The S100 calcium binding protein A3 binds directly and specifically - - PowerPoint PPT Presentation

The S100 calcium binding protein A3 binds directly and specifically to RARα and PML-RAR and modulates the ac@vity of APL cells

Giannì M., Terao M., Brunelli L., Borleri G., GaraHni E.

Aim of the work

• Iden@fica@on of novel RARα interac@ng proteins

using a differen@al proteomic approach

Experimental design

• Flag-vector and Flag-RARα were introduced in breast MDA-MB453

cells and stable cell lines named respec@vely FL53 and RA53 were selected

• Cells were treated 1hr with DMSO (vehicle) or ATRA
• Protein extracts of these cell lines were immunoprecipitated with

agarose-flag.

• To screen for RARα binding proteins, a differen@al proteomic

approach based on the quan@ta@ve SILAC (Stable Isotope Labelling with Amino acids in Cell culture) was applied

Valida@on of the interac@on between HSP27, FABP5 and S100A3 with RARα

• Prof. Rosario Donato

The Interna2onal Journal of Biochemistry & Cell Biology 33 (2001) 637–668

S100: a mul@genic family of calcium-modulated proteins of the EF - hand type with intracellular and extracellular func@onal roles

Schema'c representa'on

• f t h e s e c o n d a r y

structure of an S100 protein. S100A3 dimer 3D structure S100A3 primary sequence

10 20 30 40 50 MARPLEQAVA AIVCTFQEYA GRCGDKYKLC QAELKELLQK ELATWTPTEF 60 70 80 90 100 RECDYNKFMS VLDTNKDCEV DFVEYVRSLA CLCLYCHEYF KDCPSEPPCS Q

Ca++ domain Zn++ domain N-terminal non-canonical EF-hand C-terminal EF-hand

• Biochem. J. (2006) 396, 201–214

Calcium-dependent and -independent interac@ons of the S100 family

SKBR3 cells over-expressing S100A3 are characterized by:

• increase in RARα expression
• decrease responsiveness to ATRA

pCDNA3 pCDNA3-S100A3

S100A3 S100A3 HOECHST HOECHST RARa RARa S100A3+RARa S100A3+RARa

In COS-7 cells and in RA53 cells over-expressing RARα and S100A3

• S100A3 signal is diffused in whole cell but it accumulates on cell

membrane

• RARα signal is present only in the nucleus

In COS-7 cells, S100A3 interacts with RARα only .

• S100A3

+ S100A3

2 4 8 12 24 0 2 4 8 12 24 CHX (hrs)

β-ac@n S100A3 PML-RAR RARα

• In COS-7 cells , over-expression of S100A3 induces an increase in RARα and

PML-RAR expression

• S100A3-induced increase of RARα and PML-RAR is the result of a decreased

degrada'on of the two receptors.

S100A3 interacts directly with RARα

Iden@fica@on of the domain of RARα protein interac@ng with S100A3

GST-pull down + + +

• +
• +
• +

interac@on

Part of RARα E region interac@ng with S100A3

S100A3 stabiliza@on correlates with a decrease of RARα or PML-RAR- mediated transac@va@on

BLOOD, 6 APRIL 2017 - VOLUME 129, NUMBER 14

Effects of S100A8 and S100A9 on AML cell differen@a@on and prolifera@on

• S100A9 induces differen@a@on and growth

arrest of AML cells via TLR4.

• S100A8 regulates S100A9 ac@vity and

sustains AML immature phenotype

• ATRA–mediated expression of S100A9 in

APL cells promotes apoptosis and growth inhibi@on

NB4 cells infected with shRNA against S100A3

5 cell popula@on were selected and expanded: pGreen (empty vector) SCRA (sh scramble) SH1 (SH1-S100A3) SH2 (SH2-S100A3) SH3 (SH3-S100A3)

ATRA

In NB4 silencing of S100A3

• decrease RARα and PML-RAR expression
• increase responsiveness of different markers of NB4 ATRA-

mediated differen@a@on

Expression of granulocy@c and monocyte differen@a@on markers in S100A3 silenced NB4 cell lines

Silencing of S100A3 in NB4 cells decrease ATRA-dependent cell growth

• No. viable cells (x 106)
• ATRA

+ ATRA hrs

Synergis@c effect of Midostaurin (inhibitor of FLIT3) and ATRA in NB4 cells