Biology subjects

Keszei, A. F. A.

Publications and source records attributed to Keszei, A. F. A..

3 recordsLinked to original sources

bioRxiv2024

Unveiling the Structural Proteome of an Alzheimer's Disease Rat Brain Model

Studying native protein structures at near-atomic resolution in crowded environment presents a challenge. Consequently, understanding the structural intricacies of proteins within pathologically affected tissues often relies on mass spectrometry and proteomic analysis. In this study, we utilized electron cryomicroscopy (cryo-EM) and a specific method of analysis called Build and Retrieve (BaR) to investigate structural characteristics of protein complexes such as post-translational modification, active site occupancy, and arrested conformational state in Alzheimers Disease (AD) using brain lysate from a rat model (TgF344-AD) of the disease. Our findings reveal novel insights into the architecture of these complexes, which we corroborate through mass spectrometry analysis. Interestingly, it has been shown that the dysfunction of these protein complexes extends beyond AD, implicating them in cancer, as well as other neurodegenerative disorders such as Parkinsons disease, Huntingtons disease, and Schizophrenia. By elucidating the structural details of these complexes, our work not only enhances our understanding of disease pathology but also suggests new avenues for future approaches in therapeutic intervention.

biochemistry↗

bioRxiv2024

Cryo-EM of AKAP350 reveals fibrillar clusters and a potential association with DNA

Protein kinase A (PKA) is a promiscuous serine/threonine kinase that phosphorylates a broad-spectrum of effectors involved in vital processes such as glucose, glycogen, and lipid metabolism. Its activity is thus tightly controlled by a family of eukaryotic scaffolding proteins known as the A-kinase anchoring proteins (AKAPs) that confine PKA signaling to specific compartments in the cell. AKAP350 (the protein encoded by AKAP9) is a massive scaffolding protein that anchors PKA to the Golgi apparatus and the centrosome where it nucleates macromolecular signaling hubs that control microtubule nucleation and dynamics. Here, we have expressed and purified full-length AKAP350 from HEK293F cells in a functional conformation. Electron cryo-microscopy (cryo-EM) of the purified protein revealed polydisperse particles forming fibrillar clusters around 50 nm in diameter, and long, thin filaments that reconstructed into double-stranded DNA. Tomographic reconstruction of a tilt series of the purified protein by electron cryo-tomography (cryo-ET) further elucidated these fibrillar clusters as 3D bundles of entangled filaments. Mass spectrometry and DNA sequencing confirmed the co-purification of DNA and DNA binding proteins such as nuclear factor 1 B (NFIB) and nucleolin (NCL). Pulldown of NFIB and NCL, but not of CEP290, CDK5RAP2, and CEP170 was diminished in the presence of DNase-I, suggesting that AKAP350 interaction with these two proteins is mediated by DNA. Overall, this study has achieved a quality purification of AKAP350 from which a previously uncharacterized interaction landscape with DNA and DNA binding proteins was discovered.

biochemistry↗

bioRxiv2023

Structure and dynamics of a pentameric KCTD5/Cullin3/Gβγ E3 ubiquitin ligase complex

Heterotrimeric G proteins can be regulated by post-translational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain (CTD), engages CUL3 to form the central scaffold of a cullin- RING E3 ligase complex (CRL3KCTD5) that ubiquitylates G{beta}{gamma} and reduces G{beta}{gamma} protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/G{beta}1{gamma}2 assembly reveals a highly dynamic complex with rotations of over 60{degrees} between the KCTD5BTB/CUL3NTD and KCTD5CTD/G{beta}{gamma} moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate G{beta}{gamma} in an E3-E3 super-assembly, and extension of the structure to include full- length CUL3 with RBX1 and an ARIH1[~]ubiquitin conjugate reveals that some conformational states position the ARIH1[~]ubiquitin thioester bond to within 10 [A] of lysine-23 of G{beta} and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/G{beta}{gamma} complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex. Significance StatementIn humans, [~]600 enzyme complexes can carry out protein ubiquitylation, and the most abundant class of these are the cullin3-RING-ligase complexes (CRL3s). CRL3s are multiprotein complexes built around a BTB/cullin3 core, and the incorporation of different BTB proteins into this scaffold results in distinct architectures that ubiquitylate a wide range of substrates. In most cases, it is not known how the complexes are tuned to their substrates. We show that the BTB protein KCTD5 is the central organizer in a CRL3KCTD5 complex, and that the architecture and internal dynamics of KCTD5 are essential for positioning a G{beta}{gamma} substrate protein near an activated ubiquitin for the transfer reaction. This explains how KCTD5 targets G{beta}{gamma} for proteasomal degradation and regulates cellular activities.

biochemistry↗