Biology subjects

Grabarczyk, D. B.

Publications and source records attributed to Grabarczyk, D. B..

7 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

TRIM52 is a primate-specific player in the DNA repair process under tight proteolytic control by a triad of giant E3 ligases

Tripartite motif 52 (TRIM52) exhibits strong positive selection in humans, yet is lost in many other mammals. In contrast to what one would expect for such a non-conserved factor, TRIM52 loss compromises cell fitness. We set out to determine the cellular function of TRIM52. Genetic and proteomic analyses revealed TRIM52s involvement in resolving topoisomerase 2 (TOP2)-DNA cross-links, mitigating DNA damage and preventing cell-cycle arrest. Consistent with a fitness-promoting function, TRIM52 is upregulated in various cancers, prompting us to investigate its regulatory pathways. We found TRIM52 to be targeted for ultra-rapid proteasomal degradation by the giant E3 ubiquitin ligases BIRC6, HUWE1, and UBR4/KCMF1. BIRC6 mono-ubiquitinates TRIM52, with subsequent extension by UBR4/KCMF1. These findings underscore TRIM52s pivotal role in DNA damage repair and regulation of its own abundance through multi-ligase degradation.

molecular biology↗

bioRxiv2024

Multiple ubiquitin ligases protect human genome integrity by targeting cancer-associated APOBEC3 deaminases for degradation.

HighlightsO_LIRNA-free APOBEC3 (A3) can enter the nucleus, leading to genomic mutations. C_LIO_LIThree E3 ligases specifically bind the RNA-binding domain of nuclear A3s. C_LIO_LICancer-associated A3B and A3H-I are thereby targeted for proteasomal degradation. C_LIO_LIThese E3 ligases thus act as genome guardians by limiting A3-mediated mutagenesis. C_LI APOBEC family members play crucial roles in antiviral restriction. However, certain APOBEC3 (A3) proteins drive harmful hypermutation in humans, contributing to cancer. The cancer-associated A3 proteins are capable of transiting from the cytosol to the nucleus, where they can cause genome mutations. Here, we uncover a specific set of cellular pathways that protect genomic DNA from the major cancer-associated A3 proteins. Through genetic and proteomic screening we identify UBR4, UBR5, and HUWE1 as key ubiquitin E3 ligases marking cancer-associated A3B and A3H-I for degradation, thereby limiting A3-driven hypermutation. Mechanistically, UBR5 and HUWE1 recognize unoccupied A3 RNA-binding domains, thus promoting proteasomal degradation of APOBEC3 protein that is not engaged in its antiviral cellular function. Depletion or mutation of the E3 ligases in cells and human cancer samples increases A3-driven genome mutagenesis. Our findings reveal that UBR4, UBR5, and HUWE1 are crucial factors in a ubiquitination cascade that maintains human genome stability.

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

E3 ubiquitin ligase RNF213 employs a non-canonical zinc finger active site and is allosterically regulated by ATP

RNF213 is a giant E3 ubiquitin ligase and a major susceptibility factor of Moyamoya disease, a cerebrovascular disorder that can result in stroke or death. In the cell, RNF213 is involved in lipid droplet formation, lipotoxicity, hypoxia, and NF-{kappa}B signaling, but its exact function in these processes is unclear. Structural characterization has revealed the presence of a dynein- like ATPase module and an unprecedented but poorly understood E3 module. Here, we demonstrate that RNF213 E3 activity is dependent on ATP binding, rather than ATP hydrolysis, and is particularly responsive to the ATP/ADP/AMP ratio. Biochemical and activity-based probe analyses identify a non-canonical zinc finger domain as the E3 active site, which utilizes the strictly conserved Cys4462, not involved in zinc coordination, as the reactive nucleophile. The cryo-EM structure of the trapped RNF213:E2[~]Ub intermediate reveals RNF213 C-terminal domain as the E2 docking site, which positions the ubiquitin-loaded E2 proximal to the catalytic zinc finger, facilitating nucleophilic attack of Cys4462 on the E2[~]Ub thioester. Our findings show that RNF213 represents an undescribed type of a transthiolation E3 enzyme and is regulated by adenine nucleotide concentration via its ATPase core, possibly allowing it to react to changing metabolic conditions in the cell.

biochemistry↗

bioRxiv2020

Crystal Structure of the Tof1-Csm3 (Timeless-Tipin) Fork Protection Complex

The Tof1-Csm3 fork protection complex has a central role in the replisome - it promotes the progression of DNA replication forks and protects them when they stall, while also enabling cohesion establishment and checkpoint responses. Here, I present the crystal structure of the Tof1-Csm3 complex from Chaetomium thermophilum at 3.1 [A] resolution. The structure reveals that Tof1 is an extended alpha-helical repeat protein which is capped at its C-terminal end by Csm3, a small helical bundle protein. I also characterize the DNA binding properties of the complex and a cancer-associated peptide-binding site. This study provides the molecular basis for understanding the functions of the Tof1-Csm3 complex, its human orthologue the Timeless-Tipin complex and additionally the Drosophila circadian rhythm protein Timeless.

biochemistry↗

bioRxiv2019

A stable leading strand polymerase/clamp loader complex is required for normal and perturbed eukaryotic DNA replication

The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol {varepsilon}. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.

biochemistry↗