Biology subjects

Nadel, C. M.

Publications and source records attributed to Nadel, C. M..

4 recordsLinked to original sources

bioRxiv2025

Structures of Fab-stabilized CHIP reveal a conformational switch important in E3 ligase and chaperone functions

Carboxyl terminus of Hsc70-interacting protein (CHIP/STUB1) is a U-box E3 ligase essential for protein quality control, targeting misfolded or damaged proteins for clearance and conducting chaperone-like functions by suppressing aggregation of proteins, including tau. The previous structure of full-length CHIP identified an asymmetric homodimer in which one U-box is occluded from E2 binding, indicating an unusual half-of-sites activity. However, the flexibility of CHIP has complicated efforts to further characterize its structure and function. Here we leverage two CHIP-targeting fragment antigen-binding (Fab) antibodies to solve structures by cryo-EM. We identify one Fab binds to the CHIP U-box via interactions mimicking E2 contacts and stabilizes three distinct CHIP dimer states, revealing an asymmetric-to-symmetric conformational switch that would enable both U-box domains to be accessible for E2 binding. Conversely, the second Fab targets CHIPs coiled-coil domains, stabilizing the asymmetric dimer with a single accessible U-box. Remarkably, the Fabs exhibit opposing effects on CHIPs inhibition of tau aggregation, wherein binding to coiled-coil domains abolishes inhibition of aggregation, while binding to the U-box greatly potentiates this activity. Together, this work reveals how CHIP conformational states and binding interfaces may regulate ubiquitination cycles and chaperone-like functions.

biochemistry↗

bioRxiv2023

Phosphorylation of a Cleaved Tau Proteoform at a Single Residue Inhibits Binding to the E3 Ubiquitin Ligase, CHIP

Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimers disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to taus accumulation or what mechanisms might be involved. To explore these questions, we focused on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to block its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 revealed the mechanism of this clash and allowed design of a mutation (CHIPD134A) that partially restores binding and turnover of pS416 tauC3. We find that pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a discrete molecular mechanism for how phosphorylation at a specific site contributes to accumulation of an important tau proteoform.

biochemistry↗

bioRxiv2023

Interaction with the membrane-anchored protein CHIC2 constrains the ubiquitin ligase activity of CHIP

Maintenance of cellular health requires the proper regulation of E3 ubiquitin ligases. The E3 ligase CHIP is canonically regulated by its interactions with the molecular chaperones Hsp70 and Hsp90, which focus CHIPs ubiquitination activity on misfolded proteins. Here, we report a chaperone-independent interaction of CHIP with the membrane-anchored protein CHIC2, which strongly attenuates CHIPs ligase activity. We show that CHIC2 outcompetes abundant, cytosolic chaperones through its exquisite CHIP selectivity, rather than through enhanced affinity. In proteomic experiments, we find that CHIC2 knockout phenocopies CHIP knockout in certain cell types, implying that chaperone-independent interactions can sometimes predominate CHIPs functions. Furthermore, loss of the CHIP-CHIC2 interaction induces neurodegeneration and shortens lifespan in C. elegans, demonstrating that formation of this chaperone-independent complex is important in animals. We propose that CHIC2 attenuates CHIP activity at the membrane, offering a novel mechanism by which this ubiquitin ligase can be regulated.

biochemistry↗

bioRxiv2021

Two distinct classes of co-chaperones compete for the EEVD motif in heat shock protein 70 (Hsp70) to tune its activity

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions (PPIs) with many co-chaperones. One hotspot for co-chaperone binding is the EEVD motif that is found at the extreme C-terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat (TPR) domain co-chaperones, such as the E3 ubiquitin ligase CHIP, and Class B J-domain proteins (JDPs), such as DnaJB4. Although complexes between Hsp70-CHIP and Hsp70-DnaJB4 are both important for chaperone functions, the molecular determinants that dictate the competition between these co-chaperones are not clear. Using a collection of EEVD-derived peptides, we find that DnaJB4 binds to the IEEVD motif of Hsp70s, but not the related MEEVD motif of cytoplasmic Hsp90s. Then, we explored which residues are critical for binding to CHIP and DnaJB4, revealing that they rely on some shared features of the IEEVD motif, such as the C-terminal carboxylate. However, they also had unique preferences, especially at the isoleucine position. Finally, we observed a functionally important role for competition between CHIP and DnaJB4 in vitro, as DnaJB4 can limit the ubiquitination activity of the Hsp70-CHIP complex, while CHIP suppresses the chaperone activities of Hsp70-DnaJB4. Together, these results suggest that the EEVD motif has evolved to support diverse PPIs, such that competition between co-chaperones could help guide whether Hsp70-bound proteins are folded or degraded.

biochemistry↗