SREBP Proteolysis
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SREBP Proteolysis

Cellular Lipid homeostasis in mammalian cells is regulated through the end-product feedback regulation of Lipid synthesis by a family of membrane-bound transcription factors designated SREBPs (Sterol Regulatory Element–Binding Proteins) that control the flux of cellular metabolites into the major Lipid pathways. The mammalian cell continuously adjusts its Sterol content by regulating levels of key Sterol synthetic enzymes and levels of Lipoprotein receptors that mediate uptake of Cholesterol-laden particles. Control is brought about by SREBPs, which monitor the Sterol-regulated transcription and directly activate the expression of more than 30 relevant genes dedicated to the synthesis and uptake of Cholesterol, Fatty Acids, Triglycerides, and Phospholipids, as well as the NADPH cofactor required to synthesize these molecules. SREBP proteolysis, which refers to the site-specific cleavage of the nascent SREBP, is at the heart of each of these mechanistically distinct axes and in each case, ongoing cell biological processes are being harnessed to bring about regulation (Ref.1 & 2).

SREBPs belong to the Basic Helix-Loop-Helix–Leucine Zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the Endoplasmic Reticulum. Each SREBP precursor exists in a hairpin-like conformation containing about 1150 amino acids (~125 kDa) is organized into three domains: (a) an NH2-terminal Cytoplasmic domain of about 480 amino acids (about 68 kDa) that contains the bHLH-Zip region that functions as a transcription factor on binding DNA; (b) a Central domain containing two transmembrane segments which are linked via a short luminal loop of about 31 amino acids projected into the lumen of the Endoplasmic Reticulum; and (c) a COOH-terminal Cytoplasmic domain of about 590 amino acids that performs the essential regulatory functions, hence also called REG factor. The mammalian genome encodes three SREBP isoforms, designated SREBP1a, SREBP1c, and SREBP2. More recently, a fourth SREBP protein, an isoform of SREBP2 referred to as SREBP2gc, has been described. Unlike other SREBP isoforms, expression of SREBP2gc remains restricted to male germ cells, where it regulates transcription of spermatogenic genes in a cell- and stage-specific manner. SREBP2gc, a shortened version of the N-terminal portion of SREBP2, is constitutively active and not subject to feedback control by sterols (Ref.2, 3 & 4). All the SREBP isoforms, regardless of the type, need to be transported to the nucleus so as to function as transcriptional activators of their target genes. In order to reach the nucleus and act as a transcription factors, the NH2-terminal domain of mature SREBP is released proteolytically. This proteolysis and release of mature SREBP requires at least three additional proteins, the SCAP (SREBP Cleavage Activating Protein), S1P (Site-1 Protease) and S2P (Site-2 Protease) (Ref.2 & 3). SCAP is both an escort for SREBPs as well as a sensor of Sterols. It contains eight putative transmembrane domains, five of which represent the SSD (Sterol-Sensing Domains). Newly synthesized SREBP is tethered to the membrane of the Endoplasmic Reticulum, where it’s COOH-terminal (REG) domain binds to the COOH-terminal (WD: Aspartate-Tryptophan motif) domain of SCAPSCAP interacts with a retention protein complex comprising of SAR1A, Sec23 and Sec24, collectively designated as the COPII Complex (Ref.5).

SREBPs are activated by a classical Sterol regulated pathway. When cells are loaded with Sterols, SCAP senses the excess Cholesterol through its SSD. The Sterol mediated sensing mechanism is however a complex process, involving some other Endoplasmic Reticulum proteins as well. During high Sterol concentrations, the resident Endoplasmic Reticulum membrane proteins INSIGs (Insulin Induced Genes) exhibit Sterol-induced binding to the SCAP. INSIGs are negative regulators of SREBP proteolysis and they enhance the response to Cholesterol by promoting the binding of Cholesterol to SCAP. This type of inhibition is termed convergent inhibition (Ref.6). Human INSIG1 (Insulin Induced Gene-1) protein contains six transmembrane helices which conjugate with six (2nd to 7th) transmembrane domains of SCAP. Binding of SCAP to INSIG1 leads to membrane bound retention of SCAP. Consequently, the SCAP/SREBP complex undergoes certain conformational changes, and is sequestered in the Endoplasmic Reticulum thereby preventing the delivery of SCAP-bound SREBPs to the Endoplasmic Reticulum transport vesicles and their subsequent incorporation to the Golgi. The Sterol-mediated actions of INSIG1 prevents over-accumulation of Cholesterol or its Sterol precursors in the cells, and in the absence of any proteolytic activation of SREBP, transcriptional rates of SREBP target genes decline, leading to a reduction in Cholesterol synthesis and uptake (Ref.7).


Conversely, when cells are depleted of Sterols, the SCAP-SREBP complex dissociates from INSIG1, which in turn becomes ubiquitinated and degraded. These two levels of INSIG1 regulation (gene transcription and protein stability) are central to a process termed "convergent feedback inhibition". The mechanism for convergent feedback inhibition is provided by Gp78, an INSIG1-associated protein. The membrane-bound Gp78 is a RING finger Ubiquitin Ligase E3 that mediates the ubiquitination and degradation of INSIG1 in Sterol-depleted cells (Ref.7). Degradation of INSIG1 allows the interaction of SCAP-SREBP complex with the COPII Complex. These two complexes together form a cassette known as the COPII Vesicle that buds out of the membrane of the Endoplasmic Reticulum and later fuses with the Golgi Apparatus, where the two proteases S1P and S2P reside. Two classes of Sterols, Cholesterol and Oxysterols, block the export of SREBPs from the Endoplasmic Reticulum to the Golgi by preventing the binding of COPII-coated proteins to a hexapeptide sorting signal MELADL in SCAP. The distance of MELADL from the Endoplasmic Reticulum membrane is crucial for COPII binding. Sterols and INSIG block SREBP transport by altering the location of MELADL with respect to the membrane, rendering it inaccessible to COPII proteins (Ref.5 & 8).

In the Golgi, the first proteolytic cleavage of SREBP is accomplished by S1P, a membrane-bound Subtilisin-like Serine Protease activated by SCAP.  It contains a single putative transmembrane segment, which anchors the protease to the Golgi membrane. The activity of S1P is modulated by SCAP as the proteolytic cleavage of SREBP by S1P is dependent on the interaction of the WD repeats of SCAP with the REG domain of SREBPS1P cleaves SREBP within the intra-lumenal 31-amino-acid loop, dividing the SREBP molecule in two halves. The DSH (Asparate-Serine-Histidine) residues of S1P, which form the catalytic triad, are necessary for S1P function. The resulting two halves of SREBP remain anchored to the membrane with the help of the two trans-membrane spanning segments. Oxysterols inhibit S1P activity. The Sterol-regulated cleavage of SREBP by S1P is followed by a second, unregulated cleavage by the S2PS2P is an exceptionally hydrophobic intramembranous (Zinc) metalloproteinase containing an HEXXH active-site motif and a distal LDG motif, crucially necessary for its function. S2P cleaves SREBPs at a site which is located three amino acids into the membrane-spanning helix within the transmembrane segment that anchors the NH2-terminal domain to the membrane and results in the release of the 68-kDa NH2-terminal bHLH-Zip domain from its membrane anchor (Ref.9). Recently, it has been found that even some non-sterol lipids are also involved, either directly or indirectly, in the regulated cleavage of SREBPs. SREBP precursor proteins can also be proteolytically activated by Cysteine proteases belonging to the Caspase family. These proteases cleave SREBPs in a single step at a site located closer to the N-terminus of the protein than the recognition site for S2P; generating a SREBP fragment that is smaller than mature SREBP resulting from cellular Cholesterol depletion but that remains transcriptionally active. SREBP activation by Caspase2 occurs early in the apoptotic program and also plays a role during apoptosis. The bHLH-Zip domain representing the mature SREBP, now addressed nSREBP (nuclear-SREBP), then translocates to the nucleus as a dimer by Ipo-Beta (Importin-Beta) through interactions with the bHLH-domain, where it binds to the nonpalindromic SREs (Sterol Response Elements) in the promoter/enhancer regions of an ever expanding set of SREBP target genes directly or indirectly involved in the synthesis, metabolism, and uptake of Fatty Acids and Cholesterol. In addition to the SREs, the SREBPs also bind to the classic E-box motifs. Transcriptional activation of genes by SREBP is dependent on the interaction of SREBP with additional transcription factors, so far limited to NF-Gamma or  SP1 (Transcription Factor SP1) and to the coactivator CBP (CREB-Binding Protein) (Ref.5 & 10).

At normal levels of expression, nSREBP1a is a potent activator of presumably all SREBP-responsive genes as it binds to both the SREs and the E-box motifs. It has the capacity of triggering the synthesis of Cholesterol, Fatty Acids, and Triglycerides concurrently. However, the roles of nSREBP1c and nSREBP2 are more restricted than that of nSREBP1a. On binding to the E-box motifs, nSREBP1c preferentially enhances transcription of genes required for Saturated and Unsaturated Fatty Acids and Triglyceride biosynthesis. During this process, the Lipogenic mRNAs for Fatty Acid synthetic enzymes ACLY (ATP Citrate Lyase), ACAC (Acetyl-CoA Carboxylase), FASN (Fatty Acid Synthase), LCE (Long-Chain Fatty-Acyl Elongase) and SCD (Stearoyl-CoA Desaturase)) and rates of Fatty Acid synthesis are elevated (Ref.4 & 11). PI3K  (Phosphatidylinositde-3 Kinase)/Akt (v-Akt Murine Thymoma Viral Oncogene Homolog) play a role in lipogenesis as the PI3K  inhibitors also inhibit activation of SREBP1c in response to Insulin and Growth factors like VEGF (Vascular Endothelial Growth Factor) and PDGF (Platelet-Derived Growth Factor). Statins activate SREBP1c by inhibiting cholesterol synthesis and hence reducing the regulatory pool sensed by SCAP. Additionally, it has been found that Statins also activate PI3K/Akt pathways, indicating that the activation of SREBP by Statins may involve PI3K/Akt (Ref.12). nSREBP2 preferentially activates the transcription of the genes involved in Cholesterol biosynthesis pathway or Cholesterologenesis. Cholesterol is an important component of cell membrane, and a precursor of Oxysterols, Steroid hormones, Vitamin D3, and Biliairy Acids. Cells use varied advanced mechanisms to monitor changes in cholesterol levels and to regulate lipid metabolism. A feedback system, centered by the SREBPs, enables cholesterol and certain sterol intermediates to regulate the proteolysis and transport of specific membrane proteins. In liver cells, the homodimerised nSREBP2 forms a complex with the SREs in the promoter/enhancer regions of the genes encoding HMGCS (3-Hydroxy-3-Methylglutaryl-Coenzyme A Synthase), HMGCR (3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase), FDPS (Farnesyl Diphosphate Synthase), SS (Squalene Synthase) and DNAJA4 (DNAJ (HSP40) Homolog Subfamily-A Member-4) (Ref.2, 13 & 14). Binding of nSREBP2 to the LDLR (Low Density Lipoprotein Receptor) promoter increases the expression of LDLR on the cell surface and increases the internalization of Cholesterol-transporting LDL (Low Density Lipoprotein) particles from plasma by receptor-mediated endocytosis, increasing cellular Cholesterol levels and lowering LDL Cholesterol in the plasma with a concomitant down-regulation of SREBPs (Ref.15). nSREBP1c and nSREBP2 also combinedly activate the expression of genes encoding GPAT (Glycerol-3-Phosphate Acyltransferase), ME (Malic Enzyme), G6PD (Glucose-6-Phosphate Dehydrogenase), and PGD (6-Phosphogluconate Dehydrogenase), which are required to generate NADPH, which is consumed at multiple stages in the Lipid biosynthetic pathways (Ref.16).

The INSIG1 gene is also a target of the nSREBPs. Thus INSIG1 is itself regulated by SREBP and it can bind to SCAP under high protein concentrations independent of the Cholesterol-dependent conformation of SCAP. nSREBPs, particularly SREBP1c and SREBP2, also involve a feed-forward regulation mediated by SRE present in the enhancer/promoters of each gene to activate the transcription of their own genes. However, the transcriptional stimulation by mature SREBP is in itself a fairly weak transacting factor, and rather depends on interaction with several cofactors, and there occurs a cross-talk with Steroid Receptors, such as the Thyroid Receptor and the Estrogen Receptor. The expression and maturation of SREBPs are affected by cellular Androgens (which up-regulate both the expression and the activity of several enzymes belonging to the metabolic pathways of Fatty Acid and Cholesterol synthesis), Sphingomyelin levels, and TNF-Alpha (Tumor Necrosis Factor-Alpha) (Ref.3). As all the three SREBP isoforms are widely expressed and have the potentiality to homo- and heterodimerize, there is significant overlap of structure and function of the SREBPs. Recently, it has been suggested that there is even the possibility of selective roles for such specific homo- as well as heterodimeric SREBP complexes (Ref.2).

Mature nSREBPs are highly unstable owing to their susceptibility to Ubiquitin-dependent degradation. Sterol independent phosphorylation of nSREBPs by GSK3 (Glycogen Synthase Kinase-3) promotes their binding to the E3 Ubiquitin Ligase FBXW7 (F-Box and WD40 Domain Protein-7) followed by subsequent ubiquitylation and degradation (Ref.5). The Ubiquitin-Proteasome System regulates the degradation of active SREBPs, as fluctuations in the normal pool-strength of these factors have deleterious effects, like; increased level of SREBP2 is responsible for Hypercholesterolemia, which is observed during Chronic Renal Failure (Ref.17). This balance between regulated proteolysis and protein degradation by the Ubiquitin-Proteasome System plays an important role in regulating the activation of SREBP-dependent transcription and the synthesis of Cholesterol. Subjects with enzymatic defects in Cholesterol biosynthesis involving SREBPs are susceptible to Desmosterolosis, X-Linked Dominant Chondrodysplasia Punctata, CHILD Syndrome, Lathosterolosis, and Hydrops-Ectopic Calcification-Moth-Eaten Skeletal Dysplasia (Ref.1, 15, 18 & 19). The SREBP Proteolytic machinery thus, acts upstream of a diverse set of cellular processes and biochemical processes, including phagocytosis, cell cycle progression, oxygen sensing and survival in response to bacterial infection illustrating the wide-ranging roles that SREBPs and membrane biogenesis have in cell biology (Ref.9 & 20).

 

References

  1. The low-density lipoprotein receptor: ligands, debates and lore
  2. Disorders of cholesterol biosynthesis: prototypic metabolic malformation syndromes
  3. Selective association of sterol regulatory element-binding protein isoforms with target promoters in vivo
  4. Androgen activation of the sterol regulatory element-binding protein pathway: Current insights
  5. SREBP transcription factors: master regulators of lipid homeostasis
  6. SREBPs: sterol-regulated transcription factors
  7. SREBP in signal transduction: cholesterol metabolism and beyond
  8. Sterol-regulated degradation of Insig-1 mediated by the membrane-bound ubiquitin ligase gp78
  9. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins
  10. Maintaining cholesterol homeostasis: sterol regulatory element-binding proteins
  11. Caspase-2, a novel lipid sensor under the control of sterol regulatory element binding protein 2
  12. Acetyl-CoA carboxylase and SREBP expression during peripheral nervous system myelination
  13. Involvement of Akt in ER-to-Golgi transport of SCAP/SREBP: a link between a key cell proliferative pathway and membrane synthesis
  14. Sterol regulatory element-binding protein-2 negatively regulates low density lipoprotein receptor-related protein transcription
  15. DnaJA4 is a SREBP-regulated chaperone involved in the cholesterol biosynthesis pathway
  16. Selective up-regulation of LXR-regulated genes ABCA1, ABCG1, and APOE in macrophages through increased endogenous synthesis of 24(S),25-epoxycholesterol
  17. Increased gene expression of liver SREBP-2 in experimental chronic renal failure
  18. Modified HMG-CoA reductase and LDLr regulation is deeply involved in age-related hypercholesterolemia
  19. Dynamin is involved in endolysosomal cholesterol delivery to the endoplasmic reticulum: role in cholesterol homeostasis
  20. De novo cholesterol synthesis at the crossroads of adaptive response to extracellular stress through SREBP