Recombinant Human Monofunctional C1-tetrahydrofolate synthase, mitochondrial (MTHFD1L), partial

What is MTHFD1L and what is its function in cellular metabolism?

MTHFD1L (Methylenetetrahydrofolate Dehydrogenase 1-Like) is a monofunctional enzyme localized in mitochondria that possesses 10-formyl-tetrahydrofolate synthetase activity. Despite sharing 61% amino acid similarity with the cytoplasmic trifunctional MTHFD1, MTHFD1L is monofunctional, possessing only the 10-formyl-THF synthetase activity . It catalyzes the last step in the flow of one-carbon units from mitochondria to cytoplasm, producing formate from 10-formyl-THF . This enzyme plays a crucial role in the folate cycle and contributes to the intercompartmental pathway of one-carbon metabolism, with over 75% of one-carbon units entering the cytoplasmic methyl cycle being mitochondrially derived .

What is the tissue distribution pattern of MTHFD1L expression?

MTHFD1L is expressed in most adult tissues but shows higher levels in spleen, thymus, brain, and placenta . During embryonic development, MTHFD1L is expressed at all stages of mammalian embryogenesis and ubiquitously throughout the embryo, with localized regions of higher expression along the neural tube, the developing brain, craniofacial structures, limb buds, and the tail bud . This widespread but differential expression pattern suggests tissue-specific roles for MTHFD1L in both developmental processes and adult physiological functions.

How does MTHFD1L deficiency affect embryonic development?

Deletion of the Mthfd1l gene causes embryonic lethality with 100% penetrance, demonstrating its essential role in development . Embryos lacking Mthfd1l exhibit aberrant neural tube closure including craniorachischisis and exencephaly and/or a wavy neural tube . These neural tube defects highlight the critical importance of mitochondrial one-carbon metabolism for proper embryonic development, particularly in the formation and closure of the neural tube. The severe phenotypes observed in knockout models indicate that MTHFD1L function cannot be compensated by other enzymes during critical developmental processes.

What role does MTHFD1L play in cancer development and progression?

MTHFD1L has emerged as a significant factor in multiple cancer types. In liver hepatocellular carcinoma (LIHC), MTHFD1L is upregulated compared to normal tissue and is associated with vital status (alive vs. dead) of patients . Similarly, in tongue squamous cell carcinoma (TSCC), high MTHFD1L expression correlates with decreased disease-free survival time and poor prognosis . MTHFD1L confers redox homeostasis and promotes TSCC cell growth . Comprehensive multi-omics analysis has identified MTHFD1L as a shared biomarker across multiple cancer types, including bladder urothelial cancer (BLCA), head and neck cancer (HNSC), kidney renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), and uterine corpus endometrial carcinoma (UCEC) . The overexpression is consistent across different cancer stages, races, genders, and ages .

What experimental models are available to study MTHFD1L function?

Several experimental models have been developed to study MTHFD1L function. In vitro, cell lines with MTHFD1L knockdown or overexpression have been established to investigate its role in cellular processes . For example, CAL-27 and SCC-15 cancer cell lines have been used with shRNA-mediated knockdown of MTHFD1L to study its effects on tumor growth . In vivo, Mthfd1l knockout mice have been generated to study the developmental consequences of MTHFD1L deficiency . Additionally, xenograft models using MTHFD1L-manipulated cancer cells implanted in BALB/c nude mice have demonstrated the importance of MTHFD1L in tumorigenesis . These models provide valuable tools for investigating MTHFD1L function in different biological contexts.

How can MTHFD1L promoter activity be measured in experimental settings?

MTHFD1L promoter activity can be measured using dual fluorescent reporter gene assays. Researchers have constructed plasmids containing the MTHFD1L promoter region using specific primers (5′-CTGGTACAGCTTACCAAAC-3′ and 5′-TTCTCAGGGGACACGGAGCT-3′) . The MTHFD1L promoter fragment can be inserted into a luciferase reporter vector such as pGL4.10-Luc2 between HindIII and XhoI restriction sites . Cells of interest are then transfected with these constructs and treated with compounds of interest (e.g., rapamycin) for a specified period (typically 24h), followed by measurement of luciferase activity using standard luciferase reporter assay kits . This approach allows for quantitative assessment of factors that regulate MTHFD1L transcription.

What techniques are most effective for detecting MTHFD1L expression in tissue samples?

Multiple complementary techniques can be employed to detect MTHFD1L expression in tissue samples. Immunohistochemistry (IHC) has been successfully used to analyze MTHFD1L protein expression in both tumor tissues and paired adjacent normal tissues . For mRNA analysis, quantitative PCR (qPCR) provides a reliable method for measuring MTHFD1L expression levels . RNA sequencing (RNA-Seq) offers a comprehensive approach, as demonstrated in studies utilizing TCGA data across multiple cancer types . For validation of expression differences, techniques such as western blotting can be used to confirm protein levels . A multi-platform approach combining these methods provides the most robust assessment of MTHFD1L expression.

How can MTHFD1L be effectively knocked down or overexpressed in cellular models?

For MTHFD1L knockdown, short hairpin RNA (shRNA) approaches have proven effective. Researchers have successfully used shMTHFD1L constructs to reduce MTHFD1L expression in cancer cell lines such as CAL-27 and SCC-15 . For overexpression studies, full-length MTHFD1L cDNA can be cloned into appropriate expression vectors for transfection into target cells . Lentiviral or retroviral delivery systems can be employed for stable integration and long-term expression changes. The efficiency of knockdown or overexpression should be validated at both mRNA and protein levels using qPCR and western blotting, respectively, before proceeding with functional studies.

What are the recommended protocols for analyzing MTHFD1L in patient-derived samples?

When analyzing MTHFD1L in patient-derived samples, a standardized workflow is recommended. For tissue samples, proper collection and preservation are critical; flash-freezing in liquid nitrogen for molecular studies and formalin fixation for histological analysis are standard approaches . RNA extraction should follow validated protocols to ensure high-quality nucleic acids for expression analysis . For protein studies, extraction buffers compatible with mitochondrial proteins should be used . Patient samples should be accompanied by comprehensive clinical information to enable correlation between MTHFD1L expression and clinicopathological features . Statistical analyses including Kaplan-Meier survival analysis, Cox proportional hazards regression, and chi-square tests have been successfully applied to evaluate the relationship between MTHFD1L expression and clinical outcomes .

How does MTHFD1L contribute to tumorigenesis in xenograft models?

In xenograft models, MTHFD1L has been shown to significantly impact tumor growth. Using BALB/c nude mice injected with MTHFD1L-manipulated cancer cells, researchers have demonstrated that MTHFD1L knockdown results in slower tumor growth and lower tumor weight compared to control groups (P < 0.001) . Immunohistochemical analysis of tumor biopsies from these models showed that MTHFD1L knockdown tumors had reduced cell proliferation indices based on Ki67 staining and increased cell-apoptosis-associated indices based on cleaved caspase-3 (P < 0.05) . These findings highlight the crucial role of MTHFD1L in promoting tumorigenesis through enhancing cell proliferation and inhibiting apoptosis in vivo.

What are the clinical associations of MTHFD1L expression with tumor stage and progression?

MTHFD1L expression shows significant associations with various clinicopathological features across cancer types. In tongue squamous cell carcinoma, high MTHFD1L expression is associated with age (P = 0.024), T classification (P < 0.001), N classification (P = 0.003), and clinical TNM stage (P = 0.001) . This indicates that MTHFD1L overexpression is linked to advanced disease stages. Similar patterns have been observed in liver cancer and other malignancies . The consistent association between MTHFD1L expression and tumor stage across multiple cancer types suggests a fundamental role in tumor progression rather than a cancer-type-specific effect, making it a potentially valuable biomarker for disease monitoring and prognostication.

How might MTHFD1L serve as a therapeutic target in cancer treatment?

Based on the consistent overexpression and prognostic significance of MTHFD1L across multiple cancer types, it represents a promising therapeutic target. A constructed MTHFD1L gene-drug interaction network includes information on chemotherapeutic drugs that can reduce or enhance MTHFD1L expression levels . Potential approaches for targeting MTHFD1L include small molecule inhibitors specifically designed to block its enzymatic activity, RNA interference-based therapeutics to downregulate expression, or CRISPR-Cas9 gene editing strategies. Given MTHFD1L's role in redox homeostasis in cancer cells , combination therapies that simultaneously target MTHFD1L and other metabolic vulnerabilities could be particularly effective. Future research should focus on developing selective inhibitors and evaluating their efficacy in preclinical models.

What are the knowledge gaps in understanding MTHFD1L regulation and function?

Despite the growing body of research on MTHFD1L, several knowledge gaps remain. The precise mechanisms by which MTHFD1L promotes cancer cell survival and proliferation are not fully elucidated. Similarly, the upstream regulators controlling MTHFD1L expression in normal and disease states need further investigation. The potential role of MTHFD1L in immune modulation is suggested by its correlation with immune cell infiltration in cancers , but the mechanistic details remain unclear. Additionally, the impact of MTHFD1L variants on enzyme function and disease susceptibility requires more comprehensive analysis. Understanding these aspects will provide a more complete picture of MTHFD1L biology and its therapeutic potential.

How does MTHFD1L interact with the immune microenvironment in cancer?

Emerging evidence suggests important interactions between MTHFD1L and the tumor immune microenvironment. Analysis using the ImmuneCellAI database has revealed correlations between MTHFD1L expression and 24 immune cell types in liver cancer . Comprehensive validation combining CIBERSORT, TIMER, and ImmuneCellAI databases determined that resting dendritic cells, M0 macrophages, and M2 macrophages are closely related to MTHFD1L expression . These findings suggest that MTHFD1L may influence immune cell infiltration and function within the tumor microenvironment. Further research is needed to determine whether MTHFD1L directly modulates immune cell behavior or if these correlations reflect broader metabolic interactions between cancer cells and immune components.