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Limited proteolysis–mass spectrometry detects protein–protein interactions of integral membrane proteins

ABSTRACT: Integral membrane proteins (IMPs) are permanently embedded within the plasma membrane and execute multiple cellular functions. IMPs constitute important targets, but development of drugs that target IMPs to modulate protein–protein interactions, which mediate their functions, has been challenging. Characterizing the structural and functional properties of IMPs as well as their regulatory PPIs is a key step toward new or improved therapeutics. Functional and structural studies of IMPs are hindered by their hydrophobicity, low expression levels in cells, flexibility, and the need to use detergents to solubilize these proteins. Representative examples of IMPs are mammalian adenylyl cyclases that play a pivotal role in the 3',5'-cyclic adenosine monophosphate signaling pathway. The activity of membrane-bound adenylyl cyclases can be regulated by direct and/or indirect binding of calcium ions, for example, via calmodulin. Unstructured and highly flexible regions of adenylyl cyclases engage in PPIs. However, the principles of classical proteomics methods do not allow mapping interactions involving regions without a defined three-dimensional structure. To address this limitation, we used limited proteolysis–mass spectrometry (LiP–MS) to study the known interaction between the adenylyl cyclases AC8 and calmodulin by probing protein structural alterations in crude membrane suspensions treated with calmodulin. The LiP–MS analysis pinpointed putative binding site regions of AC8 previously shown to participate in the interaction with calmodulin, demonstrating that this method can be used to identify interaction domains in membrane proteins in a physiologically relevant context.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Aleš Holfeld

LAB HEAD: Paola Picotti

PROVIDER: PXD039520 | Pride | 2024-02-10

REPOSITORIES: Pride

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Project description:Calcium signaling is a central regulator of cardiomyocyte growth and function. Calmodulin is a critical mediator of calcium signals. Because the amount of calmodulin within cardiomyocytes is limiting, precise regulation of calmodulin expression may be an important for regulation of calcium signaling. In this study, we show for the first time that calmodulin levels are regulated post-transcriptionally in heart failure. The cardiomyocyte-restricted microRNA miR-1 inhibited translation of calmodulin-encoding mRNAs via highly conserved target sites within their 3â-untranslated regions. In keeping with its effect on calmodulin expression, miR-1 downregulated calcium-calmodulin signaling through the calcineurin to NFAT. miR-1 also negatively regulated expression of Mef2a and Gata4, key transcription factors that mediate calcium-dependent changes in gene expression. Consistent with downregulation of these hypertrophy-associated genes, miR-1 attenuated cardiomyocyte hypertrophy in cultured neonatal rat cardiomyocytes and in the intact adult heart. Our data indicate that miR-1 regulates cardiomyocyte growth responses by negatively regulating the calcium-signaling components calmodulin, Mef2a, and Gata4. We show that miR-1 is downregulated in a murine heart failure model. miRNAs expression changes were measured in calcineurin transgenic model of heart failure and control mice using a Luminex platform. Reduced miR-1 expression was associated with broad alteration in expression of predicted target genes. To test this, we measured miRs including miR-1 and genome wide transcriptome changes in vivo and in vitro system. Calcineurin transgenic heart was compared to nontransgenic heart (NTg vs. CNTg). We also investigated the gene expression changes during the course of cardiomyocytes differentiation using DMSO treated P19CL6 cell lines. Two time points (day 6 and day 10) were compared to identified the gene expression changes of predicted miR-1 targets (Day 6 vs. Day 10).

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Limited proteolysis–mass spectrometry detects protein–protein interactions of integral membrane proteins

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