Biology subjects

Benisch, R.

Publications and source records attributed to Benisch, R..

3 recordsLinked to original sources

bioRxiv2024

Structural and biochemical characterization of an encapsulin-associated rhodanesefrom Acinetobacter baumannii

Rhodanese-like domains (RLDs) represent a widespread protein family canonically involved in sulfur transfer reactions between diverse donor and acceptor molecules. RLDs mediate these transsulfuration reactions via a transient persulfide intermediate, created by modifying a conserved cysteine residue in their active sites. RLDs are involved in various aspects of sulfur metabolism, including sulfide oxidation in mitochondria, iron-sulfur cluster biogenesis, and thio-cofactor biosynthesis. However, due to the inherent complexity of sulfur metabolism caused by the intrinsically high nucleophilicity and redox sensitivity of thiol-containing compounds, the physiological functions of many RLDs remain to be explored. Here, we focus on a single domain Acinetobacter baumannii RLD (Ab-RLD) associated with a desulfurase encapsulin which is able to store substantial amounts of sulfur inside its protein shell. We determine the 1.6 [A] x-ray crystal structure of Ab-RLD, highlighting a homodimeric structure with a number of unusual features. We show through kinetic analysis that Ab-RLD exhibits thiosulfate sulfurtransferase activity with both cyanide and glutathione acceptors. Using native mass spectrometry and in vitro assays, we provide evidence that Ab-RLD can stably carry a persulfide and thiosulfate modification and may employ a ternary catalytic mechanism. Our results will inform future studies aimed at investigating the functional link between Ab-RLD and the desulfurase encapsulin.

biochemistry↗

bioRxiv2023

Targeted protein degradation systems to enhance Wnt signaling

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETSTM) to drive tissue-specific degradation of ZNRF3/RNF43 E3-ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1 and ASGR1/2 specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate binding site. Both antibodies enhanced Wnt-activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.

synthetic biology↗

bioRxiv2023

A widespread proteinaceous sulfur storage compartment in bacteria

Intracellular compartmentalization is essential for all cells and enables the regulation and optimization of metabolism1. One of the main functions of subcellular compartments is the storage of nutrients2-4. As bacteria do generally not possess membrane-bound organelles, they often have to rely on functionally analogous protein-based compartments2,5-7. Encapsulin nanocompartments are one of the most prevalent protein-based compartmentalization strategies found in prokaryotes5,8. Here we show that desulfurase encapsulins represent a novel sulfur storage compartment in bacteria able to sequester large amounts of crystalline elemental sulfur. We determined the 1.78 [A] cryo-EM structure of a 24 nm desulfurase-loaded encapsulin highlighting the molecular details of the protein shell and desulfurase encapsulation. We found that elemental sulfur crystals can be formed inside encapsulin shells in a desulfurase-dependent manner with L-cysteine acting as the sulfur donor. Intracellular sulfur accumulation can be influenced by the concentration and type of sulfur source in growth media. The selectively permeable protein shell allows the long-term intracellular storage of redox-labile elemental sulfur by excluding cellular reducing agents from its interior. We found that encapsulation substantially improves desulfurase activity and stability while also preventing substrate inhibition. These findings represent the first example of a dedicated and widespread storage system for the essential element sulfur in bacteria and provide the basis for understanding how this novel protein-based storage compartment is integrated within bacterial metabolism.

biochemistry↗