Biology subjects

Houser, J.

Publications and source records attributed to Houser, J..

7 recordsLinked to original sources

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2026

A newly identified three-domain C-type lectin associated with blood feeding in the tick Ixodes ricinus

Ixodes ricinus ticks are widely distributed throughout Europe and represent major vectors of tick-borne encephalitis virus and the Lyme borreliosis agent Borrelia burgdorferi sensu lato. In invertebrates, C-type lectins are commonly associated with innate immune functions, and several such lectins have been predicted in I. ricinus. Given the limited knowledge of lectin function in ticks, we characterized three carbohydrate-recognition domains (CRDs) of a novel C-type lectin identified in the I. ricinus transcriptome (IrCLec). The tertiary structures of CRD1, CRD2, and CRD3, predicted using the AlphaFold 3 program, corresponded to the typical structure of C-type lectins. Conserved carbohydrate-binding motifs were identified in CRD3, whereas non-canonical motifs were present in CRD1 and CRD2. Recombinant His-tagged CRDs were produced and analysed for carbohydrate-binding activity. Glycan array analysis revealed binding of all three domains to selected glycans, while hemagglutination assays demonstrated pronounced binding activity of CRD1 and CRD2 toward human erythrocyte antigens of blood groups A, B, and O. IrCLec expression was highest in the tick midgut and also detected in hemocytes, with expression levels increasing after blood feeding. RNAi-mediated silencing of IrCLec impaired blood feeding efficiency in tick nymphs. Together, these results indicate that IrCLec plays an important role in blood feeding and may additionally participate in lectin-mediated host-pathogen or host-blood component interactions.

biochemistry↗

bioRxiv2025

Distinct Mechanisms of Recognition of Phosphorylated RNAPII C-Terminal Domain by BRCT Repeats of the BRCA1-BARD1 Complex: Insights from Structural and Functional Analyses

Transcription competes with other DNA-dependent processes, such as DNA repair, for access to its substrate, DNA. However, the principles governing the interplay between these processes remain poorly understood. Evidence suggests that the BRCA1-BARD1 complex, a key player in the DNA damage response, may act as a mediator of this crosstalk. In this study, we investigated the molecular mechanism underpinning the interaction between RNA polymerase II (RNAPII) and the BRCA1-BARD1 complex, as well as its functional implications. Our findings reveal that the BRCT repeat of BRCA1 binds the Ser5-phosphorylated CTD of RNAPII, utilising a mechanism previously established for other BRCT ligands. Furthermore, we demonstrate that this interaction is critical for the organisation of RNAPII into condensates with liquid-like properties. Analysis of disease-associated variants within the BRCT repeats further supports the biological relevance of this condensation. Collectively, our results suggest that the BRCA1-BARD1 complex may coordinate transcription and DNA repair by facilitating the organisation of RNAPII into transcription factories.

biochemistry↗

bioRxiv2024

Undruggable oncoproteins cMyc and NMyc bind to mediator of transcription with superior high affinity

The overexpression of MYC genes is frequently found in many human cancers including adult and pediatric malignant brain tumors. Targeting MYC genes continues to be challenging due to their undruggable nature. The nine-amino-acid activation domain (9aaTAD) has been identified using our prediction algorithm in all four Yamanaka factors including c-Myc and showed to activate transcription as short peptides. We generated a set of c-Myc constructs (1-108, 69-108 and 98-108) in the N-terminal regions and tested their ability to initiate transcription. We discovered strong interactions in nanomolar scale between the 9aaTAD of c-Myc and N-Myc proteins with the KIX domain of CBP coactivator. The c-Myc 9aaTAD (region 98-108) was not overlapping with the MBII (region 128-143) and therefore represents TRRAP independent activation region. Next, we showed the 9aaTADs in human c-Myc and N-Myc conservation within the MYC family. Interestingly, the loss of the 9aaTAD in L-Myc paralogs was identified in higher metazoans suggesting the deletions had occurred in early tetrapod evolution. In summary, as c-Myc is largely intrinsically disordered protein and therefore difficult to target by small molecule inhibitors, our finding of the c-Myc 9aaTAD in complex with the KIX domain represents a promising druggable target for development of new peptide inhibitors in MYC-driven tumors.

biochemistry↗

bioRxiv2022

The ins and outs of membrane bending by intrinsically disordered proteins

Membrane curvature is essential to diverse cellular functions. While classically attributed to structured domains, recent work illustrates that intrinsically disordered proteins are also potent drivers of membrane bending. Specifically, repulsive interactions among disordered domains drive convex bending, while attractive interactions, which lead to liquid-like condensates, drive concave bending. How might disordered domains that contain both repulsive and attractive domains impact curvature? Here we examine chimeras that combine attractive and repulsive interactions. When the attractive domain was closer to the membrane, its condensation amplified steric pressure among repulsive domains, leading to convex curvature. In contrast, when the repulsive domain was closer to the membrane, attractive interactions dominated, resulting in concave curvature. Further, a transition from convex to concave curvature occurred with increasing ionic strength, which reduced repulsion while enhancing condensation. In agreement with a simple mechanical model, these results illustrate a set of design rules for membrane bending by disordered proteins.

biophysics↗

bioRxiv2021

A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility

Chemical synapses between axons and dendrites mediate much of the brains intercellular communication. Here we describe a new kind of synapse - the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam - scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

cell biology↗

bioRxiv2021

AHK5 mediates ETR1-initiated multistep phosphorelay in Arabidopsis

Plants, like other sessile organisms, need to sense many different signals, and in response to them, modify their developmental programs to be able to survive in a highly changing environment. The multistep phosphorelay (MSP) in plants is a good candidate for a response mechanism that integrates multiple signal types both environmental and intrinsic in origin. Recently, ethylene was shown to control MSP activity via the histidine kinase (HK) activity of ETHYLENE RESPONSE 1 (ETR1)1,2, but the underlying molecular mechanism still remains unclear. Here we show that although ETR1 is an active HK, its receiver domain (ETR1RD) is structurally and functionally unable to accept the phosphate from the phosphorylated His in the ETR1 HK domain (ETR1HK) to initiate the phosphorelay to ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTERs (AHPs), the next link downstream members in MSP signaling. Instead, ETR1 interacts with another HK ARABIDOPSIS HISTIDINE KINASE 5 (AHK5) and transfers the phosphate from ETR1HK through the receiver domain of AHK5 (AHK5RD), and subsequently to AHP1, AHP2 and AHP3, independently of the HK activity of AHK5. We show that AHK5 is necessary for ethylene-initiated, but not cytokinin-initiated, MSP signaling in planta and that it thus mediates hormonal control of root growth.

plant biology↗