NNMT Antibody

What is NNMT and what cellular functions does it serve?

NNMT (Nicotinamide N-methyltransferase) is a cytosolic enzyme that catalyzes the N-methylation of nicotinamide and other pyridines using S-adenosylmethionine (SAM) as a methyl donor. The enzyme plays a critical role in xenobiotic metabolism and is involved in the biotransformation of drugs and other foreign compounds. In normal physiology, NNMT participates in nicotinamide clearance and energy homeostasis by converting nicotinamide to 1-methylnicotinamide (MNAM) . The protein is approximately 28 kDa in size and is endogenously expressed in various human tissues, with notable expression in liver and adipose tissue .

What detection methods are available for NNMT using antibodies?

Current research primarily utilizes Western blotting (WB) as the validated application for NNMT antibody detection. Commercial antibodies are typically used at a dilution of 1:1000 for Western blot applications . Detection of NNMT has been successfully demonstrated in human kidney tissue, human lung tissue, A549 human lung carcinoma cell line, and 786-O human renal cell adenocarcinoma cell line, yielding a specific band at approximately 26 kDa under reducing conditions . Immunohistochemistry has also been employed to examine NNMT expression in breast carcinoma and paracancerous tissues, providing valuable insights into its clinical relevance .

How should researchers validate NNMT antibody specificity?

Validation of NNMT antibody specificity should include:

• Western blot analysis using both positive control tissues/cell lines (human kidney, lung, A549 cells, or 786-O cells) and negative controls

• Confirmation of the expected molecular weight band (approximately 26-28 kDa)

• Comparison with recombinant NNMT protein standards when available

• Testing in knockout or knockdown models when possible to confirm specificity

• Cross-reactivity assessment in multi-species studies noting that some antibodies are specifically reactive with human NNMT but may not cross-react with mouse or other species

What are the optimal sample preparation techniques for NNMT detection?

For optimal NNMT detection using antibody-based methods, researchers should consider the following preparation techniques:

• For Western blotting: Use PVDF membranes with appropriate reducing conditions and Immunoblot Buffer Group 1 for optimal results .

• For protein extraction: Complete cell lysis with detergent-based buffers containing protease inhibitors is essential.

• Sample storage: Freshly prepared lysates provide optimal results, but samples can be stored at -80°C with glycerol to prevent freeze-thaw damage.

• For tissue samples: Flash-freezing in liquid nitrogen followed by mechanical homogenization yields better preservation of protein integrity than chemical homogenization methods.

Researchers should avoid multiple freeze-thaw cycles as this may degrade the target protein and lead to unreliable results.

How can researchers optimize NNMT antibody performance in different applications?

Based on available research data, the following optimization strategies are recommended:

For monoclonal antibodies like the mouse anti-human NNMT (Clone #954208), researchers should use 2 μg/mL concentration followed by HRP-conjugated anti-mouse IgG secondary antibody for optimal detection .

What is known about NNMT expression patterns across cancer types?

NNMT is overexpressed in various human tumors and has been associated with cancer development and progression. Research findings indicate:

• Breast cancer: NNMT overexpression has been documented in several breast cancer cell lines and is associated with clinical outcomes. Immunohistochemistry studies have revealed significantly higher NNMT expression (53.9%) in breast carcinoma compared to paracancerous tissues .

• Ovarian and colon cancer: Studies using syngeneic ID8 ovarian and MC38 colon cancer models have demonstrated the importance of NNMT in the tumor microenvironment .

• Melanoma: Research has suggested NNMT expression may have utility as a diagnostic and prognostic biomarker in melanoma .

• Renal cell carcinoma: NNMT is expressed in 786-O human renal cell adenocarcinoma cell lines, making it a potential biomarker for this cancer type .

These expression patterns make NNMT a valuable biomarker for cancer research and potentially a therapeutic target.

How does NNMT contribute to chemoresistance mechanisms in cancer?

NNMT has been implicated in enhancing chemoresistance in cancer cells through several mechanisms:

• SIRT1-p53 Pathway: NNMT expression appears to influence the SIRT1-p53 axis, affecting cell survival in response to chemotherapeutic agents. Research has examined the relationship between NNMT, SIRT1, p53, and acetyl-p53 proteins using Western blotting .

• Epigenetic Modifications: In cancer cells, overexpression of NNMT results in excess consumption of methyl units from S-adenosylmethionine (SAM), leading to histone hypomethylation that substantially alters the epigenetic landscape . This epigenetic reprogramming may contribute to therapeutic resistance.

• Metabolic Alterations: NNMT-mediated changes in cellular metabolism, particularly through the production of 1-methylnicotinamide (MNAM), may protect cancer cells from certain chemotherapeutic agents.

• Cell Survival Pathways: Cell viability, colony formation, and apoptosis assays have demonstrated that NNMT expression affects cancer cell survival in the presence of chemotherapeutic agents .

Researchers investigating chemoresistance should consider examining these pathways when studying NNMT's role in therapeutic response.

How does NNMT function in the tumor microenvironment?

NNMT plays a complex role in shaping the tumor microenvironment through interactions with cancer-associated fibroblasts (CAFs) and immune cells:

• CAF Transformation: NNMT is highly expressed in the stroma of several malignancies and drives the transformation of resting fibroblasts into cancer-associated fibroblasts (CAFs) through epigenetic changes .

• Immune Regulation: NNMT-expressing CAFs influence the tumor-immune environment through several mechanisms:

  • IFN-γ produced by peripheral blood mononuclear cells (PBMCs) upregulates NNMT expression in fibroblasts

  • T cells are the main source of IFN-γ in the tumor microenvironment

  • Upregulation of NNMT in CAFs induces secretion of CXCL1 through promoter hypomethylation

  • CXCL1 acts as a chemoattractant for immunosuppressive myeloid-derived suppressor cells (MDSCs)

  • NNMT-expressing CAFs recruit high numbers of MDSCs through CXCL1 secretion

• Impact on Tumor Growth: Studies using whole-body NNMT-knockout mice have shown that NNMT knockout of the stroma significantly reduces MDSC abundance and increases functional CD8+ T cells, resulting in increased T cell cytotoxicity and reduced tumor burden in syngeneic cancer models .

These findings highlight the importance of considering NNMT not just in cancer cells but also in the surrounding stromal and immune compartments.

What are the current approaches for targeting NNMT activity in research?

Researchers have developed several approaches to target NNMT activity:

• Small Molecule Inhibitors: Novel tricyclic small molecule inhibitors of NNMT have been developed, with JBSNF-000028 identified as a lead molecule that inhibits both human and mouse NNMT activity. This compound reduces MNA levels in mouse plasma, liver, and adipose tissue .

• Enzymatic Assays: Both recombinant enzyme assays and cell-based assays have been established to evaluate NNMT inhibition:

  • Recombinant enzyme assays with IC50 values for human NNMT (0.033 μM), monkey NNMT (0.19 μM), and mouse NNMT (0.21 μM)

  • LC-MS/MS detection methods with an IC50 of 0.13 μM for human NNMT

  • Cellular assays measuring endogenous NNMT activity in U2OS cells after 24h treatment with inhibitors, with EC50 values typically higher (2.5 μM for JBSNF-000028) than IC50 values, likely reflecting membrane permeability limitations

• Structure-Activity Relationship Studies: Research has examined the impact of structural modifications on inhibitor potency, revealing:

  • Ring enlargement strategies improve space filling in the nicotinamide binding pocket

  • Replacement of amidine with urea renders compounds inactive

  • Sensitivity to substitution in the phenyl part of molecules with 5-membered aliphatic ring systems

These approaches provide valuable tools for researchers studying NNMT's biological functions and therapeutic potential.

How can researchers effectively measure NNMT enzymatic activity?

Researchers can employ several methodologies to measure NNMT enzymatic activity:

• Direct Enzyme Activity Assays:

  • Recombinant NNMT enzyme assays using purified protein

  • Measurement of conversion of nicotinamide to 1-methylnicotinamide (MNA)

  • Detection of S-adenosylhomocysteine (SAH) formation as a co-product

• LC-MS/MS Detection Methods:

  • Quantification of MNA via LC-MS/MS in both enzymatic and cellular systems

  • This approach allows measurement of endogenous NNMT activity without recombinant expression

• Cellular Assays:

  • Treatment of cells expressing NNMT (endogenously or through recombinant expression) with potential inhibitors

  • Measurement of MNA levels in cell culture supernatants or cell lysates

  • EC50 values from cellular assays can be compared with IC50 values from enzymatic assays to assess membrane permeability and intracellular target engagement

• In Vivo Activity Assessment:

  • Measurement of MNA levels in plasma, liver, and adipose tissue in animal models

  • Correlation of MNA levels with NNMT expression and/or inhibitor treatment

When performing these assays, researchers should be aware that EC50 values in cellular assays are typically higher than IC50 values in enzymatic assays, often by a factor of approximately 30, reflecting challenges in cellular penetration of compounds .

How does NNMT expression correlate with clinical outcomes in cancer patients?

Research has demonstrated significant correlations between NNMT expression and clinical outcomes:

• Breast Cancer: NNMT expression is significantly higher (53.9%) in breast carcinoma compared to paracancerous tissues, suggesting a potential role in breast cancer development and progression .

• Cancer Biomarker Potential: Multiple studies have suggested that NNMT expression may have utility as a diagnostic and prognostic biomarker in various cancers, including melanoma .

• Immunosuppressive Tumor Microenvironment: High NNMT expression in the tumor stroma correlates with increased recruitment of immunosuppressive myeloid-derived suppressor cells (MDSCs) and reduced numbers of functional CD8+ T cells, potentially contributing to poor clinical outcomes .

• Therapeutic Response: NNMT expression levels may predict response to certain chemotherapeutic agents, with high expression potentially conferring resistance through mechanisms involving SIRT1-p53 signaling and epigenetic modifications .

Single-cell RNA sequencing (scRNA-seq) data from various human malignancies has revealed strong correlations between NNMT and CXCL1 expression in cancer-associated fibroblasts, providing further evidence for NNMT's role in shaping the tumor microenvironment .

What are the experimental considerations for studying NNMT in patient-derived samples?

When studying NNMT in patient-derived samples, researchers should consider the following:

• Sample Collection and Processing:

  • Fresh tissue samples provide optimal results for protein and enzymatic analyses

  • Flash-freezing in liquid nitrogen is recommended for samples intended for protein extraction

  • Formalin-fixed paraffin-embedded (FFPE) samples can be used for immunohistochemistry with appropriate antigen retrieval methods

• Cell Type Heterogeneity:

  • NNMT expression varies across cell types within the tumor microenvironment

  • Consider cell isolation techniques (like laser capture microdissection) or single-cell approaches to distinguish between expression in tumor cells, fibroblasts, and immune cells

• Control Samples:

  • Include matched normal tissue when possible

  • For breast cancer studies, include paracancerous tissues as controls

  • Consider using tissue microarrays to analyze multiple samples simultaneously

• Detection Methods:

  • Western blotting is reliable for protein expression analysis using validated antibodies (1:1000 dilution)

  • Immunohistochemistry can provide spatial information about NNMT expression

  • qPCR can be used for mRNA expression analysis, particularly when protein is limited

  • Consider LC-MS/MS for measuring enzyme activity via MNA levels

• Data Interpretation:

  • Correlate NNMT expression with clinical parameters (stage, grade, treatment response)

  • Consider analyzing related proteins (SIRT1, p53, acetyl-p53) to understand pathway interactions

  • For CAF studies, examine correlations between NNMT and CXCL1 expression

Following these considerations will help ensure more reliable and clinically relevant results when studying NNMT in patient-derived samples.