Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target

Abstract

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

S100A6 Protein—Expression and Function in Norm and Pathology

“…Although the functional consequences of this interaction have not been studied, it implicates S100A6 in the structural aspects of the nucleus and, potentially, in chromatin dynamics. This assumption is reinforced by a recent report identifying high mobility group protein 20A (HMG20A), a component of the lysine-specific demethylase 1(LSD1)-REST co-repressor 1 (CoREST) complex, among S100A6 ligands [ 65 ]. Another nuclear target of S100A6, importin α, implicates it in regulation of nuclear transport.…”

“…The S100 proteins usually interact with their ligands with a 1:1 stoichiometry, which means that two ligand molecules bind per S100A6 dimer, forming a heterotetramer. Nonetheless, binding of one ligand molecule to the S100A6 dimer was postulated for CacyBP/SIP, FOR20 and HMG20A [ 61 , 65 , 99 ]. Moreover, based on structural data, the possibility of simultaneous binding of more than one ligand cannot be excluded [ 10 ].…”

The H2A.Z and NuRD associated protein HMG20A controls early head and heart developmental transcription programs

“…TEAD1, which is one of the core cardiac transcription factors in heart development 39 was also repeatedly detected in our HMG20A interactome data sets and recently described to bind to HMG20A 31 . Apart from these interacting partners that we identified, HMG20A was also shown to bind to Ca2 + /S100A6, a protein that contributes to cellular calcium signalling 53 . Very interestingly, we found that beating of Hmg20a DP cardiomyocytes was abolished or delayed.…”

The H2A.Z.1/PWWP2A/NuRD-associated protein HMG20A controls early head and heart developmental transcription programs

“…Other HMG20A interacting proteins, such as TEADs, also need to be highlighted, as TEAD1 acts as one of the core cardiac transcription factors in heart development 40 . Apart from these interacting partners we identified, it is notable that Yamamoto et al, reported the interaction of HMG20A with Ca2+/S100A6, a protein that contributes to cellular calcium signalling 51 . Very interestingly, we found that Hmg20a KO cardiomyocytes are either unable to beat or with much reduced contractility (after a delay) as compared to its WT counterparts.…”