genome-wide gene repression. Genetic analyzes of sov mutants reveal that these functions are separable: RNA-mediated transcriptional silencing is essential for piRNA pathway activity, while phase separation-dependent heterochromatin regulation is critical for stable transposon repression. We propose that Sov acts as a molecular scaffold in piRNA-guided transposon silencing, integrating transposon recognition with cotranscriptional RNA decay and chromatin-based regulatory pathways.

molecular biology↗

bioRxiv2026

Phosphorylation of TTL3 by BIK1 Functions as a Molecular Switch to Control Cellulose Biosynthesis under Salt Stress

Cellulose, a central structural component of plant cell walls, is produced by cellulose synthase complexes (CSCs) at the plasma membrane. Salinity stress is particularly damaging to cellulose biosynthesis, and therefore, plants have developed adaptive mechanisms to cope with these conditions. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are essential for growth under salt stress and show a salt-dependent association with CSCs through an as-yet unknown mechanism. Here, we identify a phosphorylation-dependent regulatory mechanism linking salt stress signaling to cellulose biosynthesis through the coordinated action of TTL3 and the receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1). Phosphorylation of Serine 93 in the N-terminal intrinsically disordered region of TTL3 controls its localization, retaining it in the cytosol, while dephosphorylation promotes association with CSCs at the plasma membrane. Biochemical and genetic analysis identified BIK1 as the kinase responsible for TTL3-S93 phosphorylation, with bik1 mutants phenocopying the phosphoablative TTL3S93A in vivo. Transcriptomic analyses reveal a strong overlap of differentially expressed genes between bik1 and a cellulose-deficient mutant, supporting a broader role for BIK1 in cell wall regulation. Notably, TTL proteins do not appear to be involved in the assayed canonical immune responses, suggesting pathway specificity downstream of BIK1. Together, these findings define a signaling module that connects salt stress perception to CSCs regulation and establish BIK1-dependent TTL3 phosphorylation as a molecular switch for maintaining cell wall integrity under abiotic stress.

Plant Biology↗

bioRxiv2026

NbRD21 protease controls receptor kinase homeostasis in Nicotiana benthamiana

RD21-like proteases are papain-like cysteine proteases with a C-terminal granulin domain that are abundant and ubiquitous in angiosperms and have often been implicated in immunity. We previously found that the activity of RD21 in Nicotiana benthamiana (NbRD21) is suppressed during infection with Pseudomonas syringae. Here, we studied the role of NbRD21 in immunity and proteome processing. NbRD21 was disrupted by genome editing and rd21 mutants were subjected to disease assays and shot-gun proteomics. Dipeptide substrate zLR-AMC was used in protease assays and agroinfiltration was used to transiently express NbRD21 and candidate substrates. Genome edited lines lacking NbRD21 develop normally but have drastically reduced zLRase activity and are significantly more susceptible to P. syringae. Shot gun proteomics revealed an increased accumulation of [~]20 diverse receptor-like kinases (RLKs) in untreated rd21 knockout lines, but their transcript levels are unaltered when compared to wild-type plants. 35S-driven GFP-tagged RLKs accumulate more upon transient expression in rd21 plants than in wild- type plants. These data indicate that NbRD21 post-translationally controls RLK homeostasis, either by directly degrading RLKs, or indirectly by regulating endocytic RLK recycling.

plant biology↗

bioRxiv2025

A clinical grade neurostimulation implant for hierarchical control of physiological activity

Bioelectronic implants for neurostimulation aim to steer disordered neurophysiological processes back towards a healthy state. However, physiology is subject to biological rhythms, including the circadian rhythm and the sleep-wake cycle. These predictable rhythms affect disease symptomatology, biomarkers used in closed-loop therapies, and a physiological systems expected response to stimulation. Therefore, therapeutic devices should incorporate feedforward elements to align algorithm parameters with predictable changes in physiological state, as a parallel of physiological rheostatic control. Here we introduce the DyNeuMo-2c, the first clinical-grade implant capable of delivering closed-loop neurostimulation while flexibly changing its functional configuration according to time of day. The device can chronically measure brain activity and motion state to track potential biomarker patterns in natural, out-of-clinic settings, allowing identification and targeting of patient-specific chronotypes. The system implements a hierarchical control flow, with baseline therapy set by a circadian scheduler, and adaptive policies layered to take effect based on specific biomarkers indicating patient and disease state. Using a benchtop validation setup, we demonstrate that the system has the required capabilities for delivering time-contingent closed-loop therapy in two established clinical use cases: Parkinsons disease and epilepsy. Next, we deploy the system in vivo to deliver closed-loop deep brain stimulation in a healthy non-human primate model of vigilance, highlighting the importance of synchronisation between device operation and physiological state in various conditions (task performance, unconstrained behaviour, and sleep). Time-of-day-dependent adaptation of closed-loop stimulation enabled modulation of both vigilance and behaviour. Overall, the novel device architecture provides a proof-of-concept for delivering time-contingent therapy in chronic therapeutic settings where biological rhythms are of key importance.

neuroscience↗

bioRxiv2025

Phosphorylation enables progressive microtubule-associated protein proteolysis and functionalisation during neural development

Neurological diseases are frequently characterised by dysregulation of the microtubule cytoskeleton, which is critical for neuronal integrity and functioning. However, the precise mechanisms by which microtubule dynamics are regulated during the development of the nervous system remain poorly understood. Here, we use global phosphoproteomic screening to identify cytoskeletal substrates of Ser-Arg Protein Kinase (SRPK), which is implicated in several neurodevelopmental and neurodegenerative conditions. We show that SRPK directly phosphorylates Microtubule Associated Protein (MAP)1S at multiple sites in a C-terminal region involved in proteolytic maturation and microtubule binding. SRPK-dependent MAP1S phosphorylation modulates the affinity of the MAP1S microtubule binding domain for microtubules and MAP1S proteolytic processing by the Calpain (CAPN)10 protease. Finally, we show that MAP1S proteolytic processing occurs progressively during neurodevelopment via a specific CAPN10 expression switch, corresponding with MAP1S acquisition of microtubule binding activity. Our results demonstrate a role for SRPK in coordinating processing and functionalisation of a key microtubule regulatory protein during neurodevelopment and provide insight into mechanisms by which the microtubule cytoskeleton may be dysregulated in neurological diseases.

biochemistry↗

bioRxiv2025

Integrative multi-omics combined with functional pharmacological profiling in patient-derived organoids identifies personalized therapeutic vulnerabilities of adult high-grade gliomas

BackgroundPrecision medicine has transformed cancer treatment by tailoring therapies to specific molecular aberrations. Integrating high-resolution multi-omics with high-throughput functional profiling in patient-derived organoids of-fers a powerful strategy to further refine patient stratification. While (epi)genetic profiling has drastically improved the classification in diffuse adult gliomas, these advances have not yet translated into effective therapeutic interventions and precision medicine approaches remain to be established. Material and MethodsWe investigated a panel of 48 patient-derived organoid and orthotopic xenograft models of adult high-grade gliomas, comprehensively characterized at genomic, epigenomic and transcriptomic levels. A functional drug screen was performed on 27 organoid models using a 202-compound library targeting cancer-related pathways and epigenetic regulators. Unsupervised multi-omics factor analysis was employed to identify patient-specific therapeutic vulnerabilities. Validation included dose-dependent drug efficacy assessments, as well as biomarker assessment in patient tumors across molecular subgroups. ResultsMulti-omics analysis revealed a broad spectrum of molecular profiles capturing the genetic, epigenetic, and transcriptomic diversity of high-grade gliomas. Multi-omics factor analysis, integrating multi-omics and drug response profiles, identified distinct subgroups associated with IDH1 mutation and MYCN amplification. IDH1 mutant grade 4 astrocytomas showed selective sensitivity to histone deacetylase 3 inhibitors, while a MYCN-amplified glioblastoma responded preferentially to histone methyltransferase inhibitors. The differential drug responses were linked to specific (epi)genetic and transcriptomic biomarkers. While other glioblastomas exhibited heterogeneous treatment responses, no robust biomarker-defined responder subgroups were identified. ConclusionOur findings highlight the value of integrating multi-omics and functional profiling to inform precision medicine strategies. This approach enables the stratification of distinct patient subgroups in preclinical models, paving the way for tailored therapeutic interventions. While we observed distinct pharmacogenomic profiles in IDH1 mutant grade 4 astrocytomas and a MYCN-amplified glioblastoma, implementing precision medicine in other glioblastoma subtypes remains a substantial challenge. Key pointsO_LIIntegrating drug screening in a panel of patient-derived organoids with multi-omics enables pharmacogenomic profiling in adult diffuse high-grade gliomas C_LIO_LIIDH1 mutant grade 4 astrocytomas are sensitive to histone deacetylase 3 inhibitors C_LIO_LIMYCN-amplified glioblastoma exhibits distinct DNA methylation pattern and drug responses C_LI Study importanceTo date, attempts to develop effective precision medicine in adult high-grade gliomas failed. Here, we provide a preclinical framework for identifying personalized therapeutic by integrating multi-omics profiling with functional drug screening in patient-derived organoids. We show that IDH1 mutant high-grade astrocytomas present distinct therapeutic vulnerabilities compared to glioblastomas, linked to sensitivity to histone deacetylase 3 inhibitors. Within glioblastomas, we identified a distinct MYCN-amplified tumor, sensitive to histone methyltransferase inhibitors. Applying pharmacogenomic approaches using novel drug libraries holds promise for uncovering additional clinically relevant patient subgroups in the future. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/675145v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@12c04f9org.highwire.dtl.DTLVardef@fa8872org.highwire.dtl.DTLVardef@141de6org.highwire.dtl.DTLVardef@b72086_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Modulation of motor cortical theta and gamma oscillations using phase-targeted, closed-loop optogenetic stimulation of local excitatory and inhibitory neurons

Theta and gamma oscillations are prominent features of cortical local field potentials (LFPs) and stimulation of the motor cortex at these frequencies can enhance motor learning. Phase-targeted closed-loop stimulation could provide a more precise and effective method to modulate these oscillations, particularly if stimulation parameters could harness the dynamics of the specific circuit mechanisms underpinning the generation of these activities. To address this question, we defined the response of theta-and gamma-frequency oscillations in the motor cortex to closed-loop optogenetic stimulation of excitatory pyramidal neurons and inhibitory interneurons transfected with Channelrhodopsin-2 in awake, head-fixed RBP4-Cre (retinol-binding-protein-4) and PV-Cre (parvalbumin) mice, respectively. Phase-targeted blue-light pulses were delivered using the OscillTrack algorithm to track theta phase in the cortical LFP in real time and trigger stimulation at one of four target theta phases. Stimulation was delivered over a quarter of the target theta cycle, either as a single continuous pulse ("continuous" protocol) or three short pulses at gamma (75Hz) frequency ("gamma" protocol). Stimulation of both neuron types, using either stimulation protocol, modulated theta power in a phase-dependent manner, with continuous stimulation of excitatory cells leading to stronger modulation. Phase-dependent amplification during stimulation of excitatory vs inhibitory neurons was offset by 90{degrees}, in line with predictions from computational models. Open-loop replay of previously recorded closed-loop stimulation patterns did not elicit the same phase-specific effects, demonstrating the necessity of the closed-loop interaction to produce these effects. Stimulation of pyramidal neurons using the gamma protocol amplified gamma power, independently of target theta phase. These findings reveal phase-dependent amplification of cortical theta power can be induced by stimulation of local excitatory or inhibitory neurons, with a phase-offset likely resulting from circuit interactions. This approach can be used to inform the development of brain stimulation methods to modulate these activities more effectively in humans.

neuroscience↗

bioRxiv2025

Early Candida - Oral Tumor Interactions Suggest miRNA-Mediated Regulation of Inflammatory and Tumor-associated processes

When simultaneously present, oral cancer and oral candidiasis are associated with high mortality. Recently, fungi-driven OSCC has emerged as a recognized phenomenon, that is triggered by Candida and sustained by several pro-tumor mechanisms. This study aimed to deepen our understanding and investigate how these mechanisms are regulated at the early stages of fungi-tumor interactions. Post-transcriptional regulation was examined through the analysis of miRNA (miR) responses in a metastatic cell line. We characterized early tumor responses to two common oral yeasts, C. albicans, frequently responsible for oral infections, and C. parapsilosis, a yeast with mostly commensal behavior in this niche. Exposed to C. albicans, tumor cells exhibited overexpression of inflammatory and pro-tumor pathways. This was also mirrored at the miR-level. Intriguingly, this species induced upregulation of oncogenic miRs, while simultaneously downregulated tumor-suppressive miRs. miR-target analyses revealed targets primarily to be involved in inflammation (TNFa signaling via NFKB), albeit tumor progression was also heavily influenced based on the targets identified within the PanCancer Progression Panel. Several miR-target pairs were identified with TRIB1-miR-30c-1-3p highlighted at the route of inflammation, and MMP3-miR-374a-3p and JUN-miR-1268b/miR-16-1-3p/miR-30c-1-3p at the route of OSCC progression. These findings highlight that C. albicans drives OSCC progression, in part, through miR regulation. Contrarily, C. parapsilosis elicited tumor responses predominantly related to hypoxia and metabolic regulation, followed by a mild activation of inflammatory and tumorigenic processes. No significant miR-regulatory responses were identified. To sum up, we added a new layer to fungi-tumor interactions and identified regulatory elements, that might hold future translational potential. ImportancePrevious studies have shown that yeast carriage in the oral cavity of oral cancer patients is significantly higher than those of healthy individuals. Additionally, the abundance of fungal cells is higher at malignant epithelial surfaces compared to the unaffected mucosal tissues. These observations indicated a direct interaction between mouth residing yeasts and oral tumorous cells. We have previously demonstrated that certain Candida species actively contribute to tumor progression in metastatic oral epithelial cells via various mechanisms including the promotion of migration, MMP activity or oncometabolite secretion. In this study, our findings demonstrate, that fungi-driven OSCC progression is also evident at the level of miRNA regulation, when C. albicans is present. In this context, the fungus might also enhance existing post-transcriptional regulatory mechanisms in the malignant oral epithelium. This behavior seems to be species-specific, as another common resident of the oral microflora, C. parapsilosis does not trigger such an effect.

microbiology↗

bioRxiv2025

HpBoRB, a helminth-derived CCP domain protein which binds RELMβ

Helminth infections persist by influencing host immunity through the release of immunomodulatory proteins which prevent immune ejection. The intestinal nematode Heligmosomoides polygyrus bakeri (Hpb) secretes multiple families of immunomodulatory proteins, many of which are composed of consecutive Complement Control Protein (CCP) domains. We hypothesized that further CCP domain proteins are secreted by the parasite to interact with the host. We identified an unusually large number of CCP domain-containing proteins in the genome of Hpb, and cloned a range of these for screening in an Avidity-based Extracellular Interaction Screening (AVEXIS) assay, focussing on interactions with host immune proteins. This screen confirmed the binding of known immunomodulators (HpBARI, TGM1) for their targets (ST2, TGFBR2) and identified a new interaction between a 2 CCP domain Hpb protein and mouse resistin-like molecule beta (RELM{beta}), a host protein demonstrated to have anti-helminth properties. This protein was named Binder of RELM{beta} (HpBoRB). This interaction was confirmed in ELISA, competition assays, size exclusion chromatography and surface plasmon resonance experiments, identifying a subnanomolar affinity interaction between HpBoRB and RELM{beta}. These data may indicate that Hpb interferes with the potent anti-helminth host protein RELM{beta} and adds to our knowledge of the host-parasite interactions mediated by Hpb secreted proteins.

immunology↗

bioRxiv2025

A Proteolytic Switch: USP5 controls SDE2 function via UBL-directed cleavage

Proteolytic processing is a critical regulatory mechanism in eukaryotic cells, yet the molecular identities and mechanisms underlying these events often remain elusive. Silencing Defective 2 (SDE2) is an essential human protein involved in multiple aspects of genome regulation, including DNA repair, ribosome biogenesis, and mRNA splicing. SDE2 is expressed with an N-terminal ubiquitin-like domain (SDE2UBL) which must be proteolytically cleaved to release the functional C-terminal domain (SDE2CT). This cleavage not only activates SDE2CT but also marks it for subsequent degradation, highlighting the importance of this tightly regulated processing. Despite the crucial role of this cleavage event, the human protease responsible has remained unknown. Here, we identify that the deubiquitinating enzyme, ubiquitin-specific protease 5 (USP5), catalyses the cleavage of SDE2. Using an integrated workflow combining biochemical assays, proteomic profiling, and mass spectrometry, we demonstrate that USP5 selectively processes SDE2 in vitro and in cell. To validate the specificity of this interaction, we engineered SDE2UBL into an activity-based probe, and developed a cellular reporter assay, both of which confirmed USP5 as the primary effector. Biophysical analysis further revealed that SDE2UBL binds to USP5 with similar characteristics to ubiquitin, albeit with reduced affinity, supporting a mechanism of substrate mimicry. Together, these findings uncover a novel regulatory axis for SDE2 function, highlighting an underappreciated role for DUBs in regulating protein maturation events. They also establish a versatile approach for identifying and validating substrate-protease interactions with broader implications for the study of post-translational regulation.

biochemistry↗

bioRxiv2025

Optimal inter-electrode distances for maximizing single unit yield per electrode in neural recordings

State-of-the-art high-density multielectrode arrays enable the recording of simultaneous spiking activity from hundreds of neurons. Although significant efforts have been dedicated to enhancing neural recording devices and developing more efficient sorting algorithms, there has been relatively less focus on the allocation of microelectrodes--a factor that undeniably affects spike sorting effectiveness and ultimately the total number of detected neurons. Here, we systematically examined the relationship between optimal electrode spacing and spike sorting efficiency by creating virtual sparser layouts from high-density recordings through spatial downsampling. We assessed spike sorting performance by comparing the quantity of well-isolated single units per electrode in sparse configurations across various brain regions (neocortex and thalamus) and species (rat, mouse, and human). Enabling the theoretical estimation of optimal electrode arrangements, we complement experimental results with a geometrical modeling framework. Contrary to the general assumption that higher electrode density inherently leads to more efficient sorting, both our theoretical and experimental results reveal a clear optimum for electrode spacing specific to species and regions. We demonstrate that carefully choosing optimal electrode distances could yield a total of 1.7 to 3 times increase in spike sorting efficiency. These findings emphasize the necessity of species- and region-specific microelectrode design optimization.

neuroscience↗

bioRxiv2024

Comprehensive multi-omics profiling of a healthy human cohort

Multi-omics approaches can offer powerful insights into personalized biomarker profiles relevant for disease diagnosis, prognosis, and therapeutics. However, separating meaningful biological variability from technical noise remains a major challenge. The EATRIS-Plus consortium analyzed blood samples from 127 healthy adults across six omics layers using twelve platforms, resulting in one of the most comprehensive multi-omics profiling datasets of healthy individuals available to date. We applied reproducible workflows to analyze and integrate these data, revealing several key findings. Sex significantly influenced all omics layers, emphasizing the importance of sex-balanced study designs. Age could be accurately predicted using epigenetic clocks, achieving high performance with our high-resolution enzymatic methylation sequencing data (R2 = 0.90), whereas candidate aging biomarkers were identified across all omics layers. The resulting dataset provides reference ranges in healthy individuals for abundance and variability of omics features, enabling robust power analyses, sample size estimations, and benchmarking of multi-omics integration methods. This resource can guide future biomarker discovery and personalized health research and was made FAIR-compliant and publicly available via the ClinData Portal (https://clindata.imtm.cz) and a Zenodo repository (https://doi.org/10.5281/zenodo.17514796).

bioinformatics↗

bioRxiv2024

Pipeline Olympics: continuable benchmarking of computational workflows for DNA methylation sequencing data against an experimental gold-standard

DNA methylation is a widely studied epigenetic mark and a powerful biomarker of cell type, age, environmental exposures, and disease. Whole-genome sequencing following selective conversion of unmethylated cytosines into thymines via bisulfite treatment or enzymatic methods remains the reference method for DNA methylation profiling genome-wide. While numerous software tools facilitate processing of DNA methylation sequencing reads, a comprehensive benchmarking study has been lacking thus far. In this study, we systematically compared complete computational workflows for processing DNA methylation sequencing data using a dedicated benchmarking dataset generated with five genome-wide profiling protocols. As an evaluation reference, we employed highly quantitative locus-specific measurements from our preceding benchmark of targeted DNA methylation assays. Based on this experimental gold-standard assessment and several comprehensive metrics, we identified workflows that consistently demonstrated superior performance and revealed major workflow development trends. To facilitate the sustainability of our benchmark, we implemented an interactive workflow execution and data presentation platform, adaptable to user-defined criteria and seamlessly expandable to future software.

bioinformatics↗

bioRxiv2024

Phospho-proteomics identifies D-group MAP kinases as substrates of the Arabidopsis tyrosine phosphatase RLPH2

Despite being one of the few bona fide plant tyrosine phosphatases, RLPH2 has no known substrates. Utilizing phospho-proteomics, we identified the activation loop phospho-tyrosine of several D-group mitogen activated protein kinases (MPKs) as potential RLPH2 substrates. All Arabidopsis D-Group MPKs possess a TDY activation loop phosphorylation motif, whereas other MPKs (Groups A, B and C) contain a TEY motif. Our findings reveal that RLPH2 has a strong preference for aspartate (D) in the TXY motif, providing specificity for RLPH2 to exclusively target and dephosphorylate the D-Group MPKs. Additionally, D-Group MPKs contain a unique activation loop insertion that conforms to a protein phosphatase 1 (PP1) binding motif, with findings presented here confirming Arabidopsis PP1 phosphatases dock at this site. Intriguingly, only D-group MPKs among all identified Arabidopsis protein kinases possess this PP1 recruiting motif. Using multiple RLPH2 deficient plant lines, we demonstrate that RLPH2 represses seed dormancy release. Overall, this work highlights the power of phospho-proteomics in identifying substrates of this novel plant tyrosine phosphatase, while also revealing new complexities in the interactions between MPK activation loops and multiple phospho-mediated cell signaling events. One sentence summaryPhospho-proteomic analysis reveals Arabidopsis tyrosine phosphatase RLPH2 dephosphorylates the activation loop of D-Group mitogen activated protein kinases.

plant biology↗

bioRxiv2024

A chemical-genetic system to rapidly inhibit the PP2A-B56 phosphatase

AO_SCPLOWBSTRACTC_SCPLOWSerine-threonine phosphatases have been challenging to study because of the lack of specific inhibitors. Their catalytic domains are druggable, but these are shared or very similar between individual phosphatase complexes, precluding their specific inhibition. Instead, phosphatase complexes achieve specificity by interacting with short-linear motifs (SLiMs) in substrates or their binding partners. We develop here a chemical-genetic system to rapidly inhibit these interactions within the PP2A-B56 family. Drug-inducible recruitment of ectopic SLiMs ("directSLiMs") is used to rapidly block the SLiM-binding pocket on the B56 regulatory subunit, thereby displacing endogenous interactors and inhibiting PP2A-B56 activity within seconds. We use this system to characterise PP2A-B56 substrates during mitosis and to identify a novel role for PP2A-B56 in maintaining kinetochore-microtubule attachments at metaphase. The directSLiMs approach can be used to inhibit any other phosphatase, enzyme or protein that uses a critical SLiM-binding interface, providing a powerful strategy to inhibit and characterise proteins once considered ."undruggable".

cell biology↗

bioRxiv2024

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

plant biology↗

bioRxiv2023

Mechanism of human PINK1 activation at the TOM complex in a reconstituted system

Loss of function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinsons disease (PD). Stabilisation of PINK1 at the Translocase of Outer Membrane (TOM) complex of damaged mitochondria is a critical step for its activation. To date the mechanism of how PINK1 is activated in the TOM complex is unclear. Herein we report co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit towards PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modelling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These molecular findings will aid in the development of small molecule activators of PINK1 as a therapeutic strategy for PD.

biochemistry↗