Potential emergence of terbinafine resistance by squalene epoxidase gene mutations: An 18-month cohort study of onychomycosis patients in the United States.

BACKGROUND: There is a concerning rise in antifungal-resistant dermatophytosis globally, with resistance to terbinafine conferred by point mutations in the squalene epoxidase (SQLE) gene. OBJECTIVES: Report changes in the prevalence and profile of SQLE mutations in onychomycosis patients in the United States. METHODS: A longitudinal cohort study of toenail samples was collected from suspected onychomycosis patients over an 18-month period from 2022 to 2023. Samples were submitted from across the United States and subjected to multiplex real-time polymerase chain reactions for dermatophyte detection, with further screening of SQLE mutations at four known hotspots (393Leu, 397Phe, 415Phe and 440His). RESULTS: A total of 62,056 samples were submitted (mean age: 57.5 years; female: 60.4%). Dermatophytes were detected in 38.5% of samples, primarily Trichophyton rubrum complex (83.6%) and T. mentagrophytes complex (10.7%). A survey of SQLE mutations was carried out in 22,610 dermatophyte samples; there was a significant increase in the prevalence of SQLE mutations between the first quarter of 2022 and the second quarter of 2023 (29.0 to 61.9 per 1000 persons). The Phe397Leu substitution was the predominant mutation; Phe415Ser and His440Tyr have also emerged which were previously reported as minor mutations in skin samples. The temporal change in mutation rates can be primarily attributed to the Phe415Ser substitution. Samples from elderly patients (>70 years) are more likely to be infected with the T. mentagrophytes complex including strains harbouring the Phe415Ser substitution. CONCLUSION: The prevalence of SQLE mutations among onychomycosis patients with Trichophyton infections may be underestimated. Older individuals may have a higher risk.

High Prevalence of Terbinafine Resistance Among Trichophyton mentagrophytes/T. interdigitale Species Complex, a Cross-Sectional Study from 2021 to 2022 in Northern Parts of Iran.

Treatment-resistant dermatophytosis caused by the members of the Trichophyton mentagrophytes/Trichophyton interdigitale species group (TMTISG) is increasing worldwide. We aimed to determine the prevalence of TMTISG in patients with dermatophytosis in two centers from north of Iran and detect the possible mutations in the squalene epoxidase (SQLE) gene in relevant terbinafine (TRB) resistant pathogenic isolates. From November 2021 to December 2022, 1960 patients suspected to dermatophytosis and referred to two mycology referral laboratories in the north of Iran were included in the study. Identification of all dermatophyte isolates was confirmed by RFLP of rDNA internal transcribed spacer (ITS) regions. Antifungal susceptibility testing against five common antifungals using the CLSI-M38-A3 protocol was performed. The TMTISG isolates resistant to TRB, were further analyzed to determine the possible mutations in the SQLE gene. Totally, 647 cases (33%) were positive for dermatophytosis of which 280 cases (43.3%) were identified as members of TMTISG. These were more frequently isolated from tinea corporis 131 (44.56%) and tinea cruris 116 (39.46%). Of 280 TMTISG isolates, 40 (14.3%) were resistant to TRB (MIC ≥ 4 µg/mL), all found to be T. indotineae in ITS sequencing. In SQLE sequencing 34 (85%) of TRB-resistant isolates had coincident mutations of Phe397Leu and Ala448Thr whereas four and two isolates had single mutations of Phe397Leu and Leu393Ser, respectively. Overall, the resistance of Iranian TMTISG isolates to TRB greatly occurred by a mutation of Phe397Leu in the SQLE gene as alone or in combination with Ala448Thr. Nevertheless, for the occurrence of in vitro resistance, only the presence of Phe397Leu mutation seems to be decisive.

Increased terbinafine resistance among clinical genotypes of Trichophyton mentagrophytes/T. interdigitale species complex harboring squalene epoxidase gene mutations.

Terbinafine resistance has become epidemic as an emerging problem in treatment of dermatohpytosis. This could be attributed in part to a point mutation in the squalene epoxidase (SQLE) gene. In this study, point mutations in the SQLE gene were studied in T. rubrum and T. mentagrophytes/T. interdigitale species complex as two main causative agents of dermatophytosis. Antifungal susceptibility of clinical isolates of T. rubrum (n = 27) and T. mentagrophytes/T. interdigitale (n = 56) was assessed using the M38-3rd edition CLSI method. The SQLE gene and ITS region were sequenced for all the fungal strains, and the mutation sites and genotypes of the terbinafine-resistant strains were characterized. The results demonstrated that, in T. rubrum, the minimum inhibitory concentration of terbinafine (MIC50 and MIC90) was 0.03 µg/ml, and the geometric mean (G mean) concentration was 0.02. For the T. mentagrophytes complex, the MIC50 and MIC90 were 0.03 and 1.0 µg/ml, respectively, and the G mean concentration was 0.04 µg/ml. Four out of the five resistant strains were T. indotineae harboring the F397L and Q408L mutations, while the last one was T. mentagrophytes genotype VII, which harbors the F397L mutation. T. indotineae was the prominent causative agent of terbinafine resistance, with 80 % of the isolates, and T. mentagrophytes genotype VII was introduced as a new genotype in the terbinafine-resistant T. mentagrophytes complex. Our findings further substantiate the importance of antifungal susceptibility testing in selecting the choice of drug for effective treatment of dermatophytosis and highlight the importance of screening dermatophyte species for point mutations responsible for newly developed resistant strains to improve the current knowledge of overcoming infections caused by resistant species.

Depletion of squalene epoxidase in synergy with glutathione peroxidase 4 inhibitor RSL3 overcomes oxidative stress resistance in lung squamous cell carcinoma.

Background: Lung squamous cell carcinoma (LUSC) lacks effective targeted therapies and has a poor prognosis. Disruption of squalene epoxidase (SQLE) has been implicated in metabolic disorders and cancer. However, the role of SQLE as a monooxygenase involved in oxidative stress remains unclear. Methods: We analyzed the expression and prognosis of lung adenocarcinoma (LUAD) and LUSC samples from GEO and TCGA databases. The proliferative activity of the tumors after intervention of SQLE was verified by cell and animal experiments. JC-1 assay, flow cytometry, and Western blot were used to show changes in apoptosis after intervention of SQLE. Flow cytometry and fluorescence assay of ROS levels were used to indicate oxidative stress status. Results: We investigated the unique role of SQLE expression in the diagnosis and prognosis prediction of LUSC. Knockdown of SQLE or treatment with the SQLE inhibitor terbinafine can suppress the proliferation of LUSC cells by inducing apoptosis and reactive oxygen species accumulation. However, depletion of SQLE also results in the impairment of lipid peroxidation and ferroptosis resistance such as upregulation of glutathione peroxidase 4. Therefore, prevention of SQLE in synergy with glutathione peroxidase 4 inhibitor RSL3 effectively mitigates the proliferation and growth of LUSC. Conclusion: Our study indicates that the low expression of SQLE employs adaptive survival through regulating the balance of apoptosis and ferroptosis resistance. In future, the combinational therapy of targeting SQLE and ferroptosis could be a promising approach in treating LUSC.

Squalene epoxidase promotes the chemoresistance of colorectal cancer via (S)-2,3-epoxysqualene-activated NF-κB.

BACKGROUND: While de novo cholesterol biosynthesis plays a crucial role in chemotherapy resistance of colorectal cancer (CRC), the underlying molecular mechanism remains poorly understood. METHODS: We conducted cell proliferation assays on CRC cells with or without depletion of squalene epoxidase (SQLE), with or without 5-fluorouracil (5-FU) treatment. Additionally, a xenograft mouse model was utilized to explore the impact of SQLE on the chemosensitivity of CRC to 5-FU. RNA-sequencing analysis and immunoblotting analysis were performed to clarify the mechanism. We further explore the effect of SQLE depletion on the ubiquitin of NF-κB inhibitor alpha (IκBα) and (S)-2,3-epoxysqualene on the binding of IκBα to beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) by using immunoprecipitation assay. In addition, a cohort of 272 CRC patients were selected for our clinical analyses. RESULTS: Mechanistically, (S)-2,3-epoxysqualene promotes IκBα degradation and subsequent NF-κB activation by enhancing the interaction between BTRC and IκBα. Activated NF-κB upregulates the expression of baculoviral IAP repeat containing 3 (BIRC3), sustains tumor cell survival after 5-FU treatment and promotes 5-FU resistance of CRC in vivo. Notably, the treatment of terbinafine, an inhibitor of SQLE commonly used as antifungal drug in clinic, enhances the sensitivity of CRC to 5-FU in vivo. Additionally, the expression of SQLE is associated with the prognosis of human CRC patients with 5-FU-based chemotherapy. CONCLUSIONS: Thus, our finding not only demonstrates a new role of SQLE in chemoresistance of CRC, but also reveals a novel mechanism of (S)-2,3-epoxysqualene-dependent NF-κB activation, implicating the combined potential of terbinafine for 5-FU-based CRC treatment.

An update on antifungal resistance in dermatophytosis.

INTRODUCTION: The reports of resistance to antifungal agents used for treating onychomycosis and other superficial fungal infections are increasing. This rise in antifungal resistance poses a public health challenge that requires attention. AREAS COVERED: This review explores the prevalence of dermatophytes and the current relationship between dermatophyte species, their minimum inhibitory concentrations (MICs) for terbinafine (an allylamine) and itraconazole (an azole), and various mutations prevalent in these species. The most frequently isolated dermatophyte associated with resistance in patients with onychomycosis and dermatophytosis was T. mentagrophytes. However, T. indotineae emerged as the most prevalent isolate with mutations in the SQLE gene, exhibiting the highest MIC of 8 µg/ml for terbinafine and MICs of 8 µg/ml and ≥ 32 µg/ml for itraconazole.Overall, the most prevalent SQLE mutations were Phe397Leu, Leu393Phe, Ala448Thr, Phe397Leu/Ala448Thr, and Lys276Asn/Leu415Phe (relatively recent). EXPERT OPINION: Managing dermatophyte infections requires a personalized approach. A detailed history should be obtained including details of travel, home and occupational exposure, and clinical examination of the skin, nails and other body systems. Relevant testing includes mycological examination (traditional and molecular). Additional testing, where available, includes MIC evaluation and detection of SQLE mutations. In case of suspected terbinafine resistance, itraconazole or voriconazole (less commonly) should be considered.

Squalene-epoxidase-catalyzed 24(S),25-epoxycholesterol synthesis promotes trained-immunity-mediated antitumor activity.

The importance of trained immunity in antitumor immunity has been increasingly recognized, but the underlying metabolic regulation mechanisms remain incompletely understood. In this study, we find that squalene epoxidase (SQLE), a key enzyme in cholesterol synthesis, is required for ß-glucan-induced trained immunity in macrophages and ensuing antitumor activity. Unexpectedly, the shunt pathway, but not the classical cholesterol synthesis pathway, catalyzed by SQLE, is required for trained immunity induction. Specifically, 24(S),25-epoxycholesterol (24(S),25-EC), the shunt pathway metabolite, activates liver X receptor and increases chromatin accessibility to evoke innate immune memory. Meanwhile, SQLE-induced reactive oxygen species accumulation stabilizes hypoxia-inducible factor 1α protein for metabolic switching into glycolysis. Hence, our findings identify 24(S),25-EC as a key metabolite for trained immunity and provide important insights into how SQLE regulates trained-immunity-mediated antitumor activity.

Age-related cholesterol and colorectal cancer progression: Validating squalene epoxidase for high-risk cases.

As people age, the risk and progression of colorectal cancer (CRC), along with cholesterol levels, tend to increase. Nevertheless, epidemiological studies on serum lipids and CRC have produced conflicting results. We previously demonstrated that the reduction of squalene epoxidase (SQLE) due to accumulated cholesterol within cells accelerates CRC progression through the activation of the ß-catenin pathway. This study aimed to investigate the mechanism by which age-related cholesterol accumulation within tissue accelerates CRC progression and to assess the clinical significance of SQLE in older individuals with elevated CRC risk. Using machine learning-based digital image analysis with fluorescence-immunohistochemistry, we assessed SQLE, GSK3ßpS9 (GSK3ß activity inhibition through serine 9 phosphorylation at GSK3ß), p53 wild-type (p53WT), and p53 mutant (p53MT) levels in CRC tissues. Our analysis revealed a significant reduction in SQLE, p53WT, and p53MT and increase in GSK3ßpS9 levels, all associated with the substantial accumulation of intra-tissue cholesterol in aged CRCs. Cox analysis underscored the significant influence of SQLE on overall survival and progression-free survival in grade 2-3 CRC patients aged over 50. SQLE and GSK3ßpS9 consistently exhibited outstanding prognostic and diagnostic performance, particularly in older individuals. Furthermore, combining SQLE with p53WT, p53MT, and GSK3ßpS9 demonstrated a robust diagnostic ability in the older population. In conclusion, we have identified that individuals aged over 50 face an increased risk of CRC progression due to aging-linked cholesterol accumulation within tissue and the subsequent reduction in SQLE levels. This study also provides valuable biomarkers, including SQLE and GSK3ßpS9, for older patients at elevated risk of CRC.

Terbinafine resistance in Trichophyton mentagrophytes and Trichophyton rubrum in the Czech Republic: A prospective multicentric study.

BACKGROUND: Terbinafine, an allylamine antifungal, is crucial for treating dermatophytosis by inhibiting squalene epoxidase (SQLE) in the ergosterol biosynthetic pathway. However, resistance is emerging, particularly in India and Southeast Asia, but reports of resistance spread worldwide. Despite this, comprehensive studies on terbinafine resistance in Trichophyton are still limited. OBJECTIVES: This research aimed to determine the prevalence of terbinafine resistance in the Czech Republic, with a focus on Trichophyton rubrum and Trichophyton mentagrophytes, and investigate the underlying molecular mechanisms. PATIENTS/METHODS: A total of 514 clinical strains of T. rubrum and 240 T. mentagrophytes collected from four Czech clinical institutions were screened for terbinafine resistance. Molecular investigations included DNA sequencing, specifically the ITS rDNA region and SQLE gene, as well as antifungal susceptibility testing following EUCAST guidelines. RESULTS: While no resistance was observed in T. rubrum, 2.5% of T. mentagrophytes strains exhibited resistance, marked by the F397L mutation in SQLE. Notably, resistance surged from 1.2% in 2019 to 9.3% in 2020 but reverted to 0% in 2021. All resistant strains were identified as T. mentagrophytes var. indotineae. Resistant strains exhibited high MICs for terbinafine (≥4 mg L-1 ) but low MICs to the other seven antifungals tested except for fluconazole. CONCLUSIONS: This study highlights the emergence of terbinafine-resistant T. mentagrophytes strains in the Czech Republic, with the F397L mutation being pivotal. Due to the relatively low resistance level, the current guidelines for dermatomycosis treatment in the Czech Republic remain effective, but ongoing surveillance is essential for timely adaptations if resistance patterns change.

Identifying squalene epoxidase as a metabolic vulnerability in high-risk osteosarcoma using an artificial intelligence-derived prognostic index.

BACKGROUND: Osteosarcoma (OSA) presents a clinical challenge and has a low 5-year survival rate. Currently, the lack of advanced stratification models makes personalized therapy difficult. This study aims to identify novel biomarkers to stratify high-risk OSA patients and guide treatment. METHODS: We combined 10 machine-learning algorithms into 101 combinations, from which the optimal model was established for predicting overall survival based on transcriptomic profiles for 254 samples. Alterations in transcriptomic, genomic and epigenomic landscapes were assessed to elucidate mechanisms driving poor prognosis. Single-cell RNA sequencing (scRNA-seq) unveiled genes overexpressed in OSA cells as potential therapeutic targets, one of which was validated via tissue staining, knockdown and pharmacological inhibition. We characterized changes in multiple phenotypes, including proliferation, colony formation, migration, invasion, apoptosis, chemosensitivity and in vivo tumourigenicity. RNA-seq and Western blotting elucidated the impact of squalene epoxidase (SQLE) suppression on signalling pathways. RESULTS: The artificial intelligence-derived prognostic index (AIDPI), generated by our model, was an independent prognostic biomarker, outperforming clinicopathological factors and previously published signatures. Incorporating the AIDPI with clinical factors into a nomogram improved predictive accuracy. For user convenience, both the model and nomogram are accessible online. Patients in the high-AIDPI group exhibited chemoresistance, coupled with overexpression of MYC and SQLE, increased mTORC1 signalling, disrupted PI3K-Akt signalling, and diminished immune infiltration. ScRNA-seq revealed high expression of MYC and SQLE in OSA cells. Elevated SQLE expression correlated with chemoresistance and worse outcomes in OSA patients. Therapeutically, silencing SQLE suppressed OSA malignancy and enhanced chemosensitivity, mediated by cholesterol depletion and suppression of the FAK/PI3K/Akt/mTOR pathway. Furthermore, the SQLE-specific inhibitor FR194738 demonstrated anti-OSA effects in vivo and exhibited synergistic effects with chemotherapeutic agents. CONCLUSIONS: AIDPI is a robust biomarker for identifying the high-risk subset of OSA patients. The SQLE protein emerges as a metabolic vulnerability in these patients, providing a target with translational potential.

Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing.

BACKGROUND: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. RESULT: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. CONCLUSION: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.

Therapeutic Updates on the Management of Tinea Corporis or Cruris in the Era of Trichophyton Indotineae: Separating Evidence from Hype-A Narrative Review.

The emergence and spread of Trichophyton indotineae (T. indotineae) has led to a sea change in the prescription practices of clinicians regarding the management of dermatophytic skin infections. An infection easily managed with a few weeks of antifungals, tinea corporis or cruris, is now often chronic and recurrent and requires prolonged treatment. Rising resistance to terbinafine, with documented squalene epoxidase (SQLE) gene mutations, and slow clinical response to itraconazole leave clinicians with limited treatment choices. However, in these testing times, it is essential that the tenets of antifungal stewardship be followed in making therapeutic decisions, and that the existing armamentarium of antifungals be used in rationale ways to counter this extremely common cutaneous infection, while keeping the growing drug resistance among dermatophytes in check. This review provides updated evidence on the use of various systemic antifungals for dermatophytic infection of the glabrous skin, especially with respect to the emerging T. indotineae species, which is gradually becoming a worldwide concern.

A New Genotype of Trichophyton quinckeanum with Point Mutations in Erg11A Encoding Sterol 14-α Demethylase Exhibits Increased Itraconazole Resistance.

Trichophyton quinckeanum, the causative agent of mouse favus, has been responsible for several infections of animal owners in recent years and showed an infection peak around 2020 in Jena, Thuringia. The isolated T. quinckeanum strains from Thuringia differ in some positions of the ITS region compared to strains from the IHEM collection as well as to Trichophyton schoenleinii. All T. quinckeanum strains of the new genotype show up to a 100-fold increased itraconazole resistance as measured by microplate laser nephelometry (MLN) assays. Analysis of genes involved in Trichophyton indotineae azole resistance, such as Erg1, which encodes squalene epoxidase, and Erg11B, one of two copies of the sterol 14-α demethylase gene, show a 100% identity between the two T. quinckeanum genotypes. In contrast, Erg11A fragments differ in 15-nucleotide positions between both T. quinckeanum genotypes, resulting in the unique amino acid substitution Ala256Ser in resistant strains. The new T. quinckeanum genotype may have evolved through interspecies mating. Mating type analysis showed a nearly 100% identity of the minus type MAT1-1-1 fragment for all T. quinckeanum isolates. The closely related Trichophyton schoenleinii belongs to the plus mating type and has 100% identical fragments of Erg1 and Erg11B. Erg11A protein sequences of T. schoenleinii and T. quinckeanum showed increased diversity.

[Recurrent tinea corporis generalisata due to Terbinafine-resistant Trichophyton rubrum strain : Long-term treatment with super bioavailability itraconazole]. / Rezidivierende Tinea corporis generalisata durch einen Terbinafin-resistenten Trichophyton-rubrum-Stamm : Langzeitbehandlung mit Super-Bioavailability-Itraconazol.

For more than 30 years, an 82-year-old man has been suffering from tinea corporis generalisata in the sense of Trichophyton rubrum syndrome. The patient received long-term treatment with terbinafine. Fluconazole had no effect. There was an increase in liver enzymes with itraconazole. Super bioavailability (SUBA) itraconazole was initially not tolerated. A therapy attempt with voriconazole was successful, but was stopped due to side effects. The Trichophyton (T.) rubrum strain isolated from skin scales was tested for terbinafine resistance using the breakpoint method and found to be (still) sensitive. Sequencing of the squalene epoxidase (SQLE) gene revealed a previously unknown point mutation of the codon for isoleucine ATC→ACC with amino acid substitution I479T (isoleucine479 threonine). Long-term therapy with terbinafine 250 mg had been given every 3 days since 2018. In addition, bifonazole cream, ciclopirox solution, and occasionally terbinafine cream were used. The skin condition was stable until an exacerbation of the dermatophytosis in 2021. There were erythematosquamous, partly atrophic, centrifugal, scaly, confluent plaques on the integument and the extremities. Fingernails and toenails had white to yellow-brown discoloration, and were hyperkeratotic and totally dystrophic. T. rubrum was cultured from skin scales from the integument, from the feet, from nail shavings from the fingernails and also toenails and detected by PCR. In the breakpoint test, the T. rubrum isolates from tinea corporis and nail samples showed a minimum inhibitory concentration (MIC) of 0.5 µg ml-1 (terbinafine resistance in vitro). Sequencing of the SQLE gene of the T. rubrum isolate revealed evidence of a further point mutation that led to amino acid substitution I479V (isoleucine 479 valine). Long-term therapy was started with SUBA itraconazole: 14 days 2â€¯× 1 capsule daily, then twice weekly administration of 2â€¯× 50 mg. During breaks in therapy, the mycosis regularly flared up again. Finally, 50 mg SUBA itraconazole was given 5 days a week, which completely suppressed the dermatophytosis. Topically, ciclopirox and miconazole cream were used alternately. In conclusion, in the case of recurrent and therapy-refractory dermatophytoses caused by T. rubrum, terbinafine resistance must also be considered in individual cases. An in vitro resistance test and point mutation analysis of the squalene epoxidase gene confirms the diagnosis. Itraconazole, also in the form of SUBA itraconazole, is the drug of choice for the oral antifungal treatment of these patients.

Squalene epoxidase/SQLE is a candidate target for treatment of colorectal cancers with p53 mutation and elevated c-MYC expression.

Elevated expression of c-MYC and inactivation of p53 represent two of the most common alterations in colorectal cancer (CRC). However, c-MYC and defective p53 are difficult to target therapeutically. Therefore, effectors downstream of both c-MYC and p53 may represent attractive, alternative targets for cancer treatment. In a bioinformatics screen we identified Squalene epoxidase/SQLE as a candidate therapeutic target that appeared to be especially relevant for cell survival in CRCs, which display elevated c-MYC expression and loss of p53 function. SQLE is a rate-limiting enzyme in the cholesterol synthesis. Here, we show that p53 supresses SQLE expression, cholesterol levels, and cell viability via the induction of miR-205, which directly targets SQLE. Furthermore, c-MYC induced SQLE expression directly and via its target gene AP4. The transcription factor AP4/TFAP4 directly induced SQLE expression and cholesterol levels, whereas inactivation of AP4 resulted in decreased SQLE expression and caused resistance to Terbinafine, an inhibitor of SQLE. Inhibition of SQLE decreased viability of CRC cells. This effect was enhanced in CRCs cells with p53 inactivation and/or enhanced c-MYC/AP4 expression. Altogether, our results demonstrate that SQLE represents a vulnerability for CRCs with p53 inactivation and elevated c-MYC activity.

Mining and modification of Oryza sativa-derived squalene epoxidase for improved ß-amyrin production in Saccharomyces cerevisiae.

ß-Amyrin is a pentacyclic triterpenoid and has anti-viral, anti-bacterial and anti-inflammatory activities. The synthetic pathway of ß-amyrin has been analyzed and its heterogeneous synthesis has been achieved in Saccharomyces cerevisiae. Squalene epoxidase (SQE) catalyzes the oxygenation of squalene to form 2,3-oxidosqualene and is rate-limiting in the synthetic pathways of ß-amyrin. The endogenous SQE in S. cerevisiae is insufficient for high production of ß-amyrin. Herein, eight squalene epoxidases derived from different plants were selected and characterized in S. cerevisiae for improved biosynthesis of ß-amyrin. Among them, the squalene epoxidase from Oryza sativa (OsSQE52) showed the best performance compared to other plant-derived sources. Through protein remodeling, the mutant OsSQE52L256R, obtained based on modeling analysis, increased the titer of ß-amyrin by 2.43-fold compared to that in the control strain with ERG1 overexpressed under the same conditions. Moreover, the expression of OsSQE52L256R was optimized with the improvement of precursor supply to further increase the production of ß-amyrin. Finally, the constructed strains produced 66.97 mg/L ß-amyrin in the shake flask, which was 6.45-fold higher than the original strain. Our study provides alternative SQEs for efficient production of ß-amyrin as well as other triterpenoids derived from 2,3-oxidosqualene.

Targeting Squalene Epoxidase Confers Metabolic Vulnerability and Overcomes Chemoresistance in HNSCC.

Cisplatin resistance poses a substantial hurdle in effectively treating head and neck squamous cell carcinoma (HNSCC). Utilizing multiple tumor models and examining an internal HNSCC cohort, squalene epoxidase (SQLE) is pinpointed as a key driver of chemoresistance and tumorigenesis, operating through a cholesterol-dependent pathway. Comprehensive transcriptomic analysis reveals that SQLE is essential for maintaining c-Myc transcriptional activity by stabilizing the c-Myc protein and averting its ubiquitin-mediated degradation. Mechanistic investigation demonstrates that SQLE inhibition diminishes Akt's binding affinity to lipid rafts via a cholesterol-dependent process, subsequently deactivating lipid raft-localized Akt, reducing GSK-3ß phosphorylation at S9, and increasing c-Myc phosphorylation at T58, ultimately leading to c-Myc destabilization. Importantly, employing an Sqle conditional knockout mouse model, SQLE's critical role in HNSCC initiation and progression is established. The preclinical findings demonstrate the potent synergistic effects of combining terbinafine and cisplatin in arresting tumor growth. These discoveries not only provide novel insights into the underlying mechanisms of SQLE-mediated cisplatin resistance and tumorigenesis in HNSCC but also propose a promising therapeutic avenue for HNSCC patients unresponsive to conventional cisplatin-based chemotherapy.

Squalene epoxidase (SE) gene related to triterpenoid biosynthesis assists to select elite genotypes in medicinal plant: Cyclocarya paliurus (Batal.) Iljinskaja.

Triterpenoids, known for their anti-inflammatory, anticancer, and hypoglycemic properties, are the major bioactive components in Cyclocarya paliurus (Batal.) Iljinskaja. Selecting elite individuals with high triterpenoids content is the basis of C. paliurus industry for medicinal use. In this study, seasonal variation patterns of total triterpenoids and five triterpene monomers accumulation for three groups with different total triterpenoid contents (TTC; H: 59.74-64.03 mg g-1; M: 47.66-57.08 mg g-1, and L: 35.26-42.22 mg g-1) were surveyed. Seasonal expression dynamics of 6 key genes relevant to triterpenoids biosynthesis, including HMGR, DXR, SQS, SE, LUS, and ß-AS, were described by quantitative real-time PCR (qRT-PCR) for three groups. The expression levels of HMGR, SE, LUS, and ß-AS genes in group H were higher than in groups M and L. In addition, Pearson correlation analysis showed that they were significantly positively correlated with triterpene accumulation, and the expression level of SE gene not only was significantly correlated with downstream genes, but also exhibited a linear relationship with TTC, especially in September. These results suggest that SE gene could serve as an effective make for screening elite individuals with high TTC from the germplasm of C. paliurus for medicinal use. Further testing on randomly selected individuals in next September proved the feasibility and reliability of SE gene in assisted selection. Also, we successfully cloned the full-length cDNA of SE. Thus, our work provides an efficient way to attain superior genotypes to develop medicinal industry of C. paliurus in practice.

Heterologous expression of Chlorophytum borivilianum Squalene epoxidase in tobacco modulates stigmasterol production and alters vegetative and reproductive growth.

KEYMESSAGE: CbSE overexpression increased stigmasterol levels and altered plant morphology. The genes upstream and downstream of CbSE were found to be upregulated, which confirms its regulatory role in the saponin biosynthetic pathway. Chlorophytum borivilianum is a high-value medicinal plant with many promising preclinical applications that include saponins as a major active ingredient. Squalene epoxidase (SE) is one of the major rate-limiting enzymes of the saponin biosynthetic pathway. Here, we functionally characterized C. borivilianum SE (CbSE) by over-expressing heterologously in Nicotiana tabacum. The heterologous expression of CbSE resulted in stunted pant growth with altered leaf and flower morphology. Next, RT-qPCR analysis of transgenic plants overexpressing CbSE revealed increased expression levels of Cycloartenol synthase (CAS), Beta amyrin synthase (ßAS), and cytochrome P450 monooxygenase 51 (CYP51) (Cytochrome P450), which encode key enzymes for triterpenoid and phytosterol biosynthesis in C. borivilianum. Further, Methyl Jasmonate (MeJa) treatment upregulated Squalene synthase (SQS), SE, and Oxidosqualene cyclases (OSCs) to a significant level. GC-MS analysis of the leaf and hairy roots of the transformants showed an increased stigmasterol content (0.5-1.0 fold) compared to wild type (WT) plants. These results indicate that CbSE is a rate-limiting gene, which encodes an efficient enzyme responsible for phytosterol and triterpenoid production in C. borivilianum.