No abstract

Metalloprotease-processed CD95L (cl-CD95L) is a soluble cytokine that implements a PI3K/Ca2+ signaling pathway in triple-negative breast cancer (TNBC) cells. Accordingly, high levels of cl-CD95L in TNBC women correlate with poor prognosis, and administration of this ligand in an orthotopic xenograft mouse model accelerates the metastatic dissemination of TNBC cells. The molecular mechanism underlying CD95-mediated cell migration remains unknown. Here, we present genetic and pharmacologic evidence that the anti-apoptotic molecules BclxL and Bcl-2 and the pro-apoptotic factors BAD and BID cooperate to promote migration of TNBC cells stimulated with cl-CD95L. BclxL was distributed in both endoplasmic reticulum (ER) and mitochondrion membranes. The mitochondrion-localized isoform promoted cell migration by interacting with voltage-dependent anion channel 1 to orchestrate Ca2+ transfer from the ER to mitochondria in a BH3-dependent manner. Mitochondrial Ca2+ uniporter contributed to this flux, which favored ATP production and cell migration. In conclusion, this study reveals a novel molecular mechanism controlled by BclxL to promote cancer cell migration and supports the use of BH3 mimetics as therapeutic options not only to kill tumor cells but also to prevent metastatic dissemination in TNBCs.

Since noncovalent protein macrocomplexes are implicated in many cellular functions, their characterization is essential to understand how they drive several biological processes. Over the past 20 years, because of its high sensitivity, mass spectrometry has been described as a powerful tool for both the protein identification in macrocomplexes and the understanding of the macrocomplexes organization. Nonetheless, stabilizing these protein macrocomplexes, by introducing covalent bonds, is a prerequisite before their analysis by the denaturing mass spectrometry technique. In this study, using the Hsp90/Aha1 macrocomplex as a model (where Hsp denotes a heat shock protein), we optimized a double cross-linking protocol with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC). This protocol takes place in a two-step process: initially, a cross-linking is performed according to a previously optimized protocol, and then a second cross-linking is performed by increasing the EDC concentration, counterbalanced by a high dilution of sample and, thus, protein macrocomplexes. Using matrix-assisted laser desorption ionization (MALDI) mass spectrometry, we verified the efficiency of our optimized protocol by submitting (or not submitting) samples to the K200 MALDI MS analysis kit containing N-succinimidyl iodo-acetate, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester), suberic acid bis(N-hydroxysuccinimide ester), disuccinimidyl tartrate, and dithiobis(succinimidyl) propionate, developed by the CovalX Company. Results obtained show that our optimized cross-linking protocol allows a complete stabilization of protein macrocomplexes and appears to be very accurate. Indeed, contrary to other cross-linkers, the "zero-length" feature of the EDC reagent prevents overdetermination of the mass of complexes, because EDC does not remain as part of the linkage.

No abstract

No abstract

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer able to self-associate in the presence of divalent cations or under heat shock. This study investigated the relationship between Hsp90 oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase). The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated by ultracentrifugation, size-exclusion chromatography coupled to multiangle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Hsp90 dimer was able to bind up to four Aha1 molecules, and Hsp90 oligomers are also able to interact with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization process. Except for Hsp90 dimer, the stoichiometry of the interaction remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to bind the Aha1 ATPase activator reinforces their role within the Hsp90 chaperone machineries.

No abstract

Proposed models of the function of Hsp90 are characterised by high flexibility of the dimeric state and conformational changes regulated by both nucleotide binding and hydrolysis, and by co-chaperone interactions. In addition to its dimeric state, Hsp90 self-associates upon particular stimuli. The Hsp90 dimer is the building block up to the hexamer that we named “cosy nest”, and the dodecamer results from the association of two hexamers. Oligomers exhibit chaperone activity, but their exact mechanism of action has not yet been determined. One of the best ways to elucidate how oligomers might operate is to study their interactions with co-chaperone proteins known to regulate the Hsp90 chaperone cycle, such as p23 and Aha1. In this review, we summarise recent results and conclude that Hsp90 oligomers are key players in the chaperone cycle. Crucible-shaped quaternary structures likely provide an ideal environment for client protein accommodation and folding, as is the case for other Hsp families. Confirmation of the involvement of Hsp90 oligomers in the chaperone cycle and a better understanding of their functionality will allow us to address some of the more enigmatic aspects of Hsp90 activity. Utilising this knowledge, future work will highlight how Hsp90 oligomers and co-chaperones cooperate to build the structures required to fold or refold numerous different client proteins.