| 1. |
- Gault, Joseph, et al.
(author)
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Mass Spectrometry Reveals the Direct Action of a Chemical Chaperone
- 2018
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In: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 9:14, s. 4082-4086
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Journal article (peer-reviewed)abstract
- Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine-N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine-N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.
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| 2. |
- Ingelshed, Katrine, et al.
(author)
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The MDM2 Inhibitor Navtemadlin Arrests Mouse Melanoma Growth In Vivo and Potentiates Radiotherapy
- 2022
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In: Cancer Research Communications. - : American Association For Cancer Research (AACR). - 2767-9764. ; 2:9, s. 1075-1088
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Journal article (peer-reviewed)abstract
- The tumor suppressor protein p53 is mutated in close to 50% of human tumors and is dysregulated in many others, for instance by silencing or loss of p14ARF. Under steady-state conditions, the two E3 ligases MDM2/MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53–MDM2/4 interaction to reactivate p53 in tumors with wild-type (WT) p53 has therefore been considered a therapeutic strategy. Moreover, studies indicate that p53 reactivation may synergize with radiation and increase tumor immunogenicity. In vivo studies of most MDM2 inhibitors have utilized immunodeficient xenograft mouse models, preventing detailed studies of action of these molecules on the immune response. The mouse melanoma cell line B16-F10 carries functional, WT p53 but does not express the MDM2 regulator p19ARF. In this study, we tested a p53-MDM2 protein–protein interaction inhibitor, the small molecule Navtemadlin, which is currently being tested in phase II clinical trials. Using mass spectrometry–based proteomics and imaging flow cytometry, we identified specific protein expression patterns following Navtemadlin treatment of B16-F10 melanoma cells compared with their p53 CRISPR-inactivated control cells. In vitro, Navtemadlin induced a significant, p53-dependent, growth arrest but little apoptosis in B16-F10 cells. When combined with radiotherapy, Navtemadlin showed synergistic effects and increased apoptosis. In vivo, Navtemadlin treatment significantly reduced the growth of B16-F10 melanoma cells implanted in C57Bl/6 mice. Our data highlight the utility of a syngeneic B16-F10 p53+/+ mouse melanoma model for assessing existing and novel p53-MDM2/MDM4 inhibitors and in identifying new combination therapies that can efficiently eliminate tumors in vivo.Significance:The MDM2 inhibitor Navtemadlin arrests mouse tumor growth and potentiates radiotherapy. Our results support a threshold model for apoptosis induction that requires a high, prolonged p53 signaling for cancer cells to become apoptotic.
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| 3. |
- Kaldmae, Margit, et al.
(author)
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Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction
- 2019
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In: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 30:8, s. 1385-1388
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Journal article (peer-reviewed)abstract
- Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation.
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| 4. |
- Leppert, Axel, et al.
(author)
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ATP-independent molecular chaperone activity generated under reducing conditions
- 2022
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In: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:8
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Journal article (peer-reviewed)abstract
- Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra- to intermolecular disulfide bond relay mechanism. Chaperone-active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol-containing compounds and proteins, and appear in parallel with reduction-induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space.Significance: Chaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction-induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.
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