Biology subjects

Janovjak, H.

Publications and source records attributed to Janovjak, H..

10 recordsLinked to original sources

bioRxiv2026

Intracellular targeting of a light-activated GPCR elicits spatially selective signalling

G protein-coupled receptors (GPCRs) initiate unique cell responses dependent on their sub-cellular location. Major advances in our understanding of sub-cellular GPCR signalling have been driven by experimental approaches that manipulate receptor trafficking, block signalling at specific intracellular sites, or compare receptor signalling in response to ligands with differing membrane permeability. Here, we describe an optogenetic platform that allows a single stimulus to activate the same GPCR at different intracellular membranes, enabling direct comparisons of spatial signalling in intact cells. To achieve this, we have directed a light-activated GPCR chimera to endosomal, mitochondrial, Golgi and nuclear membranes using C-terminal targeting sequences. We demonstrate activation of cAMP and ERK signalling in response to light at all sites; however, each targeted receptor generates a unique transcriptional fingerprint dependent on its sub-cellular location. Our engineered optogenetic platform reveals that GPCRs targeted to distinct intracellular locations are self-sufficient signalling platforms and highlights GPCR positioning as a critical driver of signalling. TeaserControlling GPCR signals by light and location reveals how intracellular positioning shapes cellular responses.

pharmacology and toxicology↗

bioRxiv2025

Gene library deep sequencing for protein super-family profiling

Unravelling protein function at scale remains challenging. Gain-of-function (GOF) screens harnessing large open reading frame (ORF) libraries enable systematic functional annotations of cell behavior drivers and drug targets. Currently, few sequencing methods are established to analyze existing ORF libraries and facilitate future library generation. Here, we developed a barcode-free deep sequencing method termed pooled plasmid library sequencing (PPLseq). PPLseq achieved high efficiency and single nucleotide accuracy, including the detection of previously undocumented variants, on a prototypical super-family ORF library (>300 human G-protein-coupled receptors (GPCRs)). We next combined PPLseq with high-throughput gene engineering to generate a new library of 246 GPCR-fluorescent protein (FP) fusions. We quantified expression of all members of this library and identified robustly expressed yet understudied receptors. Collectively, we demonstrate accessible, scalable, and sensitive ORF library sequencing towards a deeper understanding of proteome function.

synthetic biology↗

bioRxiv2023

Optimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors

Neuromodulatory signaling via G protein-coupled receptor (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. Recent efforts have led to the development of optogenetic tools to induce G protein-mediated signaling, with the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signaling in vivo remains challenging. Here we optimized the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). We validated these optoDopRs showing that they exhibit high signaling specificity and light sensitivity in vitro. In vivo we detected receptor and cell type-specific effects of dopaminergic signaling in various behaviors including the ability of optoDopRs to rescue loss of the endogenous receptors. This work demonstrates that OptoXRs can enable optical control of neuromodulatory receptor specific signaling in functional and behavioral studies.

neuroscience↗

bioRxiv2022

A F420-dependent single domain chemogenetic tool for protein de-dimerization

Protein-protein interactions (PPIs) mediate many fundamental cellular processes and their control through optically or chemically responsive protein domains has a profound impact on basic research and some clinical applications. Most available chemogenetic methods induce the association, i.e., dimerization or oligomerization, of target proteins, and the few available dissociation approaches either break large oligomeric protein clusters or heteromeric complexes. Here, we have exploited the controlled dissociation of a dimeric oxidoreductase from mycobacteria (MSMEG_2027) by its native cofactor, F420, which is not present in mammals, as a bioorthogonal monomerization switch. We found that in the absence of F420, MSMEG_2027 forms a unique domain-swapped dimer that occludes the cofactor binding site. Substantial remodelling of the intertwined N-terminal helix upon F420 binding results in the dissolution of the dimer. We then show that MSMEG_2027 can be expressed as fusion proteins in human cells and apply it as a tool to induce and release MAPK/ERK signalling downstream of a chimeric fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase. This F420-dependent chemogenetic de-dimerization tool is stoichiometric, based on a single domain and presents a novel mechanism to investigate protein complexes in situ.

synthetic biology↗

bioRxiv2022

CDK6 activity in a recurring convergent kinase network motif

More than 500 kinases phosphorylate [~]15% of all human proteins. The architecture of the phosphorylation network has been studied extensively, for instance to compile global interaction maps, trace signal transduction pathways or identify chemical intervention points. In contrast, systematic investigation of local motifs is rare but may provide a complementary understanding of network and kinase function. Here, we report on the occurrence, topology and experimental analysis of convergent kinase-substrate relationships (cKSRs) in which [≥]two kinases phosphorylate the same substrate. Through bioinformatics analysis we found that human cKSRs are frequent and involve >80% of all kinases and >24% of all substrates. We show that cKSRs occur over a wide range of stoichiometries harnessing converging kinases that are in many instances recruited from the same family subgroup and co-expressed. Experimentally, we demonstrate at the prototypical convergent CDK4/6 kinase pair how multiple inputs at the major tumor suppressor retinoblastoma protein (RB) can hamper in situ analysis of individual kinases. We hypothesized that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells that express high levels of CDK4, we confirmed this hypothesis and developed a high throughput compatible assay that sensitively quantifies genetically modified CDK6 variants and small molecule or protein inhibitors. Collectively, our work provides deeper insights into the phosphorylation network through the identification and analysis of kinase convergence.

cell biology↗

bioRxiv2021

Structure-guided optimization of light-activated chimeric G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of GPCR function is the specific coupling of activated receptors to selected downstream signaling pathways. The ability to tune this coupling would permit the development of receptors with new capabilities. GPCRs and G-proteins have been recently resolved structurally at high resolution, but this information was in only very few cases harnessed for a rational engineering of these protein complexes. Here, we demonstrate the structure-guided optimization of coupling in chimeric light-activated GPCRs (OptoXRs). Our hypothesis was that the incorporation of structural GPCR-G contacts will lead to improved receptor activity. We first evaluated structure-based alignments as complements to existing sequence-based methods for generation of chimeric receptors. We then show in a prototypical light-activated {beta}2AR that inclusion of -helical residues forming structural contacts to G resulted in receptors with 7- to 20-fold increased function compared to other design strategies. In turn, elimination of GPCR-G contacts diminished function. Finally, the efficient receptor design served as a platform for the optimization of a further light-activated receptor and spectral tuning of the photoreceptor core domain. Our work exemplifies how increased OptoXR potency and new functionalities can be achieved through structure-based design towards targeted inputs into cells and cellular networks.

molecular biology↗

bioRxiv2020

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease

Optogenetics has been harnessed to shed new mechanistic light on current therapies and to develop future treatment strategies. This has been to date achieved by the correction of electrical signals in neuronal cells and neural circuits that are affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and thereby may modify progression of degenerative disorders, has never been demonstrated in an animal disease model. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinsons disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-[kcy]B pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to overcome limitations of current strategies towards a spatio-temporal regulation of tissue repair. Significance StatementThe death of physiologically important cell populations underlies of a wide range of degenerative disorders, including Parkinsons disease (PD). Two major strategies to counter cell degeneration, soluble growth factor injection and growth factor gene therapy, can lead to the undesired activation of bystander cells and non-natural permanent signaling responses. Here, we employed optogenetics to deliver cell type-specific pro-survival signals in a genetic model of PD. In Drosophila and human cells exhibiting loss of the PINK1 kinase, akin to autosomal recessive PD, we efficiently suppressed disease phenotypes using a light-activated tyrosine kinase receptor. This work demonstrates a spatio-temporally precise strategy to interfere with degeneration and may open new avenues towards tissue repair in disease models.

neuroscience↗

bioRxiv2019

Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

Neuroligins (Nlgs) are adhesion proteins mediating trans-synaptic contacts in neurons. However, conflicting results around their role in synaptic differentiation arise from the various techniques used to manipulate Nlg expression. Orthogonally to these approaches, we triggered here the phosphorylation of endogenous Nlg1 in CA1 hippocampal neurons using a photoactivatable tyrosine kinase receptor (optoFGFR1). Light stimulation for 24 h selectively increased dendritic spine density and AMPA receptor-mediated EPSCs in wild-type neurons, but not in Nlg1 knock-out neurons or when endogenous Nlg1 was replaced by a non-phosphorylatable mutant (Y782F). Moreover, light stimulation of optoFGFR1 partially occluded LTP. Combined with computer simulations, our data support a model by which Nlg1 tyrosine phosphorylation promotes the assembly of an excitatory post-synaptic scaffold that captures surface AMPA receptors. This optogenetic strategy thus highlights the impact of Nlg1 intracellular signaling in synaptic differentiation and potentiation, while enabling an acute control of these mechanisms.\n\nImpact StatementOrthogonal to the traditional paradigms used to manipulate neuroligin expression level, the optogenetic trigger of tyrosine phosphorylation supports a strong role of endogenous neuroligin-1 in excitatory synaptic differentiation and potentiation.

neuroscience↗

bioRxiv2019

Engineering strategy and vector library for the rapid generation of modular light-controlled protein-protein interactions

Optogenetics enables the spatio-temporally precise control of cell and animal behaviour. Many optogenetic tools are driven by light-controlled protein-protein-interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPI to new target proteins is challenging because it is difficult to predict whether one the many available LSDs will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.

synthetic biology↗

bioRxiv2018

Astroglia withdraw from potentiated synapses boosting inter-synaptic cross-talk

Extrasynaptic actions of glutamate are limited by high-affinity transporters on perisynaptic astroglial processes (PAPs), which helps to maintain point-to-point transmission in excitatory circuits. Memory formation in the brain is associated with synaptic remodelling, but how this remodelling affects PAPs and therefore extrasynaptic glutamate actions is poorly understood. Here we used advanced imaging methods, in situ and in vivo, to find that a classical synaptic memory mechanism, long-term potentiation (LTP), triggers withdrawal of PAPs from potentiated synapses. Optical glutamate sensors combined with patch-clamp and 3D molecular localisation reveal that LTP induction thus prompts a spatial retreat of glial glutamate transporters, boosting glutamate spillover and NMDA receptor-mediated inter-synaptic cross-talk. The LTP-triggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca2+-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which synaptic potentiation could alter signal handling by multiple nearby connections.

neuroscience↗