Chaperone-driven proteasome assembly

Abstract Assembly of the 34-subunit, 2.5 MDa 26S proteasome is a carefully choreographed intricate process. It starts with formation of a seven-membered α-ring that serves as a template for assembly of the complementary β-ring-forming 'half-proteasomes'. Dimerization results in a latent 20S core particle that can serve further as a platform for 19S regulatory particle attachment and formation of the biologically active 26S proteasome for ubiquitin-dependent proteolysis. Both general and dedicated proteasome assembly chaperones regulate the efficiency and outcome of critical steps in proteasome biogenesis, and in complex association

Mapping the conformation of a client protein through the Hsp70 functional cycle.

International audienceThe 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein

DNAJB6 mutants display toxic gain of function through unregulated interaction with Hsp70 chaperones

Abstract Molecular chaperones are essential cellular components that aid in protein folding and preventing the abnormal aggregation of disease-associated proteins. Mutations in one such chaperone, DNAJB6, were identified in patients with LGMDD1, a dominant autosomal disorder characterized by myofibrillar degeneration and accumulations of aggregated protein within myocytes. The molecular mechanisms through which such mutations cause this dysfunction, however, are not well understood. Here we employ a combination of solution NMR and biochemical assays to investigate the structural and functional changes in LGMDD1 mutants of DNAJB6. Surprisingly, we find that DNAJB6 disease mutants show no reduction in their aggregation-prevention activity in vitro, and instead differ structurally from the WT protein, affecting their interaction with Hsp70 chaperones. While WT DNAJB6 contains a helical element regulating its ability to bind and activate Hsp70, in LGMDD1 disease mutants this regulation is disrupted. These variants can thus recruit and hyperactivate Hsp70 chaperones in an unregulated manner, depleting Hsp70 levels in myocytes, and resulting in the disruption of proteostasis. Interfering with DNAJB6-Hsp70 binding, however, reverses the disease phenotype, suggesting future therapeutic avenues for LGMDD1

Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR

INEPT-based experiments are widely used for ¹H→¹⁵N transfers, but often fail when involving labile protons due to solvent exchanges. J-based cross polarization (CP) strategies offer a more efficient alternative to perform such transfers, particularly when leveraging the Hʷᵃᵗᵉʳ ↔ Hᴺ exchange process to boost the ¹H→¹⁵N transfer process. This leveraging, however, demands the simultaneous spin-locking of both Hʷᵃᵗᵉʳ and Hᴺ protons by a strong ¹H RF field, while fulfilling the γₕB₁,ₕ=γₙB₁,ₙ Hartmann-Hahn matching condition. Given the low value of γₙ/γₕ, however, these demands are often incompatible—particularly when experiments are executed by the power-limited cryogenic probes used in contemporary high field NMR. The present manuscript discusses CP alternatives that can alleviate this limitation, and evaluates their performance on urea, amino acids, and intrinsically disordered proteins. These alternatives include new CP variants based on frequency-swept and phase-modulated pulses, designed to simultaneously fulfill the aforementioned conflicting conditions. Their performances vis-à-vis current options are theoretically analyzed with Liouville-space simulations, and experimentally tested with double and triple resonance transfer experiments.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083