Petri Kursula

MNeonGreen (mNG) is among the brightest and most photostable monomeric green fluorescent proteins and is widely used for protein tagging. Here, we present sdAb(mNG), a high-affinity single-domain antibody (sdAb) that enables biochemical capture, imaging, and manipulation of mNG-tagged proteins. A 1.26 Å crystal structure reveals an extensive interaction surface between mNG and sdAb(mNG), accounting for its high affinity (K _D = 0.39 nM) and robust target recognition across diverse experimental conditions. This allows a single sdAb to support applications that typically require multiple specialized tools. We demonstrate the utility of sdAb(mNG) in several example applications including highly specific immunoprecipitation, direct immunofluorescence, and super-resolution imaging. Importantly, sdAb(mNG) retains high-performance target recognition even in intracellular environments. When expressed as an intrabody in living mammalian cells, sdAb(mNG) enables relocalization of mNG-tagged proteins to defined compartments or visualization of synaptic vesicle transport in primary neurons. In zebrafish, fusion of sdAb(mNG) to an F-box degradation domain induces cell-autonomous depletion of an endogenous mNG-tagged transcription factor and produces a clear developmental phenotype. These findings establish sdAb(mNG) as a versatile and robust affinity reagent that converts mNG from a passive fluorescent reporter into a multifunctional handle for imaging, proteomics, and programmable manipulation of endogenous and engineered proteins.

Myelin is classically viewed as a uniform axon-insulating membrane, yet its molecular composition may differ between species and even within one species. Fatty acid binding protein-8 (FABP8/PMP2) was previously identified in CNS myelin of humans but not mice. Here we show that FABP8/PMP2 is a defining feature of CNS myelin in humans and old-world-monkeys, but absent from CNS myelin in other mammals, indicating evolutionary neofunctionalization of this lipid-binding protein in the primate lineage. In the human CNS, FABP8/PMP2 marks a subset of myelin sheaths that preferentially ensheath large-diameter axons, revealing sheath-to-sheath molecular heterogeneity correlated with axonal geometry. Chromatin is accessible at the PMP2/Pmp2 gene locus in oligodendrocytes of humans but not mice. Human oligodendrocytes intrinsically express FABP8/PMP2 when transplanted into mouse brains, demonstrating species-specific competence independent of environmental cues. "Humanized" transgenic mice expressing FABP8/PMP2 in oligodendrocytes form morphologically normal but developmentally transiently thicker myelin sheaths, and show elevated cholesterol content in purified myelin. Because FABP8/PMP2 binds cholesterol, we propose that its emergence in primate CNS myelin contributes to the cholesterol enrichment of human myelin. Thus, CNS myelin protein composition is evolvable and modular, with relevance for myelin lipids and morphology, and previously unrecognized complexity in neuron-glia co-adaptation.

Pancreatic ductal adenocarcinomas (PDACs) are aggressive, stroma-rich tumours. They are unresponsive to treatments, and patients relapse quickly on DNA-damaging chemotherapies. PDAC stroma consists of extracellular matrix proteins (ECM), secreted by cancer-associated fibroblasts (CAFs). Here we show an unexpected link between CAF-secreted ECM proteins and enhanced DNA repair. We identify NDRG1 (N-myc downstream-regulated gene 1) as a key mediator that senses signals from the ECM via adhesion receptors and serum and glucocorticoid-activated kinase. We establish NDRG1 as a DNA repair factor that physically associates with replication forks, maintains DNA replication, resolves stalled forks caused by chemotherapies and is involved in reducing R-loops, RNA-DNA hybrids known to cause genomic instability. NDRG1 is highly expressed in PDAC tumours and its high expression correlates with poor disease-specific survival and poor response to chemotherapy. In conclusion, our data reveal an unexpected role for CAF-secreted ECM proteins in promoting DNA repair via NDRG1, mechanistically linking tumour stroma to replication fork homeostasis and R-loop regulation.

The peripheral myelin protein P2 (also known as PMP2/FABP8) has two basic functions in vivo: the transport of fatty acids within the cell to the target compartments and the adhesion of lipid bilayers in the peripheral nervous system myelin to each other. In this work, we focus on the membrane‐binding properties of P2 and its Charcot–Marie–Tooth disease variants. Experiments were carried out on lipid monolayers at the air–water interface as a membrane model system, with the lipid composition of the cytoplasmic leaflet of myelin. Our study provides quantitative data on the membrane affinity of P2 and its disease‐linked or structure‐based missense variants toward the native lipid membrane and shows affinity differences due to single P2 point mutations. Phospholipid monolayer surface pressure measurements are supported by epifluorescence microscopy, which not only shows the adhesive property of all P2 variants but also the complex cross‐linking property of the wild‐type P2. An analysis of transcriptomics databases confirms expression of P2 in human, but not mouse, central nervous system nonneuronal cells. Taken together, our work further confirms the role of P2 in binding myelin‐like lipid membranes as well as its direct effects on lipid membrane properties.

Amyotrophic lateral sclerosis (ALS) and Charcot-Marie-Tooth disease (CMT) are two distinct neurodegenerative disorders. While ALS is characterised by rapidly progressive motor neuron degeneration, leading to severe complications and death, CMT as a peripheral neuropathy is less severe, and patients have a longer life span, although with a compromised quality of life. Despite their clinical differences, current knowledge suggests that familial ALS (fALS) and CMT may share common genetic and molecular mechanisms. We aimed to identify shared genes mutations and molecular pathways between fALS and CMT through a literature and database search. Thirteen genes were identified, involved in distinct cellular processes: axonal transport ( DYNC1H1 , KIF5A , SPG11 , DCTN1 ), protein homeostasis ( NEFH , VCP , SOD1 ), RNA metabolism ( GARS , SETX ), cellular stress response ( HSPB1 , FIG4 ), and mitochondrial function ( MFN2 , CHCHD10 ). While these linkages to the two diseases are rare for each gene, understanding possible mechanistic commonalities at the molecular level can initiate new research directions, help in identifying additional common genes between neurodegenerative disorders, and improve diagnostics.

AXL is a receptor tyrosine kinase with a significant role in various biological processes and important medical implications, particularly in cancer. AXL transduces signals from the extracellular environment into the cytoplasm by binding to its ligand, growth arrest-specific protein 6 (GAS6). Activation of AXL leads to autophosphorylation of its intracellular domain and subsequent activation of downstream signaling pathways involved in cell proliferation, migration, differentiation, and survival. Tilvestamab (also known as BGB149) is a first-in-class, humanized, therapeutic anti-AXL function-blocking monoclonal antibody. We carried out a structural characterization of the AXL-tilvestamab complex using both negative-stain and cryogenic transmission electron microscopy as well as synchrotron small-angle X-ray scattering. While AXL-Fc was highly elongated and formed large heterogeneous complexes with the full antibody, homogeneous samples for structural studies could be made using the monomeric soluble AXL extracellular domain, the Fab fragment of tilvestamab, and an anti-Fab nanobody. Both SAXS and cryo-EM confirmed successful complex formation between the three proteins, and a low-resolution 3D model for the tilvestamab-AXL complex is presented. The data allow for sample optimization for high-resolution structural biology, as well as designing mutations that could alter binding affinity and specificity.

Targeting AXL receptor kinase with a highly selective antibody presents a promising approach for inhibiting AXL and potentially improving cancer treatment. An essential step in antibody optimisation is the mapping of paratope residues to epitope residues. In the present study, we identify the residues of tilvestamab, a function-blocking anti-AXL monoclonal antibody, that are essential for its binding to the extracellular domain of AXL. A single-chain variable fragment (scFv) fused to osmotically inducible protein Y (osmY) was designed to enable the secretion of soluble scFv-osmY mutants, which could be directly subjected to high-throughput biolayer interferometry screening for binding to the AXL Ig1 domain. Each complementarity-determining region residue of scFv was mutated to Ala, while additional mutations were made on the basis of predicted contribution to binding. We generated AlphaFold3 predictions for the scFv (tilvestamab)-AXL Ig1 complex to gain insights into the molecular interactions of the essential residues, as determined by the experimental data. Our study reveals that tilvestamab binds to the Ig1 domain of AXL, with twelve residues on scFv (tilvestamab) contributing most to binding, likely being situated at the binding interface. Glu2 near the N-terminus of AXL is essential for binding. The data give a structural view into the AXL-tilvestamab complex and allow for further optimisation of the binding interface.

Amyotrophic lateral sclerosis (ALS) and Charcot-Marie-Tooth disease (CMT) are distinct neurodegenerative disorders. While ALS is characterised by rapidly progressive motor neuron degeneration, leading to severe complications and death, CMT as a peripheral neuropathy is less severe, and patients have a longer life span, although with a compromised quality of life. Despite their clinical differences, current knowledge suggests that ALS and CMT may share common genetic and molecular mechanisms. We aimed to identify shared genetic mutations and molecular pathways between ALS and CMT. A literature and database search was conducted to identify genes implicated in both ALS and CMT. Thirteen genes were identified: axonal transport dysfunction (DYNC1H1, KIF5A, SPG11, and DCTN1), protein homeostasis disruption (NEFH, VCP, and SOD1), defects in RNA metabolism (GARS and SETX), cellular stress response disruption (HSPB1 and FIG4), and mitochondrial dysfunction (MFN2 and CHCHD10). Understanding these mechanistic commonalities at the molecular level can initiate new research directions, help in identifying additional common genes, improve diagnostics, and promote relevant therapeutic strategies.

The proper formation and function of the myelin sheath, a proteolipid membrane multilayer, relies on the coordinated action of several key myelin proteins. We studied how proteins from the peripheral myelin cytoplasmic apposition—myelin basic protein (MBP), the cytoplasmic tail of myelin protein zero (P0ct), and peripheral myelin protein 2 (P2)—interact with each other and with myelin‐like membranes using various techniques, such as small‐angle X‐ray diffraction, differential scanning calorimetry (DSC), surface plasmon resonance (SPR), and electron and live epifluorescence microscopy. DSC revealed changes in lipid interactions depending on the protein combination, with altered membrane fluidity and stability. These results were supported by SPR, which indicated that the myelin proteins may compete for membrane surface binding. Analysis of the Bragg peaks induced by the myelin proteins in lipidic environments showed both lamellar and nonlamellar phases in protein–lipid complexes, indicating the formation of nanoscale structures that may be relevant for myelin assembly. Microscopy experiments showed the formation of new membrane structures with each of the proteins separately and together. Our data indicate both synergy and competition between the three main proteins residing in the peripheral nervous system myelin major dense line. The observed direct effects of myelin proteins on lipid membrane structure and properties may be relevant to their function in myelinating cells as well as their role in myelin disorders.