Biology subjects

Hoogerbrugge, G.

Publications and source records attributed to Hoogerbrugge, G..

4 recordsLinked to original sources

bioRxiv2025

The CAGE complex: a hollow, megadalton, protein assembly in prokaryotic and eukaryotic microbes

We report the discovery and structure of a previously unknown [~]1 MDa hollow protein assembly, identified during a survey of ciliary complexes from the ciliate Tetrahymena thermophila. By combining mass spectrometry, structure prediction, and cryo-electron microscopy, we define a homotetrameric cage-like complex with a distinctive elliptical architecture and a large internal cavity. A sequence survey revealed several thousand homologs spanning diverse unicellular eukaryotes--including green algae, fungi, amoebozoans, choanoflagellates, and SAR lineages--as well as predominantly gram-negative bacteria, indicating an ancient evolutionary origin and arguing against a eukaryote-specific function. We determined a near-atomic resolution structure of the complex from the slime mold Dictyostelium discoideum, demonstrating conservation of overall architecture and cavity despite low sequence identity. Together, these results establish the CAGE complex (Conserved Assembly in Gram-negative bacteria and Eukaryotes) as a new class of large protein cage broadly distributed across the tree of life. While its biological function remains unknown, its size, architecture, and conservation suggest possible roles in transport or protein/RNA homeostasis.

molecular biology↗

bioRxiv2025

VerteBrain reveals novel neural and non-neural protein assemblies conserved across vertebrate evolution

Protein-protein interactions underlie core brain functions, including neurotransmitter release, receptor activation, and intracellular signaling essential for learning, memory, and cognition. Here, we systematically map conserved brain protein interactions across five vertebrate species-rabbit, chicken, dolphin, pig, and mouse-using co-fractionation and immunoprecipitation mass spectrometry. From 2,197 biochemical fractions, we identify over 81,000 high-confidence interactions among 6,108 conserved proteins. This interaction map (VerteBrain) reveals both regulatory and structural complexes, including extensive synaptonemal protein associations likely involved in inter-neuronal coordination. Conservation across species underscores essential roles in neuronal and glial function, as well as in additional tissues for more widely expressed complexes. The VerteBrain dataset uncovers candidate disease mechanisms, including roles for ARHGEF1 in short stature syndromes, synaptic vesicle trafficking complexes in epilepsy, and RELCH in congenital deafness. VerteBrain provides a publicly accessible framework for investigating brain protein interactions and their relevance to human neurological disorders.

systems biology↗

bioRxiv2025

Serendipity and the slime mold: a visual survey of megadalton protein assemblies reveals the structure of the polyketide synthase Pks16

Large macromolecular assemblies are integral to most cellular processes, making their identification and structural characterization an important strategy for advancing our understanding of protein functions. In this pilot study, we investigated large multiprotein assemblies from the cytoplasm of the slime mold Dictyostelium discoideum using shotgun-electron microscopy (shotgun-EM), the combined application of mass spectrometry-based proteomics and cryo-electron microscopy (cryo-EM) to heterogenous mixtures of proteins. With its similarities in cell structure and behavior to mammalian cells, D. discoideum has long served as an invaluable model organism, particularly in the study of immune cell chemotaxis, phagocytosis, bacterial infection, and other processes. We subjected D. discoideum soluble protein complexes to two-step fractionation, performing size-exclusion chromatography followed by mixed-bed ion-exchange chromatography. Isolated fractions containing a subset of megadalton-scale protein assemblies were subsequently analyzed using mass spectrometry to identify the proteins and cryo-EM to characterize their structures. Mass spectrometry analysis revealed 299 unique proteins in the isolated fractions, then single-particle cryo-EM analysis generated distinct 2D projections of several visually distinctive protein assemblies, from which we successfully identified and reconstructed three major protein complexes: the 20S proteasome, the dihydrolipoyllysine-residue succinyltransferase (Odo2) of the mitochondrial 2-oxoglutarate dehydrogenase complex, and polyketide synthase 16 (Pks16), thought to be the primary fatty acid synthase of D. discoideum. Based on the Pks16 structure, the first of the 40 D. discoideum PKSs to be experimentally determined, models for the full set of D. discoideum PKSs were constructed with help from AlphaFold 3. Comparative analysis enabled structural characterization of their reaction chambers. Shotgun-EM thus provides a view of proteins in their native or near-native biological conformations and scaling up this approach offers an effective route to characterize new structures of multi-protein assemblies directly from complex samples.

biochemistry↗

bioRxiv2022

Integrative modeling reveals the molecular architecture of the Intraflagellar Transport A (IFT-A) complex

Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease. SummaryThe 3D structure of the six-subunit complex and its polymeric assembly gives insights into cargo transport in cilia and how specific mutations in these genes lead to ciliopathy birth defects.

biochemistry↗