Rabbit Anti-Human S100A3 (a.a. 1-101) Polyclonal antibody

All trans-retinoic acid (ATRA) is used in the treatment of acute promyelocytic leukemia (APL) and it is a promising agent also in solid tumors. The pharmacological activity of ATRA is mediated by the ligand-activated RAR and RXR transcription factors. In the present study, we define the basal and ATRA dependent RAR alpha interactome in a RAR alpha-overexpressing breast cancer cellular model, identifying 28 nuclear proteins. We focus our attention on the S100A3 calcium-binding protein, which interacts with RAR alpha constitutively. In ATRA-sensitive breast cancer cells, S100A3 binds to RAR alpha in basal conditions and binding is reduced by the retinoid. The interaction of S100A3 with RAR alpha is direct and in lung cancer, APL and acute-myeloid-leukemia (AML) cells. In APL, S100A3 interacts not only with RAR alpha, but also with PML-RAR alpha. The interaction surface maps to the RAR alpha ligand-binding domain, where the I396 residue plays a crucial role. Binding of S100A3 to RARWPML-RAR alpha controls the constitutive and ATRA-dependent degradation of these receptors. S100A3 knockdown decreases the amounts of RAR alpha in breast- and lung cancer cells, inducing resistance to ATRA-dependent antiproliferative/differentiating effects. Conversely, S100A3 knockdown in PML-RAR alpha(+) APL and PML-RAR alpha(-) AML cells reduces the amounts of RAR alpha/PML-RAR alpha and increases basal and ATRA-induced differentiation. In this cellular context, opposite effects on RAR alpha/PML-RAR alpha levels and ATRA-induced differentiation are observed upon S100A3 overexpression. Our results provide new insights into the molecular mechanisms controlling RARa activity and have practical implications, as S100A3 represents a novel target for rational drug combinations aimed at potentiating the activity of ATRA.

S100A3, a member of the EF-hand-type Ca2+-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn2+ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca2+/Zn2+ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A+C68A) abolished the Ca2+ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type Ill (PAD3), while SS2 disruption inversely increased both Ca2+ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A+C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15-1.40 angstrom resolution shows that SS1 renders the C-terminal classical Ca2+-binding loop flexible, which are essential for its Ca2+ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn2+ by (Cys)(3)His residues that is compatible with SS2 formation in S100A3. (c) 2011 Elsevier Ltd. All rights reserved.