TRDMT1 Antibody, Biotin conjugated

What is TRDMT1 and what cellular functions does it perform?

TRDMT1, formerly known as DNA methyltransferase 2 (DNMT2), functions primarily as a highly conserved cytosine-C5 methyltransferase that introduces C38 methylation in tRNA-Asp across numerous species. Despite its historical name suggesting DNA methyltransferase activity, TRDMT1 exhibits minimal methylation activity on DNA itself. Recent research has expanded our understanding of its function, demonstrating that TRDMT1 contributes to mRNA methylation in addition to its established role in tRNA modification .

The protein specifically methylates cytosine 38 in the anticodon loop of tRNA(Asp), which has significant implications for translational fidelity and cellular stress responses . TRDMT1 is essential for oxidative stress and heat shock-induced tRNA cleavage, suggesting its role in cellular adaptation mechanisms. Furthermore, TRDMT1 has emerged as a key regulator of homologous recombination (HR) in transcribed genomic regions, placing it at the intersection of RNA metabolism and DNA repair pathways .

What are the key specifications of commercially available TRDMT1 Antibody, Biotin conjugated?

Commercially available TRDMT1 Antibody with biotin conjugation typically features the following specifications:

The antibody recognizes the human TRDMT1 protein, which has several synonyms including DNA (cytosine-5)-methyltransferase-like protein 2, Dnmt2, DNA methyltransferase homolog HsaIIP, and PuMet . Researchers should note that while the primary verified application is ELISA, individual product specifications may differ between manufacturers.

How does the TRDMT1 protein structure relate to its function?

The structure-function relationship of TRDMT1 reveals important insights into its regulatory mechanisms. Research has identified that TRDMT1 is poly-ubiquitinated at Lysine 251 (K251) by the E3 ligase TRIM28 (also known as KAP-1). This post-translational modification plays a critical role in removing TRDMT1 from DNA damage sites, thereby allowing the completion of homologous recombination repair .

Interestingly, the three-dimensional structure of TRDMT1 positions K251 adjacent to Glycine 155 (G155). This spatial arrangement has significant functional implications, as evidenced by the G155V mutation which leads to hyper-ubiquitination of TRDMT1. The consequent reduction in TRDMT1 protein levels results in impaired homologous recombination capacity . This structural understanding provides researchers with valuable targets for investigating the regulation of TRDMT1 in various cellular contexts.

What distinguishes TRDMT1 from other DNA methyltransferases?

Despite its historical classification as DNMT2 (DNA methyltransferase 2), TRDMT1 differs fundamentally from other DNA methyltransferases. While it shares sequence homology with DNA methyltransferases and contains the characteristic catalytic motifs, TRDMT1 does not exhibit significant methyltransferase activity on DNA substrates .

Instead, TRDMT1 has evolved to specialize in RNA methylation, particularly adding m5C (5-methylcytosine) to position 38 in the anticodon loop of tRNA-Asp. Recent research has further expanded our understanding by demonstrating that TRDMT1 can also methylate mRNAs, which appears to be particularly important during DNA damage responses . This RNA-directed methyltransferase activity distinguishes TRDMT1 from classical DNA methyltransferases and places it in a unique category of epigenetic regulators that connect RNA modification with genomic stability.

How does TRDMT1 participate in DNA damage response pathways?

TRDMT1 has emerged as a critical factor in DNA damage response (DDR) pathways, particularly in homologous recombination (HR) repair. Research using the KillerRed (KR) system, which generates reactive oxygen species in a light-inducible manner to cause DNA strand breaks, has revealed that TRDMT1-mediated mRNA m5C modification is required for efficient repair of double-strand breaks (DSBs) through the HR pathway .

Experimental evidence indicates that cells deficient in TRDMT1 or expressing dysfunctional variants (such as G155V) exhibit increased sensitivity to DNA-damaging agents, underscoring the importance of this protein in the cellular response to genotoxic stress . These findings establish TRDMT1 as a unique link between RNA modification and genome stability maintenance.

What is the clinical significance of TRDMT1 in cancer therapy resistance?

TRDMT1 has emerged as a potential biomarker and therapeutic target in cancer, particularly in the context of platinum-based chemotherapy resistance. Research on ovarian cancer has revealed a significant correlation between TRDMT1 expression levels and treatment response. High expression of TRDMT1 in ovarian cancer patients correlates with resistance to platinum-based therapies, suggesting that TRDMT1 may confer a protective effect against DNA-damaging agents used in cancer treatment .

Conversely, genetic evidence supports this relationship from the opposite direction as well. The G155V mutation in TRDMT1, which leads to reduced protein levels and impaired function, was identified in an ovarian cancer patient who exhibited exceptional response to platinum treatment (a "super responder"). Cells expressing TRDMT1-G155V demonstrate increased sensitivity to cisplatin both in vitro and in vivo experimental models .

These observations have prompted investigation into TRDMT1 inhibition as a strategy to overcome chemotherapy resistance. Preliminary studies with TRDMT1 inhibitors have shown promise in resensitizing TRDMT1-high tumor cells to cisplatin, suggesting a potential avenue for combination therapy in resistant cancers . This represents a novel approach that targets RNA modification mechanisms to enhance the efficacy of DNA-damaging chemotherapeutics.

How does ubiquitination regulate TRDMT1 function?

The regulation of TRDMT1 through ubiquitination represents a sophisticated control mechanism that balances its activity in the context of DNA damage response. Research has identified that TRDMT1 undergoes poly-ubiquitination specifically at Lysine 251 (K251), mediated by the E3 ubiquitin ligase TRIM28 (also known as KAP-1) . This post-translational modification serves as a critical regulatory switch in the DNA damage response pathway.

The functional consequence of this ubiquitination is the removal of TRDMT1 from DNA damage sites. While TRDMT1 is initially required at these locations to promote homologous recombination through its RNA methylation activity, its persistent presence becomes inhibitory to the completion of the repair process. Therefore, the timely ubiquitination and subsequent removal of TRDMT1 allows for the proper progression and completion of homologous recombination repair .

The regulatory importance of this mechanism is highlighted by the effects of mutations that alter this ubiquitination process. The G155V mutation in TRDMT1, while not directly at the ubiquitination site, affects the protein's three-dimensional structure such that K251 becomes more accessible to TRIM28. This results in hyper-ubiquitination of TRDMT1, excessive degradation of the protein, and consequently impaired homologous recombination capacity .

What experimental approaches are used to study TRDMT1's role in cancer therapy sensitivity?

Researchers investigating TRDMT1's role in cancer therapy employ a multi-faceted experimental approach spanning molecular, cellular, and clinical studies. At the molecular level, techniques including mass spectrometry have been instrumental in identifying post-translational modifications such as the key ubiquitination at K251 by TRIM28 . Site-directed mutagenesis enables the creation of variants like G155V to study structure-function relationships.

Cell survival assays provide critical data on TRDMT1's impact on therapy response. These typically involve seeding approximately 500 cells (U2OS, TRDMT1 KO U2OS, MCF-7, HCC1954, HCC1937, or cells transfected with TRDMT1 siRNA or GFP-TRDMT1 variants) in culture dishes with or without TRDMT1 inhibitors (2.5 μM). After exposure to various treatments including cisplatin (1 μM), ATM inhibitors (KU55933, 10 μM), ATR inhibitors (AZ20, 1 μM), PARP inhibitors (Olaparib, 1 μM), or ionizing radiation, colony formation is assessed after 7-14 days to quantify survival .

In vivo xenograft models further validate findings from cell culture, while clinical correlations between TRDMT1 expression or mutation status and patient treatment responses provide translational relevance. Together, these approaches form a comprehensive framework for understanding TRDMT1's significance in therapy resistance and its potential as a therapeutic target.

How do TRDMT1 mutations affect protein function and clinical outcomes?

The G155V mutation in TRDMT1 represents a particularly intriguing case study in structure-function relationships with direct clinical implications. This mutation affects the protein's three-dimensional conformation in a manner that significantly impacts its regulation and function. Although G155 is not directly involved in the catalytic activity of TRDMT1, it is spatially positioned adjacent to K251 in the folded protein structure .

This structural arrangement becomes crucial because K251 is the site of ubiquitination by the E3 ligase TRIM28. The G155V mutation appears to alter the protein conformation in a way that makes K251 more accessible to TRIM28, resulting in hyper-ubiquitination of TRDMT1. This excessive ubiquitination leads to increased degradation of TRDMT1, resulting in reduced protein levels and consequently impaired function in homologous recombination repair .

The clinical significance of this mutation was evidenced in an ovarian cancer patient characterized as a "super responder" to platinum-based therapy. Cells expressing the TRDMT1-G155V variant show increased sensitivity to cisplatin both in laboratory experiments and in animal models . This case demonstrates how structural alterations in TRDMT1 can translate to meaningful differences in clinical outcomes, highlighting the potential value of TRDMT1 mutational status as a biomarker for predicting therapy response.

What are the optimal conditions for using TRDMT1 Antibody, Biotin conjugated in ELISA?

When performing ELISA with biotin-conjugated TRDMT1 antibody, several optimization steps can enhance specificity and sensitivity. Based on manufacturer specifications, researchers should consider the following protocol recommendations:

For antigen coating, recombinant TRDMT1 protein or cell lysates containing endogenous TRDMT1 should be diluted in carbonate-bicarbonate buffer (pH 9.6) at concentrations of 1-10 μg/mL and incubated overnight at 4°C . After blocking with 1-5% BSA in PBS for 1-2 hours at room temperature, the biotin-conjugated TRDMT1 antibody should be applied at a starting dilution of 1:1000, with optimization through titration experiments .

For detection, streptavidin-HRP conjugate (typically 1:5000-1:10000 dilution) should be used, followed by appropriate substrate (TMB for colorimetric detection). Temperature control is critical—all incubation steps should occur at room temperature (25°C) except for the initial antigen coating .

When analyzing results, researchers should include a standard curve using purified TRDMT1 protein (0-1000 ng/mL) to enable quantification. Additionally, signal-to-noise ratio optimization can be achieved by adjusting antibody concentration, incubation times (typically 1-2 hours for primary antibody), and washing stringency (PBS-Tween 0.05%, 3-5 washes per step).

What controls should be included when studying TRDMT1 in DNA damage response experiments?

When studying TRDMT1 in DNA damage contexts, time-course experiments are essential, with samples collected before damage induction (0h) and at multiple timepoints after (typically 1h, 3h, 6h, 12h, 24h) to capture the dynamic regulation of TRDMT1 . DNA damage controls should include both TRDMT1-dependent (e.g., cisplatin) and TRDMT1-independent damaging agents.

For ubiquitination studies, researchers should include the K251R mutant (ubiquitination-resistant) and co-transfection with TRIM28 siRNA to validate the specificity of the ubiquitination pathway . When assessing TRDMT1's impact on homologous recombination, established HR reporter assays with appropriate positive controls (e.g., BRCA1 siRNA) provide quantitative readouts of repair efficiency.

How can researchers optimize immunohistochemistry protocols for TRDMT1 detection in tissue samples?

Optimizing immunohistochemistry (IHC) protocols for TRDMT1 detection requires attention to several critical parameters. Based on reported protocols, paraffin-embedded tissue sections should undergo heat-mediated antigen retrieval in citrate buffer (pH 6.0) under high pressure to expose the TRDMT1 epitopes that may be masked during fixation .

Blocking is particularly important for biotin-conjugated antibodies to prevent non-specific binding. Researchers should block endogenous biotin using an avidin-biotin blocking kit, followed by blocking with 10% normal goat serum for 30 minutes at room temperature . The primary TRDMT1 antibody should be diluted in 1% BSA and incubated overnight at 4°C, with the optimal dilution typically around 1:200 for biotin-conjugated antibodies .

For detection, researchers should employ an HRP-conjugated streptavidin system rather than secondary antibodies, since the primary antibody is already biotin-conjugated. Visualization can be achieved using DAB (3,3'-diaminobenzidine) or other chromogenic substrates compatible with HRP . Counterstaining with hematoxylin provides cellular context, with careful timing (30 seconds to 2 minutes) to avoid obscuring the specific TRDMT1 signal.

Optimization should include titration experiments testing antibody dilutions from 1:100 to 1:500, as well as comparison of different antigen retrieval methods (e.g., citrate versus EDTA buffers) to determine conditions that maximize specific staining while minimizing background.

What strategies can resolve technical challenges in Western blot analysis of TRDMT1?

Western blot analysis of TRDMT1 presents several technical challenges that require specific optimization strategies. TRDMT1 has multiple reported isoforms with predicted band sizes of 45, 42, 40, 13, 8, and 7 kDa, with the most commonly observed band at approximately 45 kDa . Researchers should use gradient gels (4-15% or 4-20%) to effectively separate these different isoforms.

Protein extraction requires careful consideration—RIPA buffer with protease inhibitors, phosphatase inhibitors, and deubiquitinase inhibitors (e.g., N-ethylmaleimide) is recommended, particularly when studying TRDMT1 post-translational modifications . For ubiquitination studies, denaturing conditions with 1% SDS followed by dilution and immunoprecipitation may be necessary to disrupt protein interactions that could mask ubiquitination sites.

Transfer optimization is critical for TRDMT1 detection. Semi-dry transfer systems with 0.2 μm PVDF membranes typically yield better results than nitrocellulose. Transfer should be performed at lower voltages (10-15V) for longer periods (60-90 minutes) to ensure efficient transfer of all isoforms . For detection, researchers should block with 5% non-fat dry milk in TBST and use the antibody at a concentration of 4 μg/ml, followed by appropriate secondary antibodies or streptavidin-HRP for biotin-conjugated primary antibodies .

Common troubleshooting measures include increasing antibody concentration for weak signals, extending exposure times, and using enhanced chemiluminescence substrates with higher sensitivity for low-abundance TRDMT1 detection.

What considerations should guide experimental design for testing TRDMT1 inhibitors in cancer therapy models?

Developing robust experimental designs for evaluating TRDMT1 inhibitors in cancer therapy contexts requires careful attention to multiple parameters. Cell line selection should include pairs of TRDMT1-high and TRDMT1-low cancer models, with ovarian cancer lines being particularly relevant given the documented role of TRDMT1 in platinum resistance . Patient-derived xenografts that maintain the heterogeneity of clinical samples provide more translational insights than established cell lines alone.

When testing TRDMT1 inhibitors, researchers should establish dose-response relationships by treating cells with a range of concentrations (typically 0.1-10 μM) both as single agents and in combination with DNA-damaging therapies like cisplatin . Positive controls should include siRNA-mediated TRDMT1 knockdown or CRISPR knockout models to benchmark the inhibitor's efficacy against complete loss of TRDMT1 function.

For in vivo studies, multiple dosing schedules should be evaluated, including concurrent administration with chemotherapy versus pre-treatment with TRDMT1 inhibitors. Tumor growth inhibition, survival analysis, and mechanistic biomarkers (e.g., γH2AX foci for DNA damage, RAD51 foci for homologous recombination) should be assessed . Additionally, pharmacokinetic/pharmacodynamic relationships should be established by measuring both inhibitor concentrations in tumor tissue and functional readouts like reduced RNA m5C levels to confirm target engagement.

Researchers should also consider potential resistance mechanisms to TRDMT1 inhibition through long-term treatment studies, analyzing changes in expression of other RNA methyltransferases or DNA repair pathway components that might compensate for TRDMT1 inhibition.