Discovering protein–protein interaction stabilisers by native mass spectrometry

Bellamy‐Carter, Jeddidiah; Mohata, Manjari; Falcicchio, Marta; Basran, Jaswir; Higuchi, Yusuke; Doveston, Richard G.; Leney, Aneika C.

doi:10.1039/d1sc01450a

Cited by 19 publications

(23 citation statements)

References 31 publications

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“…14-3-3 protein binding can regulate expression of other molecules [ 88 ] and modify interaction networks. [ 8 ] Many 14-3-3 and 14-3-3 PPIs inhibitors [ 89 , 90 , 91 ] and stabilizers [ 92 , 93 , 94 , 95 ] have been regarded as potential therapeutic targets in other systems than heart [ 22 , 23 ]. Small molecules, peptides and natural products that target 14-3-3 or 14-3-3 PPIs have been applied as 14-3-3 and 14-3-3 PPI modulators [ 96 , 97 , 98 ].…”

Section: Discussionmentioning

confidence: 99%

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome

Qu¹,

Tarasov²,

Chakir³

et al. 2022

Cells

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Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein–protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.

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Section: Discussionmentioning

confidence: 99%

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome

Qu¹,

Tarasov²,

Chakir³

et al. 2022

Cells

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“…Other approaches not discussed here (e.g., microscale thermophoresis ( Centorrino et al, 2022 ; Kozeleková et al, 2022 ), differential scanning fluorimetry ( Cossar et al, 2021 ; Waløen et al, 2021 ; Joshi et al, 2022 ), analytical ultracentrifugation ( Horvath et al, 2021 ; Leysen et al, 2021 ; Neves et al, 2021 ; Pohl et al, 2021 ; Srdanović et al, 2022 ), single angle x-ray scattering ( Kast and Dominguez, 2019 ; Leysen et al, 2021 ) also contribute new understanding and support hypotheses. NMR ( Kuusk et al, 2019 ; Neves et al, 2021 ) and native mass spectrometry ( Bellamy-Carter et al, 2021 ) are also important techniques that shows great promise for studying 14-3-3 PPIs. Ultimately, a full understanding of 14-3-3 function will only be achieved by taking an interdisciplinary approach whereby high quality biophysical data complements unexplored single molecule approaches, together with structural and cellular biology studies.…”

Section: Discussionmentioning

confidence: 99%

Contemporary biophysical approaches for studying 14-3-3 protein-protein interactions

Thurairajah

Hudson

Doveston

2022

Front. Mol. Biosci.

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14-3-3 proteins are a family of regulatory hubs that function through a vast network of protein-protein interactions. Their dysfunction or dysregulation is implicated in a wide range of diseases, and thus they are attractive drug targets, especially for molecular glues that promote protein-protein interactions for therapeutic intervention. However, an incomplete understanding of the molecular mechanisms that underpin 14-3-3 function hampers progress in drug design and development. Biophysical methodologies are an essential element of the 14-3-3 analytical toolbox, but in many cases have not been fully exploited. Here, we present a contemporary review of the predominant biophysical techniques used to study 14-3-3 protein-protein interactions, with a focus on examples that address key questions and challenges in the 14-3-3 field.

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“…This work has shown that nMS can show these complexes with intact proteins, in addition to the model peptides which have previously been used. [16] nMS was able to directly show the existence of the IMiD ternary complexes, which will be beneficial in the screening of small molecule libraries for further glues that modulate this interaction.…”

Section: Bodymentioning

confidence: 99%

“…nMS is effective in separating individual species that exist in a stoichiometric mixture, capturing transient interactions, and comparing stability of complexes. [12][13][14] Whilst nMS has previously been used to predict the efficacy of PROTACs [15] and to analyse complexes formed between MGs and model peptides [16], this is the first example of its application to MGs and multimeric E3 complexes. We have demonstrated its efficacy using disease relevant drugs and targets; two E3 ligase proteins (CRBN:DDB1 and DCAF15:DDA1:DDB1), five molecular glues (lenalidomide, thalidomide, pomalidomide, E7820, and indisulam), and two POIs (GSPT1 and RBM39).…”

Section: Bodymentioning

confidence: 99%

Native Mass Spectrometry of Complexes Formed by Molecular Glues Reveals Stoichiometric Rearrangement of E3 Ligases

Jackson

Beveridge

2023

Preprint

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In this application of native mass spectrometry (nMS) to investigate complexes formed by molecular glues (MGs), we have demonstrated its efficiency in delineating stoichiometric rearrangements of E3 ligases that occur during targeted protein degradation (TPD). MGs stabilise interactions between an E3 ligase and a protein of interest (POI) targeted for degradation, and these ternary interactions are challenging to characterise. We have shown that nMS can unambiguously identify complexes formed between the CRBN:DDB1 E3 ligase and the POI GSPT1 upon the addition of lenalidomide, pomalidomide or thalidomide. Moreover, upon analysis of the DCAF15:DDA1:DDB1 E3 ligase, we uncovered that this complex self-associates into dimers and trimers when analysed alone, and dissociates into single copies of the complex upon the addition of MG (E7820 or indisulam) and POI RBM39, forming a 1:1:1 ternary complex. This work demonstrates the strength of nMS in TPD research, and reveals novel binding mechanisms of the DCAF15 E3 ligase.

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Discovering protein–protein interaction stabilisers by native mass spectrometry

Abstract: Stabilising protein–protein interactions is challenging, yet therapeutically important. Native mass spectrometry can be used to monitor binding equilibria, allowing identification and measurement of novel protein–protein interaction stabilisers.

Cited by 19 publications

References 31 publications

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome

Contemporary biophysical approaches for studying 14-3-3 protein-protein interactions

Native Mass Spectrometry of Complexes Formed by Molecular Glues Reveals Stoichiometric Rearrangement of E3 Ligases

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