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. 2010 Mar 23;49(11):2491-501.
doi: 10.1021/bi902135e.

Structural analysis and functional implications of the negative mTORC1 regulator REDD1

Silvia Vega-Rubin-de-Celis  1 , Zeina Abdallah, Lisa Kinch, Nick V Grishin, James Brugarolas, Xuewu Zhang
Affiliations

Structural analysis and functional implications of the negative mTORC1 regulator REDD1

Silvia Vega-Rubin-de-Celis et al. Biochemistry. .

Abstract

REDD1 is a conserved stress-response protein that regulates mTORC1, a critical regulator of cell growth and proliferation that is implicated in cancer. REDD1 is induced by hypoxia, and REDD1 overexpression is sufficient to inhibit mTORC1. mTORC1 is regulated by the small GTPase Rheb, which in turn is regulated by the GTPase-activating protein complex, TSC1/TSC2. REDD1 induced-mTORC1 inhibition requires the TSC1/TSC2 complex, and REDD1 has been proposed to act by directly binding to and sequestering 14-3-3 proteins away from TSC2 leading to TSC2-dependent inhibition of mTORC1. Structure/function analyses have led us to identify two segments in REDD1 that are essential for function, which act in an interdependent manner. We have determined a crystal structure of REDD1 at 2.0 A resolution, which shows that these two segments fold together to form an intact domain with a novel fold. This domain is characterized by an alpha/beta sandwich consisting of two antiparallel alpha-helices and a mixed beta-sheet encompassing an uncommon psi-loop motif. Structure-based docking and functional analyses suggest that REDD1 does not directly bind to 14-3-3 proteins. Sequence conservation mapping to the surface of the structure and mutagenesis studies demarcated a hotspot likely to interact with effector proteins that is essential for REDD1-mediated mTORC1 inhibition.

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Figures

FIGURE 1
FIGURE 1
REDD1 is both necessary and sufficient for hypoxia-induced mTORC1 inhibition. (A) Immunoblot analysis of HeLa cells in normoxia (N) or hypoxia (H). (B) Western blot analyses of U2OS HA-REDD1-inducible cells treated with tetracycline (Tet) for the indicated number of hours. Western blot analyses (C) and m7GTP pulldown assays (D) of U2OS HA-REDD1-inducible cells (or parental U2OS cells as a control). (E) Western blot of HeLa cells transfected with the stated siRNA oligos and exposed to normoxia (N) or hypoxia (H). (F) Immunoprecipitation analysis of TSC1/TSC2 binding to REDD1 in U2OS HA-REDD1-inducible cells treated with tetracycline (G) Western blot of U2OS HA-REDD1 cells transfected with the indicated amounts of Flag-Rheb (or EGFP as a control) induced (or not) with tetracycline (Tet). (H) Western blot of HeLa cells transfected with the indicated siRNA oligos and exposed to normoxia (N) or hypoxia (H).
FIGURE 2
FIGURE 2
Deletion/substitution analyses identify two regions in REDD1 required for mTORC1 inhibition, which do not function as dominant negative. Western blot analyses of HeLa cells transfected with expression vectors for HA-S6K1 and various HA-REDD1 mutants: (A) N-terminal deletions, (B) C-terminal deletions, (C) internal deletions and (D, E) substitutions with a flexible linker (NAAIRS [single letter amino acid code]) Western blot analyses of U2OS HA-REDD1-inducible cells transfected with HA-S6K1 along with either GST-HA-REDD185-193 (F) or GST-HA-REDD1207-225 (G) using GST as a control, and induced (or not) with tetracycline (Tet) to express HA-REDD1.
FIGURE 3
FIGURE 3
REDD1 exhibits a novel topology and does not oligomerize. (A) Cartoon representation of the REDD189-226Δϕ structure colored in rainbow mode from the N- to the C-terminus. The dotted line represents disordered region. The black arrowhead indicates the location of the 200FLPGF204 deletion. (B) Diagram of REDD1 topology. (C) Gel filtration chromatography of purified REDD189-226Δϕ (elution positions of molecular weight standards indicated). (D) Western blot analyses of anti-V5 immunoprecipitates (or inputs) from U2OS HA-REDD1-inducible cells transfected with V5-REDD1 (or empty vector, EV) and induced (or not) with tetracycline (Tet).
FIGURE 4
FIGURE 4
Closest structural relatives of REDD1 exhibit markedly different topologies. (A) Ribbon model of REDD189-226Δϕ (strands labeled numerically from N- to C-terminus [β1-β4] including connecting psi-loop formed by β1-β3). (B) Ribbon structure of the top dali hit (ID: 1i2l) (strand order [2’3401’] based on strands β3 and β4 corresponding to REDD1). (C) Ribbon structure of top ProSMoS hit, the YrdC-like hypothetical protein (PDB ID: 1K7J) with YrdC/RibA fold core depicted in gray. Ribbon structures of ProSMoS-identified psi-loops in a pua domain-like structure (PDB ID: 3d79; D) and a lexA-like family structure (PDB ID: 2fjr; E). In structures B-E, secondary structural elements (β strands and connecting psi-loop) corresponding to those present in REDD1 are similarly colored; unrelated structures are colored in white or gray.
FIGURE 5
FIGURE 5
Conservation mapping onto REDD1 protein surface and mutagenesis studies reveal a functionally important hotspot. (A) Conservation mapping of REDD1 surface using an increasing blue color gradient proportional to the degree of conservation and inlet stick representation of conserved residues. (B) Sequence alignment of REDD1 from different species with blue color gradient signifying conservation (as in A) and red boxes demarcating the two stretches of sequence forming the conserved surface patch. (C, D) Functional evaluation of residues involved in conserved surface patch by mutagenesis and western blot analyses in HeLa cells transfected with HA-S6K1 (EV, empty vector).
FIGURE 6
FIGURE 6
Structure-based docking studies of REDD1 binding to 14-3-3. (A) Illustration of peptides (yellow) in 14-3-3ζ binding mode I (PDB ID: 1QJB) and II (PDB ID: 1QJA) compared to the putative 14-3-3 binding motif in REDD1. (B) Depiction of unusual exoenzyme S binding to 14-3-3β (PDB ID: 2C23) and docking of REDD189-226Δϕ based on this binding mode showing multiple steric clashes. (C) Functional evaluation of putative 14-3-3-binding site by mutagenesis and western blot analyses in HeLa cells transfected with HA-S6K1 (pcDNA3 transfected HeLa cells exposed to either normoxia [N] or hypoxia [H] are shown as a control).
FIGURE 7
FIGURE 7
REDD1 does not interact with 14-3-3 proteins in vitro or in vivo. (A) Elution profile of the recombinant REDD189-226Δϕ and 14-3-3β mix and Coomasie stained SDS-PAGE analyses from corresponding fractions; purified REDD189-226Δϕ and 14-3-3β shown as controls. (B) Immunoprecipitation studies of both 14-3-3 and HA-REDD1 in U2OS HA-REDD1-inducible cells induced (or not) to express REDD1. (C) Immunoprecipitation analysis of both 14-3-3 and endogenous REDD1 in HeLa cells in which REDD1 is induced by hypoxia (H), or not (normoxia, N); in vitro translated Flag-PRAS40 [IVT] is shown as a control. TSC2 binding to 14-3-3 analysis in (D) U2OS HA-REDD1-inducible cells treated (or not) with insulin (Ins) or Tetracycline (Tet) and (E) HeLa cells exposed to normoxia (N) or hypoxia (H).
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