Biology subjects

Thalhammer, A.

Publications and source records attributed to Thalhammer, A..

4 recordsLinked to original sources

bioRxiv2025

Structural Acrobatics of IAA7: Exercising Fuzzy Regions to Aid Auxin-Mediated Catalysis

Auxin is the paragon of small molecules driving recognition of proteins for their ubiquitylation and degradation, enabling rapid decoding of external signals into transcriptional responses. Auxin functions as a molecular glue, enhancing interactions between the TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and other TIR1-like proteins with various AUXIN INDUCIBLE/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors. A Cullin RING E3 ubiquitin ligase, SCFTIR1/AFBs, mediates the ubiquitylation and degradation of AUX/IAA proteins by positioning them for ubiquitin transfer, leading to their rapid degradation by the 26S proteasome. AUX/IAAs contain a Phox Bem 1 (PB1) domain, a degron, and intrinsically disordered regions (IDRs) that influence auxin sensing and their polyubiquitylation by interacting with TIR1. However, the precise role of IDRs in AUX/IAA positioning for ubiquitylation, and the mechanism of multisite ubiquitylation within AUX/IAAs remain unclear. We used biophysical techniques and coarse-grained simulations (CGS) to study the structural behavior of IDRs in IAA7, a model AUX/IAA protein from Arabidopsis thaliana. Our findings show that IAA7 expands by approximately 2.6 nm upon binding to TIR1 in the presence of auxin, likely increasing the proximity between a lysine acceptor in IAA7 and the active site of the SCFTIR1 E3 ligase for ubiquitylation. Our data also suggest that IAA7 remains conformationally heterogeneous when bound to TIR1, forming numerous transient contacts along its IDRs and C-terminal PB1 with TIR1. We propose that these transient interactions outside the IAA7 degron interface enhance the TIR1{middle dot}IAA7 association, resulting in highly dynamic and heterogeneous complexes that facilitate IAA7 multisite ubiquitylation, targeting it for rapid proteasomal degradation. Significance StatementIn this study, we use biophysical techniques to elucidate the implications of AUX/IAA intrinsic disorder on their conformational ensemble and processing by the E3 ubiquitin ligase, SCFTIR1. Our findings demonstrate that IAA7 undergoes a structural expansion upon binding to TIR1 and remains highly dynamic, facilitating multisite ubiquitylation. These insights enhance our understanding of how ubiquitylation targets engage with their E3 receptor modules and underscore the structural advantages of intrinsic disorder in protein ubiquitylation targets.

biophysics↗

bioRxiv2022

The K+ exchange antiporter 3 senses the chloroplast energy status to synchronize photosynthesis

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed regulatory C-terminus (RCT) of the thylakoid K+-exchange antiporter KEA3. RCT-mediated control of KEA3 contributes to the balance between light capture and photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the RCT senses the energy state of the chloroplast in form of both, phosphorylation and redox potential, in a pH-dependent manner and regulates KEA3 activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via KEA3.

plant biology↗

bioRxiv2021

Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism

Plant homeodomain leucine-zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. The START domain is required for TF activity; however, its presumed role as a lipid sensor is not well understood. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative family member which controls epidermal differentiation, recruits lysophosphatidylcholines in a START-dependent manner. In vitro assays with recombinant protein verified that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally uncovered that PDF2 controls the expression of phospholipid-related target genes by binding to a palindromic octamer with consensus to a phosphate (Pi) response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments further revealed a role for START in maintaining protein levels, and Pi limitation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Overall our data provide insights towards understanding how the lipid metabolome integrates Pi availability with gene expression.

plant biology↗

bioRxiv2019

Correction of β3 integrin haplo-insufficiency by CRISPRa normalizes cortical network activity

The extracellular matrix (ECM) plays an essential role in regulating the function of neuronal networks. In many cell types, the ECM exerts its effects through the transmembrane receptors integrins. Here, we investigate whether neuronal integrins regulate network excitability. Specifically, we focus on {beta}3 integrin, which has been associated to autism spectrum disorder and whose expression in neurons is regulated by activity. We have designed CRISPRa tools to titrate {beta}3 integrin expression in neurons. By using multi-electrode arrays and Ca2+imaging, we show that {beta}3 integrin boosts network excitability and synchrony in primary cortical neurons. Crucially, CRISPRa could compensate precisely for {beta}3 integrin haplo-insufficiency, thereby rebalancing level and correlation of neuronal activity. By contrast, rescue strategies based on exogenous gene expression resulted in hyper-active networks firing in unison. Thus, regulation of {beta}3 integrin by CRISPRa provides a precise and efficient system for modulating neuronal network function.

neuroscience↗