Biology subjects

Buchner, J.

Publications and source records attributed to Buchner, J..

5 recordsLinked to original sources

bioRxiv2026

Chemical Boosting of Foldase Condensates Accelerates Oxidative Protein Folding

Dynamic biomolecular condensates play crucial roles in intracellular compartmentalization and physiological functions. While engineering tools for compartmentalization have expanded add-on functionalities, directly amplifying the inherent catalytic machinery within biological phase-separated droplets has remained elusive. Herein, we developed a phase-separated oxidative folding reaction chamber based on protein disulfide isomerase A6 (PDIA6) by chemically targeting its active site CxxC motif to enhance enzymatic activity within PDIA6 droplets. A para-substituted N-methylated pyridinylmethanethiol (pMePySH) enhanced the catalytic oxidative folding of bovine pancreatic trypsin inhibitor, proinsulin, and antibody up to 12-fold within in vitro PDIA6 droplets. Furthermore, pMePySH targeted PDIA6 foci within the endoplasmic reticulum, significantly promoting insulin secretion. These findings offer a powerful platform for the spatiotemporal manipulation of protein folding, with profound implications for the scalable manufacturing of therapeutic antibodies and other complex biopharmaceuticals.

bioengineering↗

bioRxiv2026

Sequence adaptations in the intracellular domain of Symbiosis receptor-like kinase (SymRK) promoted infection thread progression in root nodule primordia

The uptake of nitrogen-fixing bacteria into living plant cells and the intracellular accommodation of arbuscular mycorrhiza (AM) fungi requires the plasma membrane-localised Symbiosis Receptor-like Kinase (SymRK). AM is widespread across terrestrial vascular plant lineages, while the nitrogen-fixing root nodule symbiosis (RNS) is restricted to one clade within the eurosids. This distribution led to the concept that SymRK was adopted during evolution to mediate RNS. Comparative analyses revealed that SymRK orthologs from the eurosid clade support RNS while SymRK from the phylogenetically distant species Solanum lycopersicum (tomato) does not. To dissect the molecular basis for this different functionality, we carried out complementation analyses of the Lotus japonicus symrk-3 mutant which is unable to form AM or RNS. Domains swap chimera from the tomato and L. japonicus SymRK orthologs revealed that the intracellular domain of L. japonicus SymRK is necessary and for cortical infection thread (IT) and symbiosome development at 21 days post inoculation. Notably, this signalling specificity could be overcome by ectopic expression of tomato SymRK, pointing to altered protein dosage as a potential determinant of function. Consistent with this idea, SINA family E3 ubiquitin ligases interacted with and ubiquitinylated L. japonicus SymRK, but not tomato SymRK. In yeast two hybrid analysis, the interaction of SymRK with SINA2 and SINA4 depended on the C-terminal intrinsically disordered tail region of L. japonicus SymRK. We conclude that the SymRK intracellular domain evolved interaction capabilities with SINA E3 ligases which correlates with its ability to support RNS.

plant biology↗

bioRxiv2025

The essential co-chaperone Sgt1 regulates client dwell time in the Hsp90 chaperone cycle

The Hsp90 machinery is the most complex chaperone system in the eukaryotic cell. It is characterized by numerous co-chaperones that modulate the function of Hsp90. In S. cerevisiae, most of these cofactors can be deleted without affecting viability. Of the three essential ones, only the function of Sgt1 remained enigmatic. Our in vivo and in vitro experiments define key structural elements and determine the essential function of Sgt1 in the chaperoning of client proteins. We show that yeast Sgt1 exhibits a unique binding mode to Hsp90. The simultaneous interaction of Sgt1 with Hsp90 and client proteins enhances client maturation efficiency. Specifically, Sgt1 stabilizes Hsp90-client complexes and prevents their dissociation by the co-chaperone Aha1. Together, our findings reveal a distinct regulatory mechanism of the Hsp90 function, highlighting Sgt1 as a critical modulator of chaperone cycle progression.

biochemistry↗

bioRxiv2022

A network of cytosolic (co)chaperones promotes the biogenesis of mitochondrial signal-anchored outer membrane proteins

Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to varying extents. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins.

biochemistry↗

bioRxiv2020

Efficient inhibition of SARS-CoV-2 strains by a novel ACE2-IgG4-Fc fusion protein with a stabilized hinge region

The novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) enters its host cells after binding to the angiotensin-converting enzyme 2 (ACE2) via its spike glycoprotein. This interaction is critical for virus entry and virus-host membrane fusion. Soluble ACE2 ectodomains bind and neutralize the virus but the short in vivo half-lives of soluble ACE2 limits its therapeutic use. Fusion of the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain can prolong the in vivo half-life but bears the risk of unwanted Fc-receptor activation and antibody-dependent disease enhancement. Here, we describe optimized ACE2-Fc fusion constructs that avoid Fc-receptor binding by using IgG4-Fc as a fusion partner. The engineered ACE2-IgG4-Fc fusion proteins described herein exhibit promising pharmaceutical properties and a broad antiviral activity at single-digit nanomolar concentration. In addition, they allow to maintain the beneficial enzymatic activity of ACE2 and thus are very promising candidate antivirals broadly acting against coronaviruses.

immunology↗