Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1ß release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1ß maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1ß release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1ß maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.

Epithelial-to-Mesenchymal Transition Supports Ovarian Carcinosarcoma Tumorigenesis and Confers Sensitivity to Microtubule Targeting with Eribulin.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. SIGNIFICANCE: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Development of NanoLuc-targeting protein degraders and a universal reporter system to benchmark tag-targeted degradation platforms.

Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12F36V (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic NanoLuc-targeting PROTACs (NanoTACs) to hijack the CRL4CRBN complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.

Reduction of Squalene Epoxidase by Cholesterol Accumulation Accelerates Colorectal Cancer Progression and Metastasis.

BACKGROUND & AIMS: Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, is suggested as a proto-oncogene. Paradoxically, SQLE is degraded by excess cholesterol, and low SQLE is associated with aggressive colorectal cancer (CRC). Therefore, we studied the functional consequences of SQLE reduction in CRC progression. METHODS: Gene and protein expression data and clinical features of CRCs were obtained from public databases and 293 human tissues, analyzed by immunohistochemistry. In vitro studies showed underlying mechanisms of CRC progression mediated by SQLE reduction. Mice were fed a 2% high-cholesterol or a control diet before and after cecum implantation of SQLE genetic knockdown/control CRC cells. Metastatic dissemination and circulating cancer stem cells were demonstrated by in vivo tracking and flow cytometry analysis, respectively. RESULTS: In vitro studies showed that SQLE reduction helped cancer cells overcome constraints by inducing the epithelial-mesenchymal transition required to generate cancer stem cells. Surprisingly, SQLE interacted with GSK3ß and p53. Active GSK3ß contributes to the stability of SQLE, thereby increasing cell cholesterol content, whereas SQLE depletion disrupted the GSK3ß/p53 complex, resulting in a metastatic phenotype. This was confirmed in a spontaneous CRC metastasis mice model, where SQLE reduction, by a high-cholesterol regimen or genetic knockdown, strikingly promoted CRC aggressiveness through the production of migratory cancer stem cells. CONCLUSIONS: We showed that SQLE reduction caused by cholesterol accumulation aggravates CRC progression via the activation of the ß-catenin oncogenic pathway and deactivation of the p53 tumor suppressor pathway. Our findings provide new insights into the link between cholesterol and CRC, identifying SQLE as a key regulator in CRC aggressiveness and a prognostic biomarker.

The Degron Architecture of Squalene Monooxygenase and How Specific Lipids Calibrate Levels of This Key Cholesterol Synthesis Enzyme.

Cholesterol synthesis is a fundamental process that contributes to cellular cholesterol homeostasis. Cells execute transcriptional and post-translational mechanisms to control the abundance of enzymes of the cholesterol synthesis pathway, consequently affecting cholesterol production. One such highly tuned enzyme is squalene monooxygenase (SM), which catalyzes a rate-limiting step in the pathway. A well-characterized mechanism is the cholesterol-mediated degradation of SM. Notably, lipids (cholesterol, plasmalogens, squalene, and unsaturated fatty acids) can act as cellular signals that either promote or reduce SM degradation. The N-terminal region of SM consists of the shortest known cholesterol-responsive degron, characterized by atypical membrane anchoring structures, namely a re-entrant loop and an amphipathic helix. SM also undergoes non-canonical ubiquitination on serine, a relatively uncommon attachment site for ubiquitination. The structure of the catalytic domain of SM has been solved, providing insights into the catalytic mechanisms and modes of inhibition by well-known SM inhibitors, some of which have been effective in lowering cholesterol levels in animal models. Certain human cancers have been linked to dysregulation of SM levels and activity, further emphasizing the relevance of SM in health and disease.

Squalene monooxygenase: a journey to the heart of cholesterol synthesis.

Squalene monooxygenase (SM) is a vital sterol synthesis enzyme across eukaryotic life. In yeast, it is a therapeutic target for treating certain fungal infections, and in mammals it is a rate-limiting enzyme that represents a key control point in the cholesterol synthesis pathway. SM introduces an oxygen atom to squalene, which becomes the signature oxygen of the hydroxyl group in cholesterol. Our knowledge of SM has advanced tremendously since its initial cloning and characterization. Early research developed mammalian SM inhibitors to target SM for cholesterol-lowering purposes. The substrate squalene has gained considerable interest for its health benefits and in nanomedicine for delivery of drugs. More recently, SM has been implicated as a key dysregulated component in certain cancers. In this review, we summarize our present knowledge of SM, focusing on the regulation of SM and the gene encoding it, SQLE. Furthermore, we offer insights into the role of SM across different organisms and its significance in human health and disease.

A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.

Cholesterol biosynthesis is a high-cost process and, therefore, tightly regulated by both transcriptional and posttranslational negative feedback mechanisms in response to the level of cellular cholesterol. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes epoxidation of squalene. The stability of SM is negatively regulated by cholesterol via its N-terminal regulatory domain (SM-N100). In this study, using a SM-luciferase fusion reporter cell line, we performed a chemical genetics screen that identified inhibitors of SM itself as up-regulators of SM. This effect was mediated through the SM-N100 region, competed with cholesterol-accelerated degradation, and required the E3 ubiquitin ligase MARCH6. However, up-regulation was not observed with statins, well-established cholesterol biosynthesis inhibitors, and this pointed to the presence of another mechanism other than reduced cholesterol synthesis. Further analyses revealed that squalene accumulation upon treatment with the SM inhibitor was responsible for the up-regulatory effect. Using photoaffinity labeling, we demonstrated that squalene directly bound to the N100 region, thereby reducing interaction with and ubiquitination by MARCH6. Our findings suggest that SM senses squalene via its N100 domain to increase its metabolic capacity, highlighting squalene as a feedforward factor for the cholesterol biosynthetic pathway.

Valosin-containing protein mediates the ERAD of squalene monooxygenase and its cholesterol-responsive degron.

Squalene monooxygenase (SM) is an essential rate-limiting enzyme in cholesterol synthesis. SM degradation is accelerated by excess cholesterol, and this requires the first 100 amino acids of SM (SM N100). This process is part of a protein quality control pathway called endoplasmic reticulum-associated degradation (ERAD). In ERAD, SM is ubiquitinated by MARCH6, an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). However, several details of the ERAD process for SM remain elusive, such as the extraction mechanism from the ER membrane. Here, we used SM N100 fused to GFP (SM N100-GFP) as a model degron to investigate the extraction process of SM in ERAD. We showed that valosin-containing protein (VCP) is important for the cholesterol-accelerated degradation of SM N100-GFP and SM. In addition, we revealed that VCP acts following ubiquitination of SM N100-GFP by MARCH6. We demonstrated that the amphipathic helix (Gln62-Leu73) of SM N100-GFP is critical for regulation by VCP and MARCH6. Replacing this amphipathic helix with hydrophobic re-entrant loops promoted degradation in a VCP-dependent manner. Finally, we showed that inhibiting VCP increases cellular squalene and cholesterol levels, indicating a functional consequence for VCP in regulating the cholesterol synthesis pathway. Collectively, we established VCP plays a key role in ERAD that contributes to the cholesterol-mediated regulation of SM.

Consulting prostate cancer cohort data uncovers transcriptional control: Regulation of the MARCH6 gene.

Cholesterol accumulation is a hallmark of prostate cancer (PCa) enabled by the upregulation of its synthesis, which presents a potential therapeutic target. This pathway is suppressed by the E3 ubiquitin ligase membrane-associated RING-CH-type finger 6 (MARCH6); however, little is known of MARCH6 regulation, particularly at the transcriptional level. Here, we consulted large transcriptomic PCa datasets to investigate transcription factors and DNA sequence elements that regulate the MARCH6 gene. Amongst 498 primary PCa tissues of The Cancer Genome Atlas, we identified a striking positive correlation between MARCH6 and androgen receptor (AR) gene expression (r = 0.81, p < 1 × 10-117) that held in other primary tumour datasets. Two putative androgen response elements were identified in the MARCH6 gene using motif prediction and mining of publicly accessible chromatin immunoprecipitation-sequencing data. However, MARCH6 expression was not androgen-responsive in luciferase reporter and qRT-PCR assays. Instead, we established that the MARCH6-AR correlation in primary PCa is due to common regulation by the transcription factor Sp1. We located a region 100 bp downstream of the MARCH6 transcriptional start site that contains three Sp1 binding sites and strongly upregulates promoter activity. The functionality of this region, and Sp1-mediated upregulation of MARCH6, was confirmed using pharmacological and genetic inhibition of Sp1. Moreover, modulation of Sp1 activity affected the stability of squalene monooxygenase, a cholesterol biosynthesis enzyme and MARCH6 substrate. We thus establish Sp1 as the first known regulator of the MARCH6 gene and demonstrate that interrogation of transcriptomic datasets can assist in the de novo inference of transcriptional regulation.

Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase.

Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis. The region comprising the first 100 amino acids, termed SM N100, represents the shortest cholesterol-responsive degron and enables SM to sense excess cholesterol in the endoplasmic reticulum (ER) membrane. Cholesterol accelerates the ubiquitination of SM by membrane-associated ring-CH type finger 6 (MARCH6), a key E3 ubiquitin ligase involved in ER-associated degradation. However, the ubiquitination site required for cholesterol regulation of SM N100 is unknown. Here, we used SM N100 fused to GFP as a model degron to recapitulate cholesterol-mediated SM degradation and show that neither SM lysine residues nor the N terminus impart instability. Instead, we discovered four serines (Ser-59, Ser-61, Ser-83, and Ser-87) that are critical for cholesterol-accelerated degradation, with MS analysis confirming Ser-83 as a ubiquitination site. Notably, these two clusters of closely spaced serine residues are located in disordered domains flanking a 12-amino acid-long amphipathic helix (residues Gln-62-Leu-73) that together confer cholesterol responsiveness. In summary, our findings reveal the degron architecture of SM N100, introducing the role of non-canonical ubiquitination sites and deepening our molecular understanding of how SM is degraded in response to cholesterol.

The shape of human squalene epoxidase expands the arsenal against cancer.

Squalene epoxidase (also known as squalene monooxygenase, EC 1.14.99.7) is a key rate-limiting enzyme in cholesterol biosynthesis. Anil Padyana and colleagues report the long awaited structure of human squalene epoxidase (SQLE). They solved the crystal structure of the catalytic domain of human SQLE alone and in complex with two similar pharmacological inhibitors and elucidate their mechanism of action. SQLE is the target of fungicides and of increasing interest in human health and disease, particularly as a new anti-cancer target. Indeed, in a companion paper, Christopher Mahoney and colleagues performed an inhibitor screen with cancer cell lines and identified SQLE as an unique vulnerability in a subset of neuroendocrine tumours, where SQLE inhibition caused a toxic accumulation of the substrate squalene. The SQLE structure will facilitate the development of improved inhibitors. Here, we comment on these two studies in the wider context of the field and discuss possible future directions.

Mallostery: Filling a niche between quality and metabolic control.

To be, or not to be … What determines the destruction of a protein in response to metabolic cues? In the current issue of JBC, Wangeline and Hampton shed new light on this existential question by studying the classic case of HMGCR (Hmg2 in yeast), the rate-limiting step in sterol synthesis, and find a metabolic cue that causes "allosteric misfolding" and subsequent destruction of the protein, a concept they name mallostery.

Cholesterol, cancer, and rebooting a treatment for athlete's foot.

A key enzyme in cholesterol synthesis is placed firmly on the oncogenic map and demonstrated to be a potential therapeutic target in liver cancer by repurposing a common antifungal agent (Liu et al, this issue).

A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.

Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans.

Cholesterol homeostasis: How do cells sense sterol excess?

Cholesterol is vital in mammals, but toxic in excess. Consequently, elaborate molecular mechanisms have evolved to maintain this sterol within narrow limits. How cells sense excess cholesterol is an intriguing area of research. Cells sense cholesterol, and other related sterols such as oxysterols or cholesterol synthesis intermediates, and respond to changing levels through several elegant mechanisms of feedback regulation. Cholesterol sensing involves both direct binding of sterols to the homeostatic machinery located in the endoplasmic reticulum (ER), and indirect effects elicited by sterol-dependent alteration of the physical properties of membranes. Here, we examine the mechanisms employed by cells to maintain cholesterol homeostasis.

The Regulatory Domain of Squalene Monooxygenase Contains a Re-entrant Loop and Senses Cholesterol via a Conformational Change.

Squalene monooxygenase (SM) is an important control point in cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase. Although it is known to associate with the endoplasmic reticulum, its topology has not been determined. We have elucidated the membrane topology of the sterol-responsive domain of SM comprising the first 100 amino acids fused to GFP (SM N100-GFP) by determining the accessibility of 16 introduced cysteines to the cysteine-reactive, membrane-impermeable reagent PEG-maleimide. We have identified a region integrally associated with the endoplasmic reticulum membrane that is likely to interact with cholesterol or respond to cholesterol-induced membrane effects. By comparing cysteine accessibility with and without cholesterol treatment, we further present evidence to suggest that cholesterol induces a conformational change in SM N100-GFP. This change is likely to lead to its targeted degradation by the ubiquitin-proteasome system because degradation is blunted by treatment with the chemical chaperone glycerol, which retains SM N100-GFP in its native conformation. Furthermore, degradation can be disrupted by insertion of two N-terminal myc tags, implicating the N terminus in this process. Together, this information provides new molecular insights into the regulation of this critical control point in cholesterol synthesis.