Biology subjects

Bolhuis, D. L.

Publications and source records attributed to Bolhuis, D. L..

6 recordsLinked to original sources

bioRxiv2025

High-throughput protein target mapping enables accelerated bioactivity discovery for ToxCast and PFAS compounds

Chemical pollution is a global threat to human health, yet the toxicity mechanism of most contaminants remains unknown. Here, we applied an ultrahigh-throughput affinity-selection mass spectrometry (AS-MS) platform to systematically identify protein targets of prioritized chemical contaminants. After benchmarking the platform, we screened 50 human proteins against 481 prioritized chemicals, including 446 ToxCast chemicals and 35 per-and polyfluoroalkyl substances (PFAS). Among 24,050 interactions assessed, we discovered 35 novel interactions involving 14 proteins, with fatty acid-binding proteins (FABPs) emerging as the most ligandable protein family. Given this, we selected FABPs for further validation, which revealed a distinct PFAS binding pattern: legacy PFAS selectively bound to FABP1, whereas replacement compounds, PFECAs, unexpectedly interacted with all FABPs. X-ray crystallography further revealed that the ether group enhances molecular flexibility of alternative PFAS, to accommodate the binding pockets of FABPs. Our findings demonstrate that AS-MS is a robust platform for the discovery of novel protein targets beyond the scope of the ToxCast program and highlight the broader protein-binding spectrum of alternative PFAS as potential regrettable substitutes.

pharmacology and toxicology↗

bioRxiv2024

Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of [~]20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2Ds central role in proteome management.

biochemistry↗

bioRxiv2023

Proteomic Analysis Reveals a PLK1-Dependent G2/M Degradation Program and Links PKA-AKAP2 to Cell Cycle Control

Targeted protein degradation by the ubiquitin-proteasome system is an essential mechanism regulating cellular division. The kinase PLK1 coordinates protein degradation at the G2/M phase of the cell cycle by promoting the binding of substrates to the E3 ubiquitin ligase SCF{beta}TrCP. However, the magnitude to which PLK1 shapes the mitotic proteome has not been characterized. Combining deep, quantitative proteomics with pharmacologic PLK1 inhibition (PLK1i), we identified more than 200 proteins whose abundances were increased by PLK1i at G2/M. We validate many new PLK1-regulated proteins, including several substrates of the cell cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct SCF-family E3 ligases. Further, we found that the protein kinase A anchoring protein AKAP2 is cell cycle regulated and that its mitotic degradation is dependent on the PLK1/{beta}TrCP-signaling axis. Interactome analysis revealed that the strongest interactors of AKAP2 function in signaling networks regulating proliferation, including MAPK, AKT, and Hippo. Altogether, our data demonstrate that PLK1 coordinates a widespread program of protein breakdown at G2/M. We propose that dynamic proteolytic changes mediated by PLK1 integrate proliferative signals with the core cell cycle machinery during cell division. This has potential implications in malignancies where PLK1 is aberrantly regulated.

cell biology↗

bioRxiv2022

Crystal structure of the Arabidopsis SPIRAL2 C-terminal domain reveals a p80-Katanin-like domain

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 [A] resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core -helices, arranged in an -solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices 1, 3, 5, and an extended loop, the 6-7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.

biophysics↗

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↗

bioRxiv2019

A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

Ubiquitin-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitins C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin at \"exosites\". However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2 MDa E3 ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo EM structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic ubiquitin binding sites within large multiprotein complexes.\n\nSIGNIFICANCE STATEMENTUbiquitin-mediated interactions influence numerous biological processes. These are often transient or a part of multivalent interactions. Therefore, unmasking these interactions remains a significant challenge for large, complicated enzymes such as the Anaphase-Promoting Complex/Cyclosome (APC/C), a multisubunit RING E3 ubiquitin (Ub) ligase. APC/C activity regulates numerous facets of biology by targeting key regulatory proteins for Ub-mediated degradation. Using a series of Ub variants (UbVs), we identified a new Ub-binding site on the APC/C that preferentially binds to K48-linked Ub chains. More broadly, we demonstrate a workflow that can be exploited to uncover Ub-binding sites within ubiquitylation machinery and other associated regulatory proteins to interrogate the complexity of the Ub code in biology.

biochemistry↗