A novel approach is presented that combines pulse metabolic labelling with SILAC and BioID mass spectrometry to temporally resolve protein-protein interactions of a co-chaperone to gain novel insights on how co-chaperone-client interactions may occur over the lifespan of a protein to preserve proteostasis.
Protein quality control in bacteria relies on dynamic chaperone networks that rapidly respond to environmental stresses. In mycobacteria, a central Hsp70 chaperone, DnaK, and its co-chaperones, J-domain proteins (JDPs), play essential roles in the cellular response to stress and antibiotics. Conserved "J-domain" sequences of JDPs are known to mediate transient interactions with Hsp70s and other protein partners. We have previously shown that a rationally designed proteomimetic of a J-domain is toxic to stressed mycobacterial cells. Here, we aimed to trap J-domain mimetic interaction partners to potentially reveal vulnerabilities in cellular stress-response pathways. To do so, we modified a J-domain proteomimetic of the mycobacterial JDP DnaJ1 (J1C) with a photoaffinity label moiety, resulting in J1C-PAL. In heat-shocked mycobacteria, J1C-PAL crosslinked to DnaK in cells, confirming retention of canonical Hsp70-binding activity under proteotoxic stress. Chemoproteomic profiling revealed additional cellular targets, namely a conditionally essential mycobacterial protein, glutamine synthetase (GS). Biochemical assays demonstrated that J1C-PAL bound GS and inhibited its enzymatic activity. Cellular studies validated that J1C perturbed nitrogen metabolism mediated by GS in mycobacteria. Together, these findings establish J1C-PAL as a cell-permeable chemoproteomic tool for mapping protein targets and uncover a surprising link between a co-chaperone mimetic and bacterial metabolism.
Brock Nelson, Tania J. Lupoli· ChemBioChem· 0 citations
Heat shock cognate protein 70 (Hsc70) is a 71 kDa molecular chaperone belonging to the Hsp70 family of heat shock proteins. These proteins act as ATP-dependent molecular machines that assist protein folding under both physiological and stress conditions such as hypoxia, heat shock, and pH fluctuations. In addition to general chaperone functions, Hsc70 performs specialized roles, including uncoating clathrin-coated vesicles, facilitating protein transport into organelles, and targeting proteins for lysosomal degradation. Members of the Hsp70 family are known to form dimers and higher oligomers, but the structural organization and functional relevance of these assemblies remain poorly understood. Earlier studies also suggested that J-domain proteins (JDPs) can promote Hsp70 dimerization. In this study, we used chemical cross-linking, high-resolution Fourier transform mass spectrometry (FTMS), 15N isotopic labeling, and advanced data analysis to investigate the structural organization of Hsc70 dimers. Cross-link-derived distance restraints enabled structural modeling of Hsc70 monomers and dimers using AlphaLink2. Our results reveal distinct ATP- and ADP-state dimer conformations that coexist in equilibrium. In the presence of the cochaperone DnaJB1, we observed a shift in the dimer–monomer equilibrium, accompanied by enhanced ATP hydrolysis and formation of intermediate species. These findings demonstrate that the Hsc70 dimer population is structurally heterogeneous and depends on nucleotide state and cochaperone interactions.
A. Melikov, Vsevolod Viliuga, Daniel Kavan et al.· Journal of Proteome Research· 0 citations
How the regulatory networking of chaperones by cochaperones, particularly J domain proteins (JDPs; also called Hsp40 or DNAJ proteins), contributes to biocondensate formation and protein disaggregation to maintain proteostasis is discussed.
Gregory L. Blatch, Adrienne L. Edkins· 0 citations
It is shown that short nucleic acids containing Gquadruplex (G4) structure can also catalyze protein folding and uncovers a previously underappreciated role for nucleic acid in proteostasis and offers a new strategy for studying nucleic acid structure-function relationship at residue level.
DegP is a periplasmic protease-chaperone essential for protein quality control and virulence factor trafficking in Gram-negative bacteria. In its apo form, DegP adopts a dynamic ensemble of oligomers derived from trimer building blocks through two competing self-assembly pathways. Upon engaging client proteins, apo DegP oligomers redistribute into discrete cage structures inside which the clients are encapsulated. The cage ensembles formed depend on the size of the bound clients, and notably can include 12mers, 24mers, and 60mers. Previous studies mapped the DegP oligmeric landscape using dynamic light scattering, analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and electron cryomicroscopy, modalities which in general report ensemble averages and often cannot directly delineate closely related coexisting species. Here, we apply charge detection mass spectrometry (CDMS) to directly measure the masses of individual DegP ions, resolving the complete oligomeric distribution in the absence and presence of four clients of increasing size. We reveal previously undetected odd-numbered oligomers and quantify the relative abundance of each assembly. Through heat-cool cycling CDMS experiments, we track cage distribution changes and reveal client protection and refolding, providing a direct view of the chaperone capabilities of DegP. These results further establish CDMS as a powerful single-molecule tool for dissecting heterogeneous protein assembly landscapes.
Kent R. Vosper, Aizaz Humayun, Matthew S McPartlan et al.· bioRxiv· 0 citations
The 26S proteasome is the hub for regulated protein turnover in eukaryotic cells. Degradation of proteins by the Ubiquitin-Proteasome System plays critical roles in every aspect of cell biology, such as the regulation of gene transcription, the quality control of translation and protein folding, and protein transport across membranes. While mRNA levels and protein abundances can be readily measured with a robust set of established tools, only a few methodologies exist to identify proteins that are degraded by the proteasome rather than the lysosome as the second major pathway for turnover. Here, we sought to address this by using genetic code expansion to introduce a photo-crosslinkable unnatural amino acid into the yeast 26S proteasome and capture cellular protein substrates as they translocate through the proteasomal ATPase motor. In vitro biochemical experiments confirmed that these modified proteasomes are functional, which allowed us to introduce them into live yeast cells for in vivo crosslinking and the identification of enriched ATP-dependent substrates by mass spectrometry. These experiments revealed a very diverse pool of proteasomal substrates that markedly changed upon cell exposure to endoplasmic reticulum stress. Together, our results represent an exciting avenue for probing the landscape of proteasomal substrates and its changes in response to various cellular conditions and stresses.
Santiago Yori Restrepo, Andreas Martin· bioRxiv· 0 citations
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