Biology subjects

Kurzbauer, R.

Publications and source records attributed to Kurzbauer, R..

5 recordsLinked to original sources

bioRxiv2024

Architecture of the UBR4 complex, a giant E4 ligase central to eukaryotic protein quality control

Eukaryotic cells have evolved sophisticated quality control mechanisms to eliminate aggregation-prone proteins that compromise cellular health. Central to this defense is the ubiquitin-proteasome system, where UBR4 acts as essential E4 ubiquitin ligase, amplifying degradation marks on defective proteins. Our cryo-EM analysis of UBR4 in complex with its cofactors KCMF1 and CALM1 reveals a massive 1.3 MDa ring structure, featuring a central substrate-binding arena and flexibly attached catalytic units. Structural data illustrate how UBR4 binds substrate and extends K48-specific ubiquitin chains. Importantly, efficient substrate targeting depends on both pre-ubiquitination and specific N-degrons, with KCMF1 acting as key substrate filter. Furthermore, we show that the architecture of the E4 megacomplex is conserved across eukaryotes but with species specific adaptations, allowing UBR4 to perform its precisely tuned quality-control function in diverse cellular environments.

biochemistry↗

bioRxiv2024

Convergence of orphan quality control pathways at a ubiquitin chain-elongating ligase

Unassembled and partially assembled subunits of multi-protein complexes have emerged as major quality control clients, particularly under conditions of imbalanced gene expression such as stress, aging, and aneuploidy. The factors and mechanisms that eliminate such orphan subunits to maintain protein homeostasis are incompletely defined. Here, we show that the UBR4-KCMF1 ubiquitin ligase complex is required for efficient degradation of multiple unrelated orphan subunits from the chaperonin, proteasome cap, proteasome core, and a protein targeting complex. Epistasis analysis in cells and reconstitution studies in vitro show that the UBR4-KCMF1 complex acts downstream of a priming ubiquitin ligase that first mono-ubiquitinates orphans. UBR4 recognizes both the orphan and its mono-ubiquitin and builds a K48-linked poly-ubiquitin degradation signal. The discovery of a convergence point for multiple quality control pathways may explain why aneuploid cells are especially sensitive to loss of UBR4 or KCMF1 and identifies a potential vulnerability across many cancers.

cell biology↗

bioRxiv2022

Structural basis of how the BIRC6/SMAC complex regulates apoptosis and autophagy

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. BIRC6 is an exceptionally large, multidomain IAP that inhibits its targets by means of its atypical ubiquitin ligase activity and in addition, functions as an inhibitor of autophagy by depleting LC3B. Little is known of the mechanisms by which BIRC6 interacts with its targets and fulfills these two roles. Here, we determined the cryo-EM structure of BIRC6 alone and in complex with two mitochondrial pro-apoptotic proteins, HTRA2 and SMAC. We show BIRC6 is an antiparallel homodimer that forms a crescent shape that arcs around a spacious cavity. The cavity is surrounded by binding sites for client proteins, where they interact with the flexible UBC domain that mediates ubiquitin ligation. Functional data reveal that multivalent binding of SMAC in the central cavity obstructs substrate binding, impeding ubiquitination of both autophagy and apoptotic target proteins. Together our data reveal the molecular mechanisms of how SMAC specifically binds and inhibits BIRC6 to promote apoptosis, and how this regulatory mechanism also extends to autophagy substrates. The interaction sites are hot spots of cancer and atrophy mutations, highlighting the importance of carefully balancing the interplay between BIRC6 and SMAC.

biochemistry↗

bioRxiv2021

BacPROTACs mediate targeted protein degradation in bacteria

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis targeting chimeras (PROTACs). Despite their superior properties over classical inhibitors, it has so far not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, highlighting the potential of the technology to provide next generation antibiotics.

biochemistry↗

bioRxiv2020

McsB forms a gated kinase chamber to mark aberrant bacterial proteins for degradation

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labelling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labelling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The "closed" octamer interconverts with other oligomers in a phosphorylation-dependent manner and, contrary to these "open" forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher-order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.

molecular biology↗