Biology subjects

Pfuhl, M.

Publications and source records attributed to Pfuhl, M..

6 recordsLinked to original sources

bioRxiv2026

The generation and validation of a de novo-designed titin binder for use in fluorescence microscopy

Advances in the generation of proteins in silico has enabled the efficient design of such that can bind to a specified target. Here, we demonstrate the use of a fluorescently-labelled de novo-designed protein to bind its target in situ and be imaged using fluorescence microscopy, a widely used experimental technique that typically relies on antibodies or similar evolutionary derived binders to identify the presence and location of targets in their native environment. Our de novo-designed protein binds the C-terminal domain M10 (Ig-169) of the giant muscle protein titin, which spans half a sarcomere, the basic contractile unit of striated muscle. M10 antibodies suitable for fluorescence microscopy are unavailable. Confocal microscopy of muscle sections shows the binder localises to the M-band of the sarcomere - where M10 is found - and fails to label muscle in competition experiments and in mutant muscle where M10 is absent. These results demonstrate the utility of de novo-designed proteins in immunostaining-like experiments and suggest a future where targets can be routinely identified in complex biological samples by in silico-generated binders. Such an approach avoids the need to generate antibodies or similar binders either in vivo or in vitro, which can have technical, financial and ethical challenges.

bioengineering↗

bioRxiv2024

Structural characterization and inhibition of the interaction between ch-TOG and TACC3

The mitotic spindle is a bipolar array of microtubules, radiating from the poles which each contain a centrosome, embedded in pericentriolar material. Two proteins, ch-TOG and TACC3, have multiple functions at the mitotic spindle due to operating alone, together or in complex with other proteins. To distinguish these activities, we need new molecular tools to dissect their function. Here, we present the structure of the -helical bundle domain of ch-TOG that mediates its interaction with TACC3 and a structural model describing the interaction, supported by biophysical and biochemical data. We have isolated Affimer tools to precisely target the ch-TOG-binding site on TACC3 in live cells, which displace ch-TOG without affecting the spindle localization of other protein complex components. Inhibition of the TACC3-ch-TOG interaction led unexpectedly to fragmentation of the pericentriolar material in metaphase cells following the formation of a bipolar spindle and delayed mitotic progression; uncovering a novel role of TACC3-ch-TOG in maintaining pericentriolar material integrity during mitosis to ensure timely cell division.

cell biology↗

bioRxiv2022

Differential Effects of Mutations of Popeye Domain Containing Proteins on Heteromeric Interaction and Membrane Trafficking

BackgroundThe Popeye domain containing (POPDC) genes encode sarcolemma-localised cAMP effector proteins. Mutations in BVES (POPDC1) and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. MethodsBiopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein interaction. FindingsPatients carrying a novel homozygous variant, BVES (c.547G>T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. This is in contrast to variants such as BVES p.Q153X or POPDC2 p.W188X, which were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1-POPDC2 complex. InterpretationMutations in POPDC proteins that cause an impairment in membrane localisation affect POPDC complex formation while mutations which leave the protein interaction intact likely affect some other essential function of POPDC proteins. FundingThis study was funded by an EPSRC/British Heart Foundation co-funded Imperial Institute of Chemical Biology (ICB) Centre for Doctoral Training (CDT) PhD studentship (EP/S023518/1), a project grant of the British Heart Foundation (PG19/13/34247) and the Deutsche Forschungsgemeinschaft (DE1482/9-1). Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSSeveral biallelic missense and nonsense variants in BVES (POPDC1) have been described and are associated with heart and skeletal muscle disease. Skeletal muscle biopsies of homozygous carriers of these variants display a loss of sarcolemmal localisation of POPDC1 and POPDC2. Added value of this studyWe demonstrate that POPDC1 and POPDC2 form a heteromeric complex and that complex formation is required for plasma membrane trafficking of POPDC proteins. Transfection of different disease variants in HEK293 cells replicates their defective membrane targeting observed in biopsy material. Structural modelling and site-directed mutagenesis identifies an interface of strongly conserved hydrophobic residues in POPDC proteins, which likely mediate the interaction of POPDC proteins. Implications of all the available evidenceThese data provide novel insight into the membrane targeting requirements of POPDC proteins. We recommend testing the membrane targeting properties of any novel variant in POPDC isoforms using a newly developed co-transfection assay in HEK293 cells to characterise its pathogenicity. Our novel insight into the requirement of heterodimerization for proper membrane targeting may also offer novel opportunities to treat patients carrying mutations in POPDC proteins.

genetics↗

bioRxiv2022

Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro

Obscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in cells, in vitro characterization of these interactions using biophysical techniques has been hampered by the intrinsic instability of obscurin GEF domains. To study substrate specificity, mechanism and regulation of obscurin GEF function by individual domains, we successfully optimized recombinant production of obscurin GEF domains and found that MST-family kinases phosphorylate the obscurin DH domain at Thr5798. Despite extensive testing of multiple GEF domain fragments, we did not detect any nucleotide exchange activity in vitro against 9 representative small GTPases. Bioinformatic analyses show that obscurin differs from other Trio-subfamily GEFs in several important aspects. While further research is necessary to evaluate obscurin GEF activity in vivo, our results indicate that obscurin has atypical GEF domains that, if catalytically active at all, are subject to complex regulation.

biochemistry↗

bioRxiv2021

Development of a yeast-based vaccine for Theileria parva infection in cattle

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called "Infection and Treatment Method" (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain specific, MHC class I restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length native Tp2 and Tp9 as well as fragments of native Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNy responses using PBMCs from ITM-animals, while Tp2 and Tp9 induced IFNy responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induce Tp2 specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cows will further contribute to the development of a yeast-based vaccine for ECF.

immunology↗

bioRxiv2019

Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase

The PARP enzyme and scaffolding protein tankyrase (TNKS, TNKS2) uses its ankyrin repeat clusters (ARCs) to bind a wide range of proteins and thereby controls diverse cellular functions. A number of these are implicated in cancer-relevant processes, including Wnt/{beta}-catenin signaling and telomere maintenance. The ARCs recognise a conserved tankyrase-binding peptide motif (TBM). All currently available tankyrase inhibitors target the catalytic domain and inhibit tankyrases poly(ADP-ribosyl)ation function. However, there is emerging evidence that catalysis-independent \"scaffolding\" mechanisms contribute to tankyrase function. Here we report a fragment-based screening program against tankyrase ARC domains, using a combination of biophysical assays, including differential scanning fluorimetry (DSF) and nuclear magnetic resonance (NMR). We identify fragment molecules that will serve as starting points for the development of tankyrase substrate binding antagonists. Such compounds will enable probing the scaffolding functions of tankyrase, and may, in the future, provide potential alternative therapeutic approaches to inhibiting tankyrase activity in cancer and other conditions.

biophysics↗