Biology subjects

Dreier, B.

Publications and source records attributed to Dreier, B..

5 recordsLinked to original sources

bioRxiv2023

Designed Ankyrin Repeat Proteins for detecting prostate-specific antigen expression in vivo

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with the hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide 68Ga. [68Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [68Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [68Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 {+/-} 0.58 %ID g-1 (n = 3; 2 h post-administration) and was reduced by [~]50% in the blocking group (2.47 {+/-} 0.42 %ID g-1; n = 3; P-value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

biochemistry↗

bioRxiv2022

Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by the Helicobacter pylori type IV secretion system

The bacterial human pathogen Helicobacter pylori produces a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. The cagT4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shaped N-terminal domains (CagIN) and prolonged by globular C-terminal domains (CagIC). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagIC with subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagIC were found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagIC plays a key role in cagT4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of the cagT4SS, a crucial risk factor for gastric cancer development. Author summaryHelicobacter pylori is a bacterial pathogen that colonises the human stomach in half of the worlds population. The most virulent strains use the cag- type IV secretion system (cagT4SS), a molecular nanomachine capable of injecting the oncoprotein CagA into gastric cells. How CagA is delivered is unknown, but the cagT4SS produces an external appendage referred to as pilus, which interacts with host cell receptors, mediating CagA translocation from the cytoplasm of the bacteria to the inner membrane of the host cell. In this study we have investigated the structural and functional properties of CagI, a protein long-thought to be associated with the cagT4SS pilus but with yet unknown function. We found that CagI displays a unique dimeric structure and that its C-terminal domain is involved in interaction with the host cell. Designed Ankyrin Repeat Proteins were selected against CagI and found to interact with its C-terminal moiety with high affinity. DARPin binding was able to prevent CagI interaction with the host cell and inhibited CagA translocation by H. pylori. Our study reveals the role of CagI in cagT4SS interaction with gastric cells and provides a first example of a small protein binder inhibiting the cagT4SS activity.

microbiology↗

bioRxiv2022

Nuclear myosin VI maintains replication fork stability

The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous (F-) actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase WRNIP1 in protecting these structures from DNA2- mediated nucleolytic attack. Using nuclear localization sequence (NLS) and ubiquitin E3-fusion DARPins to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.

cell biology↗

bioRxiv2022

A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

Neuroscience currently requires the use of antibodies to study synaptic proteins, where antibody binding is used as a correlate to define the presence, plasticity, and regulation of synapses. Gephyrin is an inhibitory synaptic scaffolding protein used to mark GABAergic and glycinergic postsynaptic sites. Despite the importance of gephyrin in modulating inhibitory transmission, its study is currently limited by the tractability of available reagents. Designed Ankyrin Repeat Proteins (DARPins) are a class of synthetic protein binder derived from diverse libraries by in vitro selection, and tested by high-throughput screening to produce specific binders. In order to generate a functionally diverse toolset for studying inhibitory synapses, we screened a DARPin library against gephyrin mutants representing both phosphorylated and dephosphorylated states. We validated the robust use of anti-gephyrin DARPin clones for morphological identification of gephyrin clusters in rodent neuron culture and brain tissue, discovering previously overlooked clusters. This DARPin-based toolset includes clones with heterogenous gephyrin binding modes that allowed for identification of the most extensive gephyrin interactome to date, and defined novel classes of putative interactors, creating a framework for understanding gephyrins non-synaptic functions. This study demonstrates anti-gephyrin DARPins as a versatile platform for studying inhibitory synapses in an unprecedented manner.

neuroscience↗

bioRxiv2021

Structural basis of adenylyl cyclase 9 activation

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPS, (iv) bound to Gs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation. One Sentence SummaryCryo-EM reveals activator-induced conformational changes in adenylyl cyclase AC9

biochemistry↗