Biology subjects

Hodakova, Z.

Publications and source records attributed to Hodakova, Z..

4 recordsLinked to original sources

bioRxiv2024

A multivalent adaptor mechanism drives the nuclear import of proteasomes

Nuclear protein homeostasis, including the turnover of transcription factors, critically depends on nuclear proteasomes. After each cell division, proteasomes need to be re-imported into the newly formed nucleus in a highly dynamic process that requires the largely unstructured protein AKIRIN2. However, how AKIRIN2 orchestrates this process and, more generally, how large protein complexes are translocated into the nucleus remains poorly understood. Here, we have used an integrated approach combining protein-wide saturation mutagenesis screens, cryoEM, and biochemical reconstitution to characterize AKIRIN2 as a scaffold protein that coordinates the stepwise assembly of an importin cluster around the proteasome. Through surveying every possible single amino acid substitution in AKIRIN2 using FACS- and microscopy-based genetic screens, we establish a comprehensive map of functionally relevant residues and binding interfaces in structured and disordered protein regions. Integrating these results with cryoEM analysis reveals a wing helix in a disordered region of AKIRIN2 that plays a crucial role in stabilizing proteasome interactions. Upon primary binding, AKIRIN2 homodimers recruit the importin IPO9, which in turn facilitates the binding of a second AKIRIN2 homodimer that recruits additional importins. Together, this multivalent molecular assembly amplifies the number of nuclear localisation signals and, thereby, triggers efficient proteasome translocation into the nucleus. Inside the nucleus, RanGTP rapidly dissociates importins, and AKIRIN2 is degraded by the proteasome in a ubiquitin-independent manner. Beyond mechanistically resolving the nuclear import of proteasomes, we propose that multivalent adaptor proteins like AKIRIN2 orchestrate the import of other macromolecular complexes and thereby dynamically control the composition of the nuclear proteome.

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

Cryo-EM structure of the chain-elongating E3 ligase UBR5

UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT E3 ligase has recently been identified as an important regulator of oncogenes, e.g., MYC, but little is known about its structure or mechanisms of substrate engagement and ubiquitination. Here, we present the cryo-EM structure of the human UBR5, revealing a building block of an antiparallel dimer which can further assemble into larger oligomers. The large helical scaffold of the dimer is decorated with numerous protein-interacting motifs for substrate engagement. Using cryo-EM processing tools, we observe the dynamic nature of the domain movements of UBR5, which allows the catalytic HECT domain to reach engaged substrates. We characterise the proteasomal nuclear import factor AKIRIN2 as an interacting protein and propose UBR5 as an efficient ubiquitin chain elongator. This preference for ubiquitinated substrates permits UBR5 to function in several different signalling pathways and cancers. Together, our data expand on the limited knowledge of the structure and function of HECT E3s.

biochemistry↗

bioRxiv2020

CRYO-EM STRUCTURE OF THE DELTA-RETROVIRAL INTASOME IN COMPLEX WITH THE PP2A REGULATORY SUBUNIT B56gamma

The Retroviridae delta-retrovirus genus includes the most oncogenic pathogen- human T-cell lymphotropic virus type 1 (HTLV-1)(1). Many of the ~20 million people infected with HTLV-1 will develop severe leukaemia (2) or an ALS-like motor disease (3) unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the viral integrase (IN) enzyme. Here we used X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of functional delta-retroviral IN assembled on viral DNA ends and bound the B56{gamma} subunit of its human host factor, the protein phosphatase 2A (4). The structure reveals a tetrameric IN assembly bound to the phosphatase via a conserved short linear motif found within the extended linker connecting the catalytic core (CCD) and C-terminal (CTD) IN domains. Unexpectedly, all four IN subunits are involved in B56{gamma} binding, taking advantage of the flexibility of the CCD-CTD linkers. Our results fill the current gap in the structural understanding of the delta-retroviral integration machinery. Insight into the interactions between the delta-retroviral intasome and the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.

biochemistry↗