Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target

Sakane, Kyohei; Nishiguchi, Miyu; Denda, Miwako; Yamagchi, Fuminori; Kanayama, Naoki; Morishita, Ryo; Tokumitsu, Hiroshi

doi:10.1016/j.bbrc.2017.07.161

Cited by 10 publications

(17 citation statements)

References 28 publications

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“…They are generally considered as relatively low specificity proteins, with dozens of interaction partners, among them they are unable to maintain a high selectivity [15]. In this study we determined the interaction profile of the full human S100 family (termed here as the S100ome) against a set of diverse known S100 partners (and some of their paralogs) systematically, including kinases such as RSK1 [16] and its paralogs MK2 and MNK1; cytoskeletal elements such as CapZ [17] (commonly known as TRTK12), NMIIA [18], ezrin [19], FOR20 and its paralog FOP [20]; membrane proteins such as NCX1 [15] and TRPM4 [21]; and other signaling proteins such as the tumor suppressor p53 [22][23][24], SIP [15] and MDM4 [23].…”

Section: Introductionmentioning

confidence: 99%

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

Simon

Gógl

Ecsédi

et al. 2019

Preprint

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The calcium-binding, vertebrate-specific S100 protein family consists of 20 paralogs in humans (referred as the S100ome), with several clinically important members. To assess their interactome, high-throughput, systematic analysis is indispensable, which allows one to get not only qualitative but quantitative insight into their protein-protein interactions (PPIs). We have chosen an unbiased assay, fluorescence polarization (FP) that revealed a partial functional redundancy when the complete S100ome (n=20) was tested against numerous model partners (n=13). Based on their specificity, the S100ome can be grouped into two distinct classes: promiscuous and orphan. In the first group, members bound to several ligands (>4-5) with comparable high affinity, while in the second one, the paralogs bound only one partner weakly, or no ligand was identified (orphan). Our results demonstrate that in vitro FP assays are highly suitable for quantitative ligand binding studies of selected protein families. Moreover, we provide evidence that PPI-based phenotypic characterization can complement the information obtained from the sequence-based phylogenetic analysis of the S100ome, an evolutionary young protein family. Author summaryFunctional similarity among a protein family can be essential in order to understand proteomic data, to find biomarkers, or in inhibitor design. Proteins with similar functions can compensate the loss-offunction of the others, their expression can co-vary under pathological conditions, and simultaneous targeting can lead to better results in the clinics. To investigate this property one can use sequencebased approaches. However, this path can be difficult. In the case of the vertebrate specific, evolutionary young, S100 family, phylogenetic approaches lead to ambiguous results. To overcome this problem, we applied a high-throughput biochemical approach to experimentally measure the binding affinities of a large number of S100 interactions. We performed unbiased fluorescence polarization assay, involving the complete human S100ome (20 paralogs) and 13 known interaction partners. We used this measured 20x13 (260) protein-protein interaction array to reveal the functional relationships within the family. Our work provide a general framework for studies focusing on phenotype-based domain classification.

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Section: Introductionmentioning

confidence: 99%

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

Simon

Gógl

Ecsédi

et al. 2019

Preprint

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“…Protein array plates (Protein Active Array ® , 1536-well format × 27 plates) containing 19,676 recombinant human proteins in duplicate, generated by wheat germ cell-free protein synthesis, were constructed by CellFree Sciences [17], and the screening for protein-protein interactions was performed by incubation with 0.36 µg/mL biotinylated S100A6, followed by a streptavidin-horseradish peroxidase-mediated enhanced chemiluminescence (ECL) detection of S100A6 binding with the ChemiDoc™ XRS (Bio-Rad Laboratories, Hercules, CA, USA), as previously reported [18].…”

Section: Screening Of S100a6 Targets Using Human Protein Arraymentioning

confidence: 99%

“…To comprehensively analyze target proteins of EF-hand Ca 2+ -binding proteins, including calmodulin and S100 proteins, we developed a novel genome-wide screening method based on the protein-protein interactions using the Protein Active Array ® (CellFree Sciences, Ehime, Japan), carrying 19,676 recombinant glutathione s-transferase (GST)-fusion proteins [17]. The use of this method resulted in the identification of the striated muscle activator of Rho signaling as a calmodulin interactant, and FOR-20 (FOP-related protein of 20 kDa) as a novel S100A6-interacting protein [18]. In this study, during the course of human protein array screening with biotinylated S100A6 as a ligand, we discovered HMG20A (high-mobility group protein 20A) as a novel S100A6-binding protein.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…These latter ligands implicate S100A6 in nuclear transport and the maintenance of nuclear structure but up to now there are no functional data concerning these interactions. Quite recently three centrosomal proteins, possibly involved in cilia formation, namely FOR20, FOP and OFD have been added to the list of S100A6 ligands [Sakane et al, 2017]. Last but not least, research concerning S100A6 revealed a structurally similar but functionally diverse group of S100A6 ligands consisting of TPR or CS domain -containing proteins.…”

Section: S100a6 Ligandsmentioning

confidence: 99%

Aktualny pogląd na komórkową funkcję S100A6 i jego ligandów, CacyBP/SIP i Sgt1

Filipek

2018

Postepy Biochem

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S100A6 jest białkiem wiążącym wapń, którego gen zidentyfikowano po raz pierwszy jako gen indukowany czynnikami wzrostu. Od tego czasu przyjmuje się, że S100A6 jest białkiem biorącym udział w proliferacji i pokrewnych procesach komórkowych. Podczas gdy struktura S100A6 i kinetyka wiązania Ca2+ zostały dość wcześnie określone to badania nad mechanizmem działania S100A6 są prowadzone stosunkowo od niedawna. Niemniej wydaje się oczywiste, że S100A6 pełni swoją funkcję w komórce poprzez oddziaływanie w wieloma ligandami, z których większość została zidentyfikowana w naszym zespole. Ostatnio nasze badania koncentrują się na dwóch ligandach S100A6, to jest białkach CacyBP/SIP i Sgt1, które posiadają też swoje własne białka docelowe. Długa lista ligandów S100A6, CacyBP/SIP i Sgt1 wskazuje, że białka te są składnikami złożonej sieci interakcji i mogą być zaangażowane nie tylko w proliferację ale również w wiele innych procesów komórkowych, z których różnicowanie czy odpowiedź na stres wydają się być najlepiej udokumentowane.

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Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target

Cited by 10 publications

References 28 publications

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

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

Aktualny pogląd na komórkową funkcję S100A6 i jego ligandów, CacyBP/SIP i Sgt1

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