Origin and Isoform Specific Functions of Exchange Proteins Directly Activated by cAMP: A Phylogenetic Analysis

Ni, Zhuofu; Cheng, Xiaodong

doi:10.3390/cells10102750

Cited by 5 publications

(2 citation statements)

References 83 publications

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“…Lysine 561 is located within the RA domain, which displays significant sequence diversity between the two isoforms (33). RA domain folds into a ubiquitin alpha/beta roll superfold (34) and has been found in various proteins to function mainly as a protein interaction scaffold (35).…”

Section: Discussionmentioning

confidence: 99%

Protein SUMOylation promotes cAMP-independent EPAC1 activation

Yang,

Mei,

Lin

et al. 2024

Preprint

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Exchange protein directly activated by cAMP (EPAC1) mediates the intracellular functions of a critical stress-response second messenger, cAMP. Herein, we report that EPAC1 is a cellular substrate of protein SUMOylation, a prevalent stress-response posttranslational modification. Site-specific mapping of SUMOylation by mass spectrometer leads to identifying K561 as a primary SUMOylation site in EPAC1. Sequence and site-directed mutagenesis analyses reveal a functional SUMO-interacting motif required for cellular SUMOylation of EPAC1. SUMO modification of EPAC1 mediates its heat shock-induced Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

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Section: Discussionmentioning

confidence: 99%

Protein SUMOylation promotes cAMP-independent EPAC1 activation

Yang,

Mei,

Lin

et al. 2024

Preprint

Self Cite

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“…It also plays a crucial role in altering the host immune system at the time of infection. 4, 5 Intriguingly, the adaptability of the cAMP-binding domain has evolved in such a way that it can perform diverse physiological functions in response to a broad range of signals, [6][7][8] with the best and the most studied prototype proteins of this family being c-AMP receptor protein (CRP) and fumarate and nitrate reduction regulatory protein (FNR) of E. coli, which are known to control genes in hypoxia and starvation, respectively. 7 Mycobacterium tuberculosis H37Rv also has a CRP/FNR homolog, CRP Mt (Rv3676) the protein is encoded by the gene rv3676.…”

Section: Introductionmentioning

confidence: 99%

Detailed Stability and Unfolding Study of Mycobacterium Global Transcription Regulator Protein

Saha,

Chakravarty,

Chakraborti

2023

Gene Expr

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Background and objectives:The cyclic adenosine monophosphate Receptor Protein of Mycobacterium tuberculosis (CRP Mt ) (Rv3676), is a global transcriptional regulator and plays a pivotal role in the survival and infection of Mycobacterium. This signaling protein (CRP Mt ) shares several common structural and functional features with the CRP from Escherichia coli. Structurally, CRP Mt is a homodimer that undergoes allosteric changes upon cyclic AMP binding. This binding also triggers the activation of several genes responsible for various physiological processes in this bacterium. Despite the importance of CRP for mycobacterial survival, limited information is available regarding the stability and unfolding properties of the protein. The main objective of this study is to study stability, unfolding and dynamics of CRP Mt in terms of its structure. Methods:In this study, we monitored the stability and unfolding of CRP Mt using various biophysical and computational techniques. Results:We experimentally studied protein unfolding in the presence of chemical denaturants [urea and guanidine hydrochloride (GdnHCl)]. The results from these chemical-induced unfolding studies suggest that CRP Mt follows a two-state transition and that chemical-induced protein denaturation is reversible. According to circular dichroism and activity data, CRP Mt structure and function were restored upon refolding. We also studied the stability and unfolding of the CRP Mt protein against temperature variations and protease action (trypsin). Limited proteolysis experiments provide insights into the minimum domain structure requirement for CRP Mt activity. Interestingly, temperature-induced CRP Mt unfolding was completely different compared to chemical-induced unfolding. The thermal unfolding of CRP Mt was found to be irreversible, leading to the formation of insoluble aggregates at elevated temperatures. To understand why the thermal unfolding of the protein differed from chemically induced unfolding, we carried out a detailed molecular dynamics simulation analysis of the protein at three different temperatures. The results from these molecular dynamics simulations mechanistically validate the significant differences between chemical and temperature-induced CRP Mt unfolding. Conclusion:Our study provides detailed insights into the stability and folding/unfolding properties of CRP Mt , which could be useful in developing new anti-mycobacterial medicines.

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Protein SUMOylation promotes cAMP-independent EPAC1 activation

Yang,

Mei,

Lin

et al. 2024

Cell. Mol. Life Sci.

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Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

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Origin and Isoform Specific Functions of Exchange Proteins Directly Activated by cAMP: A Phylogenetic Analysis

Cited by 5 publications

References 83 publications

Protein SUMOylation promotes cAMP-independent EPAC1 activation

Protein SUMOylation promotes cAMP-independent EPAC1 activation

Detailed Stability and Unfolding Study of Mycobacterium Global Transcription Regulator Protein

Protein SUMOylation promotes cAMP-independent EPAC1 activation

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