Biology subjects

Furkert, D.

Publications and source records attributed to Furkert, D..

3 recordsLinked to original sources

bioRxiv2024

NUDIX Hydrolases Target Specific Inositol Pyrophosphates and Regulate Phosphate and Iron Homeostasis, and the Expression of Defense Genes in Arabidopsis

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher-order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange (SAX)-HPLC, PAGE, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer-selective and Mg2+-dependent. Specifically, subclade I NUDTs preferentially hydrolyze 4-InsP7, while subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher-order mutants of subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wildtype (WT) and subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we found that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

plant biology↗

bioRxiv2024

Proteome-wide quantification of inositol pyrophosphate-protein interactions

Inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs) constitute a group of highly phosphorylated molecules that are involved in many cellular signaling processes. To characterize discrete signaling events of these structurally closely related molecules, a mass spectrometry approach was developed to derive apparent binding constants for these ligands on a proteome-wide scale. The method employed a series of chemically synthesized, biotinylated affinity reagents for inositol hexakisphosphate (InsP6), and the inositol pyrophosphates 1PP-InsP5, 5PP-InsP5 and 1,5(PP)2-InsP4 (also termed InsP8). Application of these affinity reagents at different concentrations, in combination with tandem mass tag (TMT) labeling, provided binding data for thousands of proteins from a mammalian cell lysate. Investigation of different enrichment conditions, where Mg2+ ions were either available or not, showcased a strong influence of Mg2+ on the protein binding capacities of PP-InsPs. Gene ontology analysis closely linked PP-InsP-interacting proteins to RNA processing in the nucleus and nucleolus. Subsequent data analysis enabled a targeted search for protein pyrophosphorylation among PP-InsP interactors, and identified four new pyrophosphorylated proteins. The data presented here constitute a valuable resource for the community, and application of the method reported here to other biological contexts will enable the exploration of PP-InsP dependent signaling pathways across species.

biochemistry↗

bioRxiv2024

Structural basis of dual BACH1 regulation by SCFFBXO22 and SCFFBXL17

BTB and CNC homolog 1 (BACH1) is a master transcriptional regulator of the cellular oxidative stress response and pro-metastatic oncogene. Post-translational stability of BACH1 is tightly regulated by distinct F-box ubiquitin ligases, including SCFFBXO22 and SCFFBXL17. However, the molecular details have been elusive. Here, we reveal a structural switch in FBXO22 that controls the recognition of a three-dimensional degron in the BACH1 BTB domain, thus explaining its specificity for dimeric BACH1. We describe how cancer-associated mutations in FBXO22 modulate binding and ubiquitylation of BACH1. Further, we reveal that cancer-related mutations or cysteine-modifications destabilize the BTB domain and redirect BACH1 to FBXL17, where it is recognized as a monomer. This explains how complementary ligases post-translationally regulate BACH1 depending on the state of its BTB domain. Our findings provide mechanistic insights into the regulation of the oxidative stress response and may spur therapeutic strategies to targeting oxidative stress-related disorders and metastatic cancers.

biophysics↗