Biology subjects

Toth, R.

Publications and source records attributed to Toth, R..

51 records · Page 3Linked to original sources

bioRxiv2020

Epilepsy kinase CDKL5 is a DNA damage sensor which controls transcriptional activity at DNA breaks

Mutation of the CDKL5 kinase gene leads to the seizure-prone neurodevelopmental condition CDD (CDKL5 deficiency disorder) and is the most common genetic cause of childhood epilepsy. However, the phospho-targets and roles of CDKL5 are poorly understood, especially in the nucleus. We reveal CDKL5 as a sensor of DNA damage in actively transcribed regions of the nucleus, which phosphorylates transcriptional regulators such as Elongin A (ELOA) on a specific consensus motif. Recruitment of CDKL5 and ELOA to DNA damage sites, and subsequent ELOA phosphorylation, requires both active transcription and synthesis of poly-ADP ribose to which CDKL5 can bind. Critically, CDKL5 is essential for transcriptional control at DNA breaks. Therefore, CDKL5 is a DNA damage-sensing regulator of transcription, with implications for CDKL5-related human diseases. One sentence summaryCDKL5 is a DNA damage-sensing kinase that modulates transcriptional activity near DNA breaks.

cell biology↗

bioRxiv2020

Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro inhibition by nanobodies

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

biochemistry↗

bioRxiv2020

Structural basis of FANCD2 deubiquitination by USP1-UAF1

Ubiquitin-Specific Protease 1 (USP1), together with the cofactor UAF1, acts during DNA repair processes to specifically to remove mono-ubiquitin signals. The mono-ubiquitinated FANCI-FANCD2 heterodimer is one such substrate and is involved in the repair of DNA interstrand crosslinks via the Fanconi Anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin, and bound to mono-ubiquitinated FANCI-FANCD2 substrate. Crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1. A cryoEM reconstruction of USP1-UAF1 in complex mono-ubiquitinated FANCI-FANCD2, highlights a highly orchestrated deubiquitination process with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for their requirement despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.

biochemistry↗

bioRxiv2020

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Much attention has focused on LRRK2, as autosomal dominant missense mutations that enhance its kinase activity cause inherited Parkinsons disease. LRRK2 regulates biology by phosphorylating a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. In this study we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that the endogenous Rab7A protein, phosphorylated at Ser72 was most impacted by LRRK1 knock-out. This residue is not phosphorylated by LRRK2 but lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinsons mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous Type-2 tyrosine kinase inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. Finally, we show that interaction of Rab7A with its effector RILP is not affected by high stoichiometry LRRK1 phosphorylation. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology.

biochemistry↗

bioRxiv2020

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

Spreading depolarizations (SD) indicate infarct maturation and predict worse clinical outcome in ischemic stroke. We demonstrate here in rodents that brain edema formation upon ischemic stroke impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic glutamate accumulation predisposes an extensive bulk of tissue for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices under hypo-osmotic stress that SiD is the pathological expansion of prior SD foci, is associated with astrocyte swelling and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl- co-transporters, or volume-regulated anion channels mitigated slice edema, glutamate accumulation and SiD occurrence. Reversal of slice edema by hyperosmotic treatment counteracted glutamate accumulation and prevented SiD. In contrast, paralysis of astrocyte metabolism or inhibition of astrocyte glutamate uptake reproduced the SiD phenotype. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in ischemic stroke.

neuroscience↗

bioRxiv2020

DyNeuMo Mk-1: A Fully-Implantable, Motion-Adaptive Neurostimulator with Configurable Response Algorithms

There is growing interest in using adaptive neuro-modulation to provide a more personalized therapy experience that might improve patient outcomes. Current implant technology, however, can be limited in its adaptive algorithm capability. To enable exploration of adaptive algorithms with chronic implants, we designed and validated the Picostim DyNeuMo Mk-1 (DyNeuMo Mk-1 for short), a fully-implantable, adaptive research stimulator that titrates stimulation based on circadian rhythms (e.g. sleep, wake) and the patients movement state (e.g. posture, activity, shock, free-fall). The design leverages off-the-shelf consumer technology that provides inertial sensing with low-power, high reliability, and relatively modest cost. The DyNeuMo Mk-1 system was designed, manufactured and verified using ISO 13485 design controls, including ISO 14971 risk management techniques to ensure patient safety, while enabling novel algorithms. The system was validated for an intended use case in movement disorders under an emergency-device authorization from the Medicines and Healthcare Products Regulatory Agency (MHRA). The algorithm configurability and expanded stimulation parameter space allows for a number of applications to be explored in both central and peripheral applications. Intended applications include adaptive stimulation for movement disorders, synchronizing stimulation with circadian patterns, and reacting to transient inertial events such as posture changes, general activity, and walking. With appropriate design controls in place, first-in-human research trials are now being prepared to explore the utility of automated motion-adaptive algorithms.

bioengineering↗

bioRxiv2020

Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. Ser-Arg Repeat Protein Kinase (SRPK) is one such conserved eukaryotic kinase, which controls mRNA splicing. Surprisingly, we show that SRPK has acquired a novel function in regulating a neurodevelopmental ubiquitin signalling pathway. In mammalian embryonic stem cells, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of transcription factor substrates, thereby acting to restrain a neural gene expression programme that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signalling that ensures correct regulation of neurodevelopmental gene expression.

developmental biology↗

bioRxiv2020

Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL

BackgroundIn cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset and progression. The cancer epigenome is composed of the epigenetic patterns present in the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground-state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations. MethodsWe developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation. ResultsMethyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level. ConclusionsMethyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.

cancer biology↗

bioRxiv2019

Reference-free deconvolution of complex DNA methylation data - a detailed protocol

Epigenomic profiling enables unique insights into human development and diseases. Often the analysis of bulk samples remains the only feasible option for studying complex tissues and organs in large patient cohorts, masking the signatures of important cell populations in convoluted signals. DNA methylomes are highly cell type-specific, and enable recovery of hidden components using advanced computational methods without the need for reference profiles. We propose a three-stage protocol for reference-free deconvolution of DNA methylomes comprising: (i) data preprocessing, confounder adjustment and feature selection, (ii) deconvolution with multiple parameters, and (iii) guided biological inference and validation of deconvolution results. Our protocol simplifies the analysis and integration of DNA methylomes derived from complex samples, including tumors. Applying this protocol to lung cancer methylomes from TCGA revealed components linked to stromal cells, tumor-infiltrating immune cells, and associations with clinical parameters. The protocol takes less than four days to complete and requires basic R skills.

bioinformatics↗

bioRxiv2019

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

Loss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

biochemistry↗

bioRxiv2019

Phagocytic predation by the fungivorous amoeba Protostelium aurantium targets metal ion and redox homeostasis

Predatory interactions among microbes are considered to be a major evolutionary driving force for biodiversity and the defense against phagocytic killing. The fungivorous amoeba Protostelium aurantium has a wide fungal food spectrum but strongly discriminates among major pathogenic members of the Saccharomycotina. While C. albicans is not recognized, C. glabrata is rapidly internalized, but remains undigested. Phagocytic killing and feeding by P. aurantium is highly effective for the third major fungal pathogen, C. parapsilosis. Here we show that the different prey patterns of the three yeasts were reflected by distinct transcriptional responses, indicating fungal copper and redox homeostasis as primary targets during intracellular killing of C. parapsilosis. Gene deletions in this fungus for the highly expressed copper exporter Crp1 and the peroxiredoxin Prx1 confirmed their role in copper and redox homeostasis, respectively and identified methionine biosynthesis as a ROS sensitive metabolic target during predation. Both, intact Cu export and redox homeostasis contributed to the survival of C. parapsilosis not only when encountering P. aurantium, but also in the presence of human macrophages. As both genes were found to be widely conserved within the entire Candida clade, our results suggest that they could be part of a basic tool-kit to survive phagocytic attacks by environmental predators.

microbiology↗

bioRxiv2019

Hypoxia-Induced PIM Kinase and Laminin-Activated Integrin α6 Mediate Resistance to PI3K Inhibitors in Bone-Metastatic CRPC

Bone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin 6{beta}1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. In vitro and in vivo models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin 6{beta}1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.

cancer biology↗

bioRxiv2019

Random forest-based modelling to detect biomarkers for prostate cancer progression

The clinical course of prostate cancer (PCa) is highly variable, demanding an individualized approach to therapy and robust prognostic markers for treatment decisions. We present a random forest-based classification model to predict aggressive behaviour of PCa. DNA methylation changes between PCa cases with good or poor prognosis (discovery cohort with n=70) were used as input. The model was validated with data from two large independent PCa cohorts from the \"International Cancer Genome Consortium\" (ICGC) and \"The Cancer Genome Atlas\" (TCGA). Ranking of cancer progression-related DNA methylation changes allowed selection of candidate genes for additional validation by immunohistochemistry. We identified loss of ZIC2 protein expression, mediated by alterations in DNA methylation, as a promising novel prognostic biomarker for PCa in >12,000 tissue micro-array tumors. The prognostic value of ZIC2 proved to be independent from established clinico-pathological variables including Gleason grade, tumor stage, nodal stage and PSA. In summary, we have developed a PCa classification model, which either directly or via expression analyses of the identified top ranked candidate genes might help in decision making related to the treatment of prostate cancer patients.

cancer biology↗

bioRxiv2018

Specificity for deubiquitination of monoubiquitinated FANCD2 is driven by the N-terminus of USP1

The DNA damage response depends on ubiquitin signalling to orchestrate DNA repair. The Fanconi Anemia pathway for interstrand crosslink repair, and the translesion synthesis pathway for DNA damage tolerance, both require cycles of monoubiquitination and deubiquitination. The ubiquitin specific protease USP1 regulates both these pathways by deubiquitinating monoubiquitinated PCNA, FANCD2 and FANCI. Loss of USP1 activity gives rise to chromosomal instability. While many USPs hydrolyse ubiquitin-ubiquitin linkages, USP1 targets ubiquitin-substrate conjugates at specific sites. The molecular basis of USP1s specificity for multiple substrates is poorly understood. Here we show that the molecular determinants for substrate deubiquitination by USP1 reside within the highly conserved and extended N-terminus. We find that the N-terminus of USP1 harbours a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2. In contrast, the N-terminus is not required for PCNA or FANCI deubiquitination. Furthermore, we show that the N-terminus of USP1 is sufficient to engineer specificity in a more promiscuous USP.

biochemistry↗

bioRxiv2018

Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals.

In eukaryotes, DNA damage repair is implemented by a host of proteins that are coordinated by defined molecular signals. One such signal that transpires during the Fanconi Anemia (FA) - interstrand crosslink (ICL) repair pathway is the site-specific monoubiquitination of FANCD2 and FANCI proteins by a large, multi-protein FA core complex. The mechanics for this exquisitely specific monoubiquitin signal has been elusive. Here we show FANCL, the RING E3 module of the FA core complex, allosterically activates its cognate E2 Ube2T for monoubiquitination by a mechanism distinct from the typical RING-based catalysis. FANCL triggers intricate re-wiring of Ube2Ts intra-residue network thus activating the E2 for precision targeting. This network is intrinsically regulated by conserved gates and loops which can be engineered to yield Ube2T variants that enhance FANCD2 ubiquitination by ~30-fold without compromising on target specificity. Finally, we also uncover allosteric networks in other ubiquitin E2s that can be leveraged by RING E3 ligases to drive specific ubiquitination.

biochemistry↗