Sterol Regulatory Element-binding Protein Cleavage Research Articles

Abstract 4122301: The Role of Hepatic CDS2 in Phosphatidylinositol Homeostasis and Lipid Metabolism

Background: Recent studies in both human genetics and mouse models have highlighted the significance of phosphatidylinositol (PI) in the progression of fatty liver disease (FLD). Despite this, the precise mechanisms underlying PI's role remain elusive. Aims: This study aimed to elucidate the involvement of PI in FLD by disrupting hepatic PI biosynthesis through modulation of CDP-diacylglycerol synthase 2 (CDS2) expression, the primary enzyme in PI biosynthesis (A). Methods: We generated liver-specific knockouts of CDS2 and SCAP (SREBP cleavage-activating protein) using adeno-associated virus (AAV) expressing sgRNA against each gene. Phospholipid species were analyzed and quantified by LC/MS. Results: CDS2 liver-specific knockout mice (B, C) exhibited a pronounced development of fatty liver, characterized by elevated triglyceride and cholesterol levels (C). Notably, liver PI levels were significantly reduced in CDS2 knockout mice (E), accompanied by dysregulated activation of sterol regulatory element-binding protein (SREBP) even during fasting conditions (F), leading to increased de novo lipogenesis. Intraperitoneal administration of exogenous PI effectively normalized SREBP cleavage (G), highlighting PI's critical role in SREBP regulation. Furthermore, simultaneous knockout of SCAP with CDS2 restored the normal liver phenotype, implicating SREBP activation as a central mechanism underlying fatty liver development in CDS2 knockout mice (H, I). Conclusion: Deletion of CDS2 in the liver reduced PI levels, subsequently inducing significant fatty liver due to dysregulated SREBP activation. This study underscores the importance of CDS2 in maintaining PI homeostasis, which in turn regulates SREBP activity and lipid metabolism. Our findings provide valuable insights into the pathogenesis of metabolic disorders and offer potential therapeutic strategies for targeting FLD.

SARS-CoV-2 nucleocapsid protein promotes TMAO-induced NLRP3 inflammasome activation by SCAP–SREBP signaling pathway

The sterol regulatory element-binding protein (SREBP) activation and cytokine level were significantly increased in coronavirus disease-19. The NLRP3 inflammasome is an amplifier for cellular inflammation. This study aimed to elucidate the modulatory effect of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP) on trimethylamine N-oxide (TMAO)-induced lipogenesis and NLRP3 inflammasome activation and the underlying mechanisms in vascular smooth muscle cells (VSMCs). Our data indicated that SARS-CoV-2 NP activates the dissociation of the SREBP cleavage activating protein (SCAP) from the endoplasmic reticulum, resulting in SREBP activation, increased lipogenic gene expression, and NLRP3 inflammasome activation. TMAO was applied to VSMC-induced NLRP3 inflammasome by promoting the SCAP–SREBP complex endoplasmic reticulum-to-Golgi translocation, which facilitates directly binding of SARS-CoV-2 NP to the NLRP3 protein for NLRP3 inflammasome assembly. SARS-CoV-2 NP amplified the TMAO-induced lipogenic gene expression and NLRP3 inflammasome. Knockdown of SCAP–SREBP2 can effectively reduce lipogenic gene expression and alleviate NLRP3 inflammasome-mediated systemic inflammation in VSMCs stimulated with TMAO and SARS-CoV-2 NP. These results reveal that SARS-CoV-2 NP amplified TMAO-induced lipogenesis and NLRP3 inflammasome activation via priming the SCAP–SREBP signaling pathway.

Abstract PR-009: Targeting the sterol regulatory element-binding protein pathway in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with limited diagnostic and therapeutic options. Due to its proliferative nature and thick, desmoplastic stroma, PDAC tumor cells are challenged with meeting a high demand for lipids in a hypoxic, lipid-poor environment. Cancer cells respond to lipid conditions through sterol regulatory element-binding proteins (SREBPs). SREBPs are master transcriptional regulators of lipid homeostasis and require SREBP cleavage activating protein (SCAP) during signaling. While the SREBP pathway has been implicated in several cancers, its role in PDAC has not been examined. In this study, we assess the requirement of this pathway using both in vitro and in vivo model systems. Furthermore, we target the SREBP pathway using a novel combination therapy of two FDA-approved compounds: dipyridamole and statins. Methods: We acquired four patient-derived pancreatic adenocarcinoma cell lines containing mutations in both Kras and p53 and knocked out SCAP in each cell line followed by gene-based rescue. Functional growth assays were performed in both lipid-poor and lipid-rich media conditions. Subcutaneous and pancreatic orthotopic xenografts were performed in nude mice using these same cell lines. A previously established mouse line, LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1 Cre (KPC) on a C57Bl/6 background, was utilized as a PDAC model. KPC mice lacking Scap in one or both alleles (Sfl/+ or Sfl/fl) were generated. Cohort survival and histologic analysis were performed for all mice, and results plotted on a Kaplan-Meier survival curve. In each patient cell line, the following drugs were applied individually and in combination: Dipyridamole (DP), tetramethyl-DP (TMDP), Fluvastatin, and Simvastatin. Cell viability post-treatment was assessed, and synergy calculated for each combination using SynergyFinder. Results: In lipid-poor conditions, SCAP knockout cells showed significantly reduced growth when compared to wild type or SCAP rescued cells. In tumor xenograft models, SCAP knockout cells exhibited reduced tumor growth and tumor volume when compared to wild-type cells. In the genetically engineered mouse models, KPC mice exhibited a median survival time of 289 days, while KPCSfl/+ mice show a significantly increased median survival time of 425 days. Additionally, KPCSfl/fl mice did not develop invasive PDAC. Cells treated individually with DP, TMDP and both statins exhibit lipid-dependent growth defects in all cell lines tested. In combination, DP with either statin as well as TMDP with either statin demonstrate synergy in lipid-poor conditions. Conclusions: Our results demonstrate that loss of SCAP in PDAC tumor cells alters the growth capability both in vitro and using mouse xenograft models. Additionally, heterozygous loss of Scap in the KPC mouse model significantly increased survival. Finally, dipyridamole works in synergy with statins to alter growth of PDAC tumor cells. These findings suggest that targeting the SREBP pathway has significant therapeutic potential in PDAC. Citation Format: Stephanie Myers, Meredith McGuire, Wei Shao, Chine Liu, Theodore Ewachiw, Zeshaan Rasheed, William Matsui, Toni Sepalla, Richard Burkhart, Peter Espenshade. Targeting the sterol regulatory element-binding protein pathway in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-009.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

SREBP Plays a Regulatory Role in LH/hCG Receptor mRNA Expression in Human Granulosa-Lutein Cells

LH receptor (LHR) expression has been shown to be regulated posttranscriptionally by LHR mRNA binding protein (LRBP) in rodent and human ovaries. LRBP was characterized as mevalonate kinase. The gene that encodes mevalonate kinase is a member of a family of genes that encode enzymes involved in lipid synthesis and are regulated by the transcription factor sterol regulatory element binding proteins (SREBPs). The current study examined the regulation of LHR mRNA expression in human granulosa-lutein cells in response to alterations in cholesterol metabolism. Using atorvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase to inhibit cholesterol biosynthesis, we examined its effect on LHR mRNA expression. The effect of atorvastatin on SREBP and mRNA expression as well as LHR mRNA binding protein expression was examined. Finally, the effect of atorvastatin on human chorionic gonadotropin (hCG)-stimulated progesterone production and the expression of key steroidogenic enzymes was also examined. Statin treatment reduced LHR mRNA expression by increasing the levels of SREBP1a and SREBP2, leading to an increase in LRBP. RNA gel shift assay showed that increased binding of LHR mRNA to LRBP occurred in response to atorvastatin, leading to LHR mRNA degradation. The granulosa-lutein cells pretreated with atorvastatin also showed decreased responsiveness to hCG by decreasing the mRNA and protein expression of steroidogenic enzymes. Atorvastatin also attenuated LH/hCG-induced progesterone production. These results imply that LHR mRNA expression by the human granulosa-lutein cells is regulated by cholesterol, through a mechanism involving SREBP and SREBP cleavage activating protein serving as the cholesterol sensor.

Requirement of Sterol Regulatory Element‐Binding Protein Pathway in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with limited diagnostic and therapeutic options. Due to its proliferative nature, PDAC tumor cells have a high demand for lipid synthesis. However, this tumor is known to be poorly vascularized and nestled within a hypoxic environment. As lipid synthesis is a highly oxygen‐consumptive process, neoplastic cells are challenged with meeting the demand for lipids. Cells, including cancer cells, respond to lipid conditions through sterol regulatory element‐binding proteins (SREBPs). SREBPs are master regulators of lipid homeostasis and require SREBP cleavage activating protein (SCAP) during signaling to regulate lipid synthesis. While the SREBP pathway has been implicated in several cancers, its role in PDAC has not been examined. Here, we acquired four patient‐derived pancreatic adenocarcinoma cell lines, knocked out SCAP, then followed with a rescue. We performed functional growth assays using both lipid‐poor and lipid‐rich conditions as well as subcutaneous and orthotopic xenografts in nude mice. In lipid‐poor conditions, SCAP knockout cells showed significantly reduced growth when compared to wildtype or SCAP rescued cells. In subcutaneous tumor xenografts, SCAP knockout cells exhibited reduced tumor volume in 3 out of 4 cell lines when compared to parent wildtype tumor cells. Similarly, SCAP knockout cells grew poorly in pancreatic orthotopic xenograft models. Our results demonstrate that loss of SCAP in PDAC tumor cells alters growth both in vitro and in vivo. These findings suggest SCAP may be useful as a therapeutic target in combating PDAC.Support or Funding InformationNIH T32 OD011089 (SM); Allegheny Health Network Cancer Grant (PJE); Sol Goldman Pancreatic Cancer Research Center Grant from Sidney Kimmel Comprehensive Center (PJE); NIH F31LB131185 (MM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast

Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA+) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe, generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe.

Physiological Suppression of Lipotoxic Liver Damage by Complementary Actions of HDAC3 and SCAP/SREBP

Liver fat accumulation precedes non-alcoholic steatohepatitis, an increasing cause of end-stage liver disease. Histone deacetylase 3 (HDAC3) is required for hepatic triglyceride homeostasis, and sterol regulatory element binding protein (SREBP) regulates the lipogenic response to feeding, but the crosstalk between these pathways is unknown. Here we show that inactivation of SREBP by hepatic deletion of SREBP cleavage activating protein (SCAP) abrogates the increase in lipogenesis caused by loss of HDAC3, but fatty acid oxidation remains defective. This combination leads to accumulation of lipid intermediates and to an energy drain that collectively cause oxidative stress, inflammation, liver damage, and, ultimately, synthetic lethality. Remarkably, this phenotype is prevented by ectopic expression of nuclear SREBP1c, revealing a surprising benefit of de novo lipogenesis and triglyceride synthesis in preventing lipotoxicity. These results demonstrate that HDAC3 and SCAP control symbiotic pathways of liver lipid metabolism that are critical for suppression of lipotoxicity.

A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP.

Hypoxic growth of fungi requires sterol regulatory element-binding protein (SREBP) transcription factors, and human opportunistic fungal pathogens require SREBP activation for virulence. Proteolytic release of fission yeast SREBPs from the membrane in response to low oxygen requires the Golgi membrane-anchored Dsc E3 ligase complex. Using genetic interaction arrays, we identified Rbd2 as a rhomboid family protease required for SREBP proteolytic processing. Rbd2 is an active, Golgi-localized protease that cleaves the transmembrane segment of the TatA rhomboid model substrate. Epistasis analysis revealed that the Dsc E3 ligase acts on SREBP prior to cleavage by Rbd2. Using APEX2 proximity biotinylation, we demonstrated that Rbd2 binds the AAA-ATPase Cdc48 through a C-terminal SHP box. Interestingly, SREBP cleavage required Rbd2 binding of Cdc48, consistent with Cdc48 acting to recruit ubiquitinylated substrates. In support of this claim, overexpressing a Cdc48-binding mutant of Rbd2 bypassed the Cdc48 requirement for SREBP cleavage, demonstrating that Cdc48 likely plays a role in SREBP recognition. In the absence of functional Rbd2, SREBP precursor is degraded by the proteasome, indicating that Rbd2 activity controls the balance between SREBP activation and degradation.

Fatostatin blocks ER exit of SCAP but inhibits cell growth in a SCAP-independent manner

Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipid homeostasis and activate expression of genes required for fatty acid, triglyceride, and cholesterol synthesis and uptake. SREBP cleavage activating protein (SCAP) plays an essential role in SREBP activation by mediating endoplasmic reticulum (ER)-to-Golgi transport of SREBP. In the Golgi, membrane-bound SREBPs are cleaved sequentially by the site-1 and site-2 proteases. Recent studies have shown a requirement for the SREBP pathway in the development of fatty liver disease and tumor growth, making SCAP a target for drug development. Fatostatin is a chemical inhibitor of the SREBP pathway that directly binds SCAP and blocks its ER-to-Golgi transport. In this study, we determined that fatostatin blocks ER exit of SCAP and showed that inhibition is independent of insulin-induced gene proteins, which function to retain the SCAP-SREBP complex in the ER. Fatostatin potently inhibited cell growth, but unexpectedly exogenous lipids failed to rescue proliferation of fatostatin-treated cells. Furthermore, fatostatin inhibited growth of cells lacking SCAP Using a vesicular stomatitis virus glycoprotein (VSVG) trafficking assay, we demonstrated that fatostatin delays ER-to-Golgi transport of VSVG. In summary, fatostatin inhibited SREBP activation, but fatostatin additionally inhibited cell proliferation through both lipid-independent and SCAP-independent mechanisms, possibly by general inhibition of ER-to-Golgi transport.

New insight into the cholesterol-lowering effect of phytosterols in rat cardiomyocytes

The exact mechanisms for cholesterol-lowering effect of phytosterols (PS) are not well defined yet. Dietary cholesterol minimally affects blood cholesterol, and regulation of cholesterolaemia could not exclusively rely on modification in intestinal absorption. Activation of sterol regulatory element-binding proteins (SREBPs), master regulators of lipid homeostasis, is tightly regulated through a sterol-sensing domain (SSD); in cholesterol-depleted cells, SREBP activation upregulates the expression of genes involved in cholesterol synthesis and trafficking. Since PS structure is very similar to cholesterol, they could be sensed by SSD, so preventing SREBP cleavage. To verify this hypothesis primary cultures of rat cardiomyocytes were supplemented with PS (sitosterol, campesterol, and brassicasterol, separately or in a mixture), cholesterol or mevastatin.PS were actively incorporated by cardiac cells, and caused a significant decrease in cholesterol cellular content. Genes encoding for SREBP2, HMGCR and LDLR were upregulated in cells treated with mevastatin, while no increase in their transcription was detected in PS-supplemented cardiomyocytes, although the reduction of cholesterol was similar in PS- and mevastatin-treated cells. According to herein reported data, it is conceivable that PS are sensed by the SSD so preventing the activation of the nuclear receptors and consequent upregulation of genes connected with cholesterol metabolism.

The signal peptide peptidase SppA is involved in sterol regulatory element-binding protein cleavage and hypoxia adaptation in Aspergillus nidulans.

Using forward genetics, we revealed that the signal peptide peptidase (SPP) SppA, an aspartyl protease involved in regulated intramembrane proteolysis (RIP), is essential for hypoxia adaptation in Aspergillus nidulans, as well as hypoxia-sensitive mutant alleles of a sterol regulatory element-binding protein (SREBP) srbA and the Dsc ubiquitin E3 ligase complex dscA-E. Both null and dead activity [D337A] mutants of sppA failed to grow in hypoxia, and the growth defect of ΔsppA was complemented by nuclear SrbA-N381 expression. Additionally, SppA interacted with SrbA in the endoplasmic reticulum, where SppA localized in normoxia and hypoxia. Expression of the truncated SrbA-N414 covering the SrbA sequence prior to the second transmembrane region rescued the growth of ΔdscA but not of ΔsppA in hypoxia. Unlike ΔdscA and ΔdscA;ΔsppA double mutants, in which SrbA cleavage was blocked, the molecular weight of cleaved SrbA increased in ΔsppA compared to the control strain in immunoblot analyses. Overall, our data demonstrate the sequential cleavage of SrbA by Dsc-linked proteolysis followed by SppA, proposing a new model of RIP for SREBP cleavage in fungal hypoxia adaptation. Furthermore, the function of SppA in hypoxia adaptation was consistent in Aspergillus fumigatus, suggesting the potential roles of SppA in fungal pathogenesis.

Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity

Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP)

Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipogenesis. Release of membrane-bound SREBP requires SREBP cleavage-activating protein (SCAP) to escort SREBP from the endoplasmic reticulum (ER) to the Golgi for cleavage by site-1 and site-2 proteases. SCAP then recycles to the ER for additional rounds of SREBP binding and transport. Mechanisms regulating ER-to-Golgi transport of SCAP-SREBP are understood in molecular detail, but little is known about SCAP recycling. Here, we have demonstrated that SCAP Golgi-to-ER transport requires cleavage of SREBP at site-1. Reductions in SREBP cleavage lead to SCAP degradation in lysosomes, providing additional negative feedback control to the SREBP pathway. Current models suggest that SREBP plays a passive role prior to cleavage. However, we show that SREBP actively prevents premature recycling of SCAP-SREBP until initiation of SREBP cleavage. SREBP regulates SCAP in human cells and yeast, indicating that this is an ancient regulatory mechanism.

Ginsenoside F2 Reduces Hair Loss by Controlling Apoptosis through the Sterol Regulatory Element-Binding Protein Cleavage Activating Protein and Transforming Growth Factor-β Pathways in a Dihydrotestosterone-Induced Mouse Model

This study was conducted to test whether ginsenoside F2 can reduce hair loss by influencing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and the transforming growth factor beta (TGF-β) pathway of apoptosis in dihydrotestosterone (DHT)-treated hair cells and in a DHT-induced hair loss model in mice. Results for ginsenoside F2 were compared with finasteride. DHT inhibits proliferation of hair cells and induces androgenetic alopecia and was shown to activate an apoptosis signal pathway both in vitro and in vivo. The cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of DHT-treated human hair dermal papilla cells (HHDPCs) and HaCaTs increased by 48% in the ginsenoside F2-treated group and by 12% in the finasteride-treated group. Western blot analysis showed that ginsenoside F2 decreased expression of TGF-β2 related factors involved in hair loss. The present study suggested a hair loss related pathway by changing SCAP related apoptosis pathway, which has been known to control cholesterol metabolism. SCAP, sterol regulatory element-binding protein (SREBP) and caspase-12 expression in the ginsenoside F2-treated group were decreased compared to the DHT and finasteride-treated group. C57BL/6 mice were also prepared by injection with DHT and then treated with ginsenoside F2 or finasteride. Hair growth rate, density, thickness measurements and tissue histotological analysis in these groups suggested that ginsenoside F2 suppressed hair cell apoptosis and premature entry to catagen more effectively than finasteride. Our results indicated that ginsenoside F2 decreased the expression of TGF-β2 and SCAP proteins, which have been suggested to be involved in apoptosis and entry into catagen. This study provides evidence those factors in the SCAP pathway could be targets for hair loss prevention drugs.

Subunit Architecture of the Golgi Dsc E3 Ligase Required for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast

The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

Structural Requirements for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast

Sterol regulatory element-binding proteins (SREBPs) are central regulators of cellular lipid synthesis and homeostasis. Mammalian SREBPs are proteolytically activated and liberated from the membrane by Golgi Site-1 and Site-2 proteases. Fission yeast SREBPs, Sre1 and Sre2, employ a different mechanism that genetically requires the Golgi Dsc E3 ligase complex for cleavage activation. Here, we established Sre2 as a model to define structural requirements for SREBP cleavage. We showed that Sre2 cleavage does not require the N-terminal basic helix-loop-helix zipper transcription factor domain, thus separating cleavage of Sre2 from its transcription factor function. From a mutagenesis screen of 94 C-terminal residues of Sre2, we isolated 15 residues required for cleavage and further identified a glycine-leucine sequence required for Sre2 cleavage. Importantly, the glycine-leucine sequence is located at a conserved distance before the first transmembrane segment of both Sre1 and Sre2 and cleavage occurs in between this sequence and the membrane. Bioinformatic analysis revealed a broad conservation of this novel glycine-leucine motif in SREBP homologs of ascomycete fungi, including the opportunistic human pathogen Aspergillus fumigatus where SREBP is required for virulence. Consistent with this, the sequence was also required for cleavage of the oxygen-responsive transcription factor Sre1 and adaptation to hypoxia, demonstrating functional conservation of this cleavage recognition motif. These cleavage mutants will aid identification of the fungal SREBP protease and facilitate functional dissection of the Dsc E3 ligase required for SREBP activation and fungal pathogenesis.

Protein kinase A (PKA) interferes with the processing and activity of sterol regulatory element binding proteins (SREBPs)

Several kinases are known to modulate the activity of the sterol regulatory element binding proteins (SREBPs), which are transcription factors that activate lipid biosynthesis. The inactive SREBP precursor forms are bound to the endoplasmic reticulum (ER) and require the SREBP cleavage activating protein (SCAP) to shuttle SREBPs to the Golgi, where proteases cleave and release the active SREBPs, which can then enter the nucleus and activate transcription. SREBPs are known to be down‐regulated by conditions where PKA activity is higher such as by glucagon action in the liver; however, a mechanism to explain this effect is lacking. Here, we show that PKA can block processing of SREBPs in various cells types. We used forskolin to induce PKA activity and found lower levels of mature SREBP‐1 in BCR‐ABL transformed bone marrow. We also used H‐89, a PKA inhibitor, and found higher levels of mature SREBPs in the BCR‐ABL transformed bone marrow, HeLa cells, and mouse primary hepatocytes. Preliminary results showed that H‐89 injection into mice can blunt the negative effect of glucagon on mature SREBP accumulation in the liver. In preliminary studies, we found higher amounts of SCAP in the Golgi when H‐89 is added to Chinese hamster ovary (CHO) cells that stably express GFP‐SCAP. There is a conserved putative PKA phosphorylation motif in SCAP and many phosphoproteomic studies show the site is phosphorylated in many different settings. Thus, our recent discovery points to a novel role for PKA in SREBP trafficking and activation, possibly via direct PKA phosphorylation of SCAP.

Structure‐function studies of the Golgi Dsc E3 ligase complex required for SREBP activation in yeast

Fungal sterol regulatory element‐binding protein (SREBP) transcription factors are oxygen‐responsive transcription factors required for adaptation to hypoxia. Our recent studies identified five genes (dsc1–5, defective for SREBP cleavage) that code for subunits of the Golgi Dsc E3 ligase complex that is required for fission yeast SREBP activation. Dsc proteins have domains that are predicted to function in the ubiquitin proteasome pathway: Dsc1 is a ubiquitin E3 ligase with a RING domain, Dsc2 has a predicted ubiquitin‐associated (UBA) domain and Dsc5 contains a ubiquitin regulatory X (UBX) domain that binds Cdc48.To begin to define the mechanism for Sre1 cleavage in fission yeast, we performed structure‐function studies to elucidate the organization of this multi‐subunit complex. Using co‐immunoprecipitation analysis and Blue native PAGE, we found that Dsc1–5 form a stable, detergent solubilized complex with a discrete architecture with distinct subcomplexes. Parallel studies in budding yeast, which lacks SREBP homologs, demonstrate that the Dsc E3 ligase complex is structurally conserved and localizes to the Golgi. Future studies will focus on the identification of additional Dsc E3 ligase substrates and the function of this complex in Golgi protein degradation.Studies were supported by the National Institutes of Health (HL‐077588).

Yeast Sterol Regulatory Element-binding Protein (SREBP) Cleavage Requires Cdc48 and Dsc5, a Ubiquitin Regulatory X Domain-containing Subunit of the Golgi Dsc E3 Ligase

Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.