Biology subjects

Dohnalek, J.

Publications and source records attributed to Dohnalek, J..

6 recordsLinked to original sources

bioRxiv2026

Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes.

Signalling via the epidermal growth factor receptor (EGFR) is indispensable for morphogenesis and tissue homeostasis. It is activated by extracellular ligands, typically released from transmembrane precursors by proteolysis. Ligand shedding activity is provided by the conserved rhomboid intramembrane serine proteases in Drosophila, but by the unrelated ADAM family metalloproteases in mammals, leaving the functions of mammalian non-mitochondrial rhomboids underexplored. Using quantitative proteomics, we show that EGFR is the main endogenous substrate of the human rhomboid protease RHBDL2 in keratinocytes. By shedding the EGFR ectodomain, thus producing a decoy receptor, RHBDL2 suppresses EGFR signalling, limiting cell migration and invasion. Conspicuously, RHBDL2 activity is upregulated by elevated intracellular calcium concentration, a condition typical for keratinocyte differentiation. These effects are recapitulated in primary human keratinocytes, and human skin equivalents deficient in RHBDL2 display incomplete differentiation and are morphologically disordered compared to wild type cells. We propose that context-specific fine-tuning of EGFR signalling and sensitivity to cross-talk from other signalling pathways could be important and hitherto overlooked roles of rhomboid proteases in mammals.

cell biology↗

bioRxiv2026

Rhomboid protease Rhbdl2 regulates macrophage recruitment and wound regeneration in zebrafish

Tissue regeneration requires tight control of immune cell behavior, yet the mechanisms that restrain immune-driven regenerative responses remain poorly defined. Here, we identify the rhomboid intramembrane serine protease Rhbdl2 as a critical regulator of regeneration in zebrafish. We generated rhbdl2 mutants by CRISPR Cas9 and found that it does not affect normal development, but triggers enhanced regenerative growth following injury, accompanied by increased macrophage accumulation at the wound site, which is accompanied by increased early apoptosis and proliferation. Proteomic analyses reveal increased Rac2 protein levels in rhbdl2 mutants, indicating dysregulated immune signaling. Functionally, Rac2 morpholino oligonucleotides mediated knockdown in rhbdl2 mutant larvae suppresses the elevated macrophage recruitment and enhanced tissue regenerative phenotype. Together, these findings uncover Rhbdl2 as an immune checkpoint that constrains macrophage driven enhanced regeneration, with vast implications for inflammatory disease, fibrosis, and tumor immune interactions.

immunology↗

bioRxiv2024

Light-dependent flavin redox and adduct states control the conformation and DNA binding activity of the transcription factor EL222

The activity of the transcription factor EL222 is regulated through protein-chromophore adduct formation, interdomain dynamics, oligomerization and protein-DNA interactions, all triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV/visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using non-canonical amino acids. The observation of only subtle atomic displacements between crystal structures of EL222 with and without blue-light back-illumination, was confirmed by NMR data indicating no major changes in secondary structure and fold compactness. Kinetic experiments in solution provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently-bound N5 protonated, and non-covalently-bound hydroquinone forms of FMN, respectively. Molecular modeling revealed differential dynamics and domain separation times arising from the three FMN states (oxidized, adduct, and reduced). Furthermore, while the dark state is largely monomeric, both lit states undergo slow monomer-dimer exchange. The photoinduced loss of -helicity, seen by infrared difference spectroscopy, was ascribed to dimeric EL222 species. Unexpectedly, NMR revealed that all three EL222 species (dark, lit1, lit2) can associate with DNA to some extent, but only under illumination a high population of stable complexes is obtained. Overall, we propose a refined model of EL222 photo-activation where photoinduced changes in the oxidation state of FMN and thioadduct formation shift the population equilibrium towards an open conformation that favors self-association and DNA-binding. Significance StatementFlavin-binding light-oxygen-voltage (LOV) proteins constitute a prominent example of highly evolved chromophore-containing proteins that convert light into biochemical changes in the cell. However, it is not well understood how blue-light orchestrates changes in LOV structure and function. Here we show that the dynamics, oligomerization and DNA-binding properties of the photocontrolled transcription factor EL222 are dependent on both the flavin redox state and thioadduct formation. In the dark, monomeric EL222 forms transient encounter complexes with DNA. Under illumination, two distinct lit states are sequentially generated, termed lit1 and lit2, that are both able to assemble into EL222:DNA (2:1) complexes. Our results reveal the coupling between flavin photochemistry (protonation and covalent linkage) and fold stability in EL222 and potentially other flavoproteins.

biochemistry↗

bioRxiv2021

Crystal structure of the human NKR-P1 bound to its lymphocyte ligand LLT1 reveals receptor clustering in the immune synapse

Human NKR-P1 (CD161, KLRB1) and its ligand LLT1 (CLEC2D) are a prototypical inhibitory C-type lectin-like receptor:ligand pair of NK cells with a critical role in homing lymphocytes to immune-privileged sites, particularly in multiple sclerosis, rheumatoid arthritis, and Crohns disease. Furthermore, NKR-P1:LLT1 inhibitory signaling is associated with glioblastoma, non-Hodgkins lymphoma, breast, and prostate cancer. However, the lack of structural data on the formation of the NKR-P1:LLT1 complex limits our understanding of this signaling. We thus solved the crystal structures of NKR-P1 and the NKR-P1:LLT1 complex. NKR-P1 forms a homodimer with an unexpected arrangement that enables LLT1 binding in two modes, bridging two LLT1 molecules, thereby forming interaction clusters suggestive of an inhibitory immune synapse. Moreover, observing the formation of these clusters by SEC-SAXS analysis in solution and by dSTORM super-resolution microscopy on the cell surface, and following their role in receptor signaling using in vitro cytotoxicity assay with freshly isolated NK cells, we show how NKR-P1:LLT1 clustering allows these proteins to overcome the weak affinity of C-type lectin-like receptors to their ligands. Furthermore, only the ligation of both primary and secondary LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. Therefore, our findings show how inhibitory receptor cross-linking and clustering work together to trigger signal transduction upon cellular contact in the immune synapse.

molecular biology↗

bioRxiv2020

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation {sigma} factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

molecular biology↗

bioRxiv2020

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

Electron crystallography of sub-micron sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realising 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix, surrounding the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, we experimentally verified the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB) can be used to remove excess host lipidic mesophase matrix, and then thin the crystals to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic mesophase of monoolein were used as a model system. LCP from a part of a 50-μm thick crystal, which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210-nm thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope (cryo-EM), and a nanovolume of ∼1400×1400×210 nm3 of the BR lamella was exposed to 200-kV electrons at a fluence of ∼0.06 e−/Å2. The resulting electron diffraction peaks were detected beyond 2.7-Å resolution (with mean signal-to-noise ratio <I/σ(I)> of >7) by a CMOS-based Ceta 16M camera. The results demonstrate, that cryo-FIB milling produces high quality lamellae from crystals grown in lipidic mesophases, and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media.Synopsis Electron diffraction experiments on crystals of membrane proteins grown in lipidic mesophases have not been possible due to a thick layer of viscous crystallisation medium around the crystals. Here we show that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can remove the viscous layer, and demonstrate high-quality electron diffraction on a FIB-milled lamella of a bacteriorhodopsin 3D crystal.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗