SREBPs: protein interaction and SREBPsRyuichiro Sato Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan Introductio
Trang 1SREBPs: protein interaction and SREBPs
Ryuichiro Sato
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan
Introduction
The sterol regulatory element-binding protein
(SREBP) family members SREBP-1 and SREBP-2
are localized on the endoplasmic reticulum (ER) as
membrane proteins after being synthesized Once the
intracellular cholesterol level is decreased, the
SREBPs subsequently move in vesicles to the Golgi
complex, where they are processed sequentially by
two proteases These cleavage steps release the
mature forms of SREBPs, which enter the nucleus
and activate genes related to cholesterol and fatty
acid metabolism [1,2] In both the cytoplasm and
nucleus, SREBPs associate with a variety of proteins
This interaction determines their intracellular
translo-cation and stability, and also regulates their activities
as transcriptional factors
Protein interaction on the ER and in the cytosol
SREBPs are localized on the ER membrane, associat-ing with another ER membrane protein, SREBP cleav-age-activating protein (SCAP) (Fig 1) SCAP has two distinct domains The N-terminal domain has eight transmembrane helices, which include the so-called ste-rol-sensing domain This domain resembles sequences
in three other proteins that are postulated to interact with sterols: HMG-CoA reductase, the Niemann–Pick C1 protein, and Patched [3] The C-terminal domain of SCAP contains five WD repeats, which are sequences
of 40 amino acids found in many proteins involved
in protein–protein interactions The WD repeat domain is the region of SCAP that forms a complex with the C-terminal domain of SREBPs [4] When cells
Keywords
ATF6; HNF-4; importin; LRH-1; PGC-1; S1P;
S2P; SCAP; SREBPs
Correspondence
R Sato, Department of Applied Biological
Chemistry, Graduate School of Agricultural
and Life Sciences, The University of Tokyo,
Tokyo 113-8657, Japan
Fax: +81 3 5841 5136
Tel: +81 3 5841 8029
E-mail: aroysato@mail.ecc.u-tokyo.ac.jp
(Received 6 August 2008, revised 22
October 2008, accepted 24 October
2008)
doi:10.1111/j.1742-4658.2008.06807.x
Sterol regulatory element-binding proteins (SREBPs) are tightly controlled
by various mechanisms, including intracellular localization, protein process-ing, limited proteolysis, post-translational modifications and interaction with associated proteins Here, I review the regulatory mechanisms of SREBP activity through the interaction with various kinds of protein
Abbreviations
AFF6, activating transcription factor-6; ARC, activator-recruited co-factor; CBP, CREB-binding protein; ER, endoplasmic reticulum; HNF-4, hepatocyte nuclear factor-4; LDL, low-density lipoprotein; LRH-1, liver receptor homolog-1; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1, peroxisome proliferator-activated receptor-c coactivator-1; S1P, site 1 protease; S2P, site 2 protease; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein; SUMO, small ubiquitin-like modifier.
Trang 2become depleted in cholesterol, SCAP escorts SREBPs
from the ER to the Golgi apparatus, where two
proteases, designated site 1 protease (S1P) and site 2
protease (S2P), reside In the Golgi apparatus, S1P, a
membrane-bound serine protease, cleaves SREBPs in
the luminal loop between their two
membrane-span-ning segments The N-terminal domain is then released
from the membrane by S2P, a membrane-bound zinc
metalloproteinase In the cytoplasm, the N-terminal
cleaved forms of SREBPs interact with importin-b, an
escort protein of nuclear proteins, and thereafter are
transported into the nucleus [5] It is a quite
character-istic transport pathway, in that the nuclear import
occurs in the absence of importin-a Furthermore, the
dimerization of SREBPs via the leucine zipper domain
is required for the interaction with importin-b [6] In
the nucleus, SREBPs detach from importin-b, and
their transcription factor activities are regulated
through interaction with a variety of nuclear proteins
Interaction with the ubiquitous
transcription factors Sp1 and NF-Y
in the nucleus
SREBPs were first discovered as transcription factors
that stimulate low-density lipoprotein (LDL) receptor
gene expression [7] In the promoter of the LDL
recep-tor gene, a pair of essential elements exists to which a
ubiquitous transcription factor, Sp1, binds An
SREBP-binding site, SRE, is closely located between
these two Sp1-binding sites, and all of these sites are
required for full activation of the LDL receptor
pro-moter Similarly, a number of promoters of the
SREBP target genes contain the SREs and certain proximal elements to which another ubiquitous tran-scription factor, NF-Y, binds [8–10] SREBPs physi-cally interact with both Sp1 and NF-Y, thereby synergistically augmenting the transcription of the target genes [11,12]
Coactivators directly bind to SREBPs, thereby inducing the transcription of target genes
Transcription factors, which bind to specific nucleotide sequences in the promoter region, require coactivators that facilitate access of the transcriptional machinery
to a nucleosomal template One of the well-known coactivators, CREB-binding protein (CBP), interacts with the N-terminal region of SREBP-1 [13], thereby supporting high levels of synergistic activation by SREBPs CBP possesses acetyltransferase activity, and therefore is thought to be involved in the acetylation
of histones and alteration of chromatin structure A recent study revealed that the activator-recruited co-factor (ARC)–mediator coactivator complex, a large complex associating with RNA polymerase II, interacts with SREBPs through structurally related motifs in both CBP and the ARC105 subunit of the ARC–mediator coactivator complex [14] How SREBPs recruit both of these coactivators on target genes remains unclear
The peroxisome proliferator-activated receptor-c coactivator-1 (PGC-1) family of coactivators is of particular importance in the control of liver metabo-lism PGC-1a stimulates mitochondrial biogenesis and
Fig 1 SREBPs interact with SCAP or
importin-b in the cytoplasm SREBPs
local-ized on the ER membrane associate with
another ER membrane protein, SCAP This
complex is transported to the Golgi
appara-tus, where SREBPs are processed
sequen-tially by two proteases, S1P and S2P The
cleaved forms of SREBPs, as homodimers,
interact with importin-b, which escorts them
to the nucleus.
Trang 3respiration, and modulates hepatic gluconeogenesis In
contrast, PGC-1b, a transcriptional coactivator closely
related to PGC-1a, is highly induced in response to
short-term high-fat feeding in mice PGC-1b interacts
with SREBPs, thereby inducing a broad program of
lipid metabolism, including de novo lipogenesis and
lipoprotein secretion [15] This suggests, at least in
part, a mechanism through which dietary saturated
fats stimulate hyperlipidemia and atherogenesis
SREBPs dock on PGC-1b at a domain that has no
counterpart in PGC-1a, and hence, PGC-1a does not
coactivate the SREBPs
Protein modification of SREBPs
regulates their activities
In the nucleus, SREBPs are unstable and rapidly
degraded by the ubiquitin–proteasome pathway [16]
The rapid turnover of nuclear SREBPs is not affected
by the intracellular sterol levels, and the half-life is
estimated to be 3 h In the presence of proteasome
inhibitors, nuclear SREBPs become stable and enhance
the transcription of endogenous target genes
Polyubiq-uitination is the rate-limiting step in protein
degrada-tion, and involves a three-step cascade of ubiquitin
transfer reactions The ubiquitin ligase (E3) required
for the final reaction of SREBP ubiquitination is a
complex consisting of Skp1, Cul1, Rbx1, and one of a
family of F-box proteins, Fbw7 [17] Glycogen
syn-thase kinase-3b phosphorylates serine and⁄ or threonine
residues near the ubiquitination site in SREBP-1 and
SREBP-2, and SREBP ubiquitination is enhanced in a
manner dependent on the phosphorylation SREBPs
do interact with the Fbw7 protein when the
phosphor-ylation is enhanced by glycogen synthase kinase-3b
SREBPs are modified by another protein, small
ubiquitin-like modifier (SUMO) SUMO-1 is a 101
amino acid protein having 18% identity with ubiquitin,
but with a remarkably similar secondary structure
With the increase in the number of proteins modified
by SUMO, it has become obvious that the effects of
SUMO conjugation are diverse and largely depend on
the function of the protein targeted for sumoylation
Sumoylation of transcription factors, including
SREBPs, is prone to result in attenuation of their
tran-scriptional activities [18–20] Sumoylation requires a
multistep reaction similar to that of ubiquitination, but
the specific enzymes are distinct from those involved in
ubiquitination Ubc9 is a SUMO-conjugating enzyme
(E2) that directly interacts with most sumoylated
pro-teins, including SREBPs [20] In some cases, ubc9 itself
plays, to a certain extent, a SUMO E3-like role in the
absence of any E3 ligases Unlike ubiquitination,
which requires phosphorylation near the ubiquitination site, sumoylation competes with the phosphorylation near the sumoylation site, which occurs in response to growth factor stimuli [21] This implies that growth factor stimuli interfere with sumoylation, thereby enhancing SREBP transcriptional activities, and lipid synthesis required for cell growth Sumoylated SREBPs recruit a corepressor complex containing his-tone deacetylase 3 to suppress their transcriptional activities [21] Histone deacetylase 3 is unable to directly interact with SREBPs, but a certain subunit in the corepressor complex, which is not yet identified, is considered to be involved in the interaction
SREBPs interact with activating transcription factor-6 (ATF6) and nuclear receptors to regulate their transcriptional activities
ATF6 is an ER membrane-bound transcription factor activated in response to ER stress During the quies-cent state, the C-terminus of ATF6 resides in the ER lumen, with its N-terminus projecting into the cytosol Once unfolded or misfolded proteins accumulate in the
ER, ATF6 moves from the ER to the Golgi, where both ATF6 and SREBPs are cleaved by S1P and S2P Such proteolytic cleavage causes the nuclear localiza-tion of the N-terminal leucine zipper transcriplocaliza-tion factor to direct the transcriptional activation of the chaperone molecules and enzymes essential for protein folding [22,23] Overexpression of the cleaved form of ATF6, and also glucose depletion, which causes ATF6 cleavage, suppresses the transcription of SREBP target genes, suggesting that the interaction between ATF6 and SREBPs inhibits SREBP-mediated transcription Indeed, ATF6 interacts with SREBP-2 via its leucine zipper domain, thereby reducing the transcriptional activity of SREBP through the recruitment of histone deacetylase 1 [24]
In the liver, several types of nuclear receptor orches-trate glucose and lipid metabolism Among them, hepatocyte nuclear factor-4 (HNF-4), which was ini-tially identified as a transcription factor essential for liver-specific gene expression, activates the expression
of the gluconeogenic genes encoding phosphoenolpyr-uvate carboxykinase (PEPCK) and glucose-6-phospha-tase At the same time, HNF-4 also regulates the expression of certain crucial genes for lipid metabo-lism, including the genes encoding apolipoprotein B and microsome triglyceride transfer protein [25] Over-expression of SREBP-1 severely reduces the hepatic expression level of PEPCK in SREBP-1a transgenic and SREBP-1c adenovirus-infected mice SREBP-1
Trang 4does not directly bind to the PEPCK promoter, but
the HNF-4-binding site is responsible for the SREBP-1
inhibition SREBPs and HNF-4 physically interact
through the N-terminal transactivation domain of
SREBP and the C-terminal ligand-binding domain of
HNF-4 [26] HNF-4 recruits a coactivator, PGC-1a,
for its transcriptional activation SREBPs interfere
with this recruitment of PGC-1a Under fasting
condi-tions, HNF-4 and PGC-1a vigorously activate the
expression of gluconeogenic genes In contrast, under
feeding conditions, SREBP-1c, the expression of which
is highly enhanced by insulin, might negatively regulate
HNF-4 transcriptional activity by competing with
PGC-1a, leading to a reduction of gluconeogenesis
In contrast to the above findings, the transcriptional
activity of SREBPs is augmented by HNF-4 [27]
Overexpression of HNF-4 enhances the expression of
SREBP target genes in culture cells, but not through
the direct binding of HNF-4 to the promoters HNF-4
interaction with SREBPs probably augments their
transcriptional activities due to HNF-4-mediated
recruitment of several coactivators, which are not
recruited by SREBPs alone, including PGC-1a In the
liver and intestine, where lipid biosynthesis is quite
active and HNF-4 is exclusively expressed, the
syner-gistic activity of SREBPs and HNF-4 might cause
lipids to be distributed to other tissues that do not
have the capacity to biosynthesize sufficient lipids on
their own
In a study aimed at identifing other nuclear receptor
family members affecting SREBP transcriptional
activ-ities, liver receptor homolog-1 (LRH-1) was found to
suppress them [28] (Fig 2) Unlike other nuclear
recep-tor family members, LRH-1 acts as a monomer
transcription factor to regulate the expression of genes related to cholesterol metabolism, including the genes encoding CYP7A1, a rate-limiting enzyme for bile acid synthesis, and apolipoprotein A-1, a high-density lipo-protein lipo-protein The basic helix–loop-helix leucine zipper domain in SREBPs binds to the ligand-binding domain in LRH-1, thereby reciprocally suppressing their transcriptional activities SREBPs interfere with the recruitment of a coactivator of LRH-1, PGC-1a, resulting in the inhibition of LRH-1 activity When human hepatoma HepG2 cells are cultured with an HMG-CoA reductase inhibitor, statin, the reduction of intracellular cholesterol levels activates SREBPs, and eventually suppresses the expression of LRH-1 target genes, including the genes encoding CYP7A1, apolipo-protein A-I, and the small heterodimer partner Although most nuclear receptors are activated only when their specific ligands are present, HNF-4 and LRH-1 appear to be exceptional, in that they are con-stitutively active in the abundance of their endogenous ligands, acyl-CoA and phospholipids, respectively [29,30] The small heterodimer partner, one of the nuclear receptor family members, acts as a negative regulator of both HNF-4 and LRH-1 by suppressing their transcriptional activities SREBPs might consti-tute another group of inhibitory nuclear factors modu-lating the activity of these nuclear receptors in response to a wide variety of physiological changes
Conclusions
SREBPs are translocated from the ER to the Golgi complex, where they are processed, and then trans-ported into the nucleus In this pathway, two
interact-Fig 2 HNF-4 stimulates and LRH-1 suppresses the transcriptional activities of SREBP-1a and SREBP-2 HEK293 cells were transfected with either 0.1 lg of pGAL4–SREBP1a (Gal4–DBD–SREBP1a) or pGAL4–SREBP2 (Gal4–DBD–SREBP2), 0.2 lg of pG5Luc containing five copies
of the Gal4-binding sites, and 10 ng of phRL-TK, together with increasing amounts of an expression vector for HNF-4a or LRH-1 (0.2 and 0.6 lg); they were then cultured in a medium containing 10% fetal bovine serum for 48 h Luciferase assays were performed The promoter activities in the absence of pGAL4–SREBP1a or pGAL4–SREBP2 are represented as 1 All data are presented as means ± SD.
Trang 5ing proteins, SCAP and importin-b, play an important
role in determining the fate of SREBPs In the nucleus,
multiple nuclear proteins form a complex with
SREBPs on their target gene promoters to regulate the
transcriptional activity In addition, SREBPs interact
with ubiquitin- or SUMO-transfer enzymes, thereafter
being rapidly degraded or inactivated, respectively
Some nuclear receptors and transcription factors also
associate with SREBPs in the nucleus This association
exerts considerable physiological influence on the
expression of their target genes Further studies will be
required to elucidate the more complex network
among the numerous transcription factors that
regu-late lipid and energy metabolism
Acknowledgements
The author is grateful to K Boru of Pacific Edit for
reviewing the manuscript
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