LCMT1 Human

What is LCMT1 and what is its primary function in human cells?

LCMT1, or Leucine carboxyl methyltransferase 1, is an enzyme encoded by the LCMT1 gene located on human chromosome 16 . It functions primarily as a methyltransferase that catalyzes the post-translational methylation of protein phosphatase 2A catalytic subunit (PP2Ac). This methylation is essential for proper PP2A holoenzyme assembly and function. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor for its methyltransferase activity . Understanding LCMT1's function requires protein expression and purification methodologies, followed by in vitro methyltransferase assays using recombinant proteins.

What protein interactions have been identified for LCMT1?

LCMT1 has been shown to interact with FXR2 (Fragile X mental retardation syndrome-related protein 2) . Additionally, LCMT1 interacts with PP2Ac, as evidenced by methyltransferase assays performed using recombinant human LCMT-1 (rhLCMT-1) and recombinant human PP2Ac (rhPP2Ac) at a 2:1 molar ratio . To study these interactions, researchers typically employ co-immunoprecipitation techniques, yeast two-hybrid screening, or pull-down assays combined with mass spectrometry to identify binding partners. Experimental design should include appropriate controls to distinguish specific from non-specific interactions.

How is LCMT1 expression altered in cancer, particularly hepatocellular carcinoma?

LCMT1 is significantly upregulated in hepatocellular carcinoma (HCC) tissues compared to adjacent normal liver tissues, as demonstrated by both laboratory experiments and bioinformatic analyses of multiple datasets . This upregulation correlates with poor prognosis in HCC patients. Western blotting and RT-PCR analyses of 34 pairs of human primary liver tumors and adjacent normal tissues revealed consistently higher LCMT1 expression in tumor samples . This finding was further supported by RNA-seq data from The Cancer Genome Atlas (TCGA) comparing 50 normal liver specimens to 369 HCC patients .

A meta-analysis of six GEO datasets (GSE29721, GSE50579, GSE57555, GSE101685, GSE101728, and GSE121248) provided strong evidence for LCMT1 upregulation in HCC, with a standardized mean difference of 1.21 (95% CI [0.91, 1.50]) . To study LCMT1 expression patterns, researchers should employ multiple techniques including qRT-PCR, western blotting, and immunohistochemistry, complemented by mining public databases such as TCGA and GEO.

What clinical correlations exist between LCMT1 expression and patient outcomes?

• Asian race (p = 0.008)

• Tumor histologic grade (p = 0.006)

• Tumor size (p = 0.016)

• Family history (p = 0.010)

Higher LCMT1 expression is also observed in advanced tumor stages . Interestingly, LCMT1 expression was not significantly associated with other clinicopathological characteristics including age, gender, alcohol consumption, HBV infection, and vascular invasion (p > 0.050) . When investigating clinical correlations, researchers should employ multivariate Cox regression analysis to account for confounding factors, and validate findings across independent cohorts.

What methodologies are most effective for studying LCMT1 methyltransferase activity in vitro?

For studying LCMT1 methyltransferase activity in vitro, researchers have employed several effective methodologies:

• Recombinant protein-based assays: Using recombinant human LCMT-1 (rhLCMT-1) and its substrate rhPP2Ac at a 2:1 molar ratio in reaction buffer containing 20 mM Tris (pH 8.0), 50 mM NaCl, and 1 mM EDTA . The reaction typically requires 20 μM S-adenosylmethionine (SAM) as the methyl donor and incubation at 37°C for 30 minutes .

• Dot immunoblot detection: When studying inhibitors that may interfere with colorimetric or bioluminescent methyltransferase assays, dot immunoblot techniques offer an alternative approach. Reactions are applied to nitrocellulose membranes, blocked with tris-buffered saline containing 5% nonfat dry milk, then probed with antibodies specific to the methylation state of the substrate (e.g., antibody directed to demethylated PP2Ac) .

• Genetic manipulation approaches: Overexpression using mammalian expression vectors (such as pCMV6-LCMT1) or silencing with specific siRNAs, followed by functional assays to assess methyltransferase activity .

These methodologies should be complemented with appropriate controls, including reactions without SAM as negative controls and known LCMT1 inhibitors like sinefungin (a SAM analogue) as positive controls for inhibition studies .

How does LCMT1 expression influence cellular metabolism, particularly glycolysis?

LCMT1 has a significant impact on cellular metabolism, particularly glycolysis. Research using LCMT1 silencing and overexpression models has revealed:

• Glycolytic reprogramming: In HCC cell lines (HepG2 and Huh7), silencing LCMT1 reduces glucose uptake, intracellular lactate production, and the enzymatic activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH) . Conversely, overexpression of LCMT1 in Hep3B cells increases these glycolytic parameters .

• Regulation of glycolytic gene expression: LCMT1 silencing in Huh7 and HepG2 cells reduces the mRNA levels of key glycolytic genes including GLUT1, HK2, PFK2, and MCT4, while LCMT1 overexpression in Hep3B cells increases their expression .

• Metabolite accumulation: LCMT1 silencing induces cellular accumulation of pyruvate, which contributes to inhibition of HCC cell proliferation .

To study LCMT1's influence on cellular metabolism, researchers should employ a combination of:

• Metabolic flux analysis using isotope-labeled glucose

• Enzymatic activity assays for key glycolytic enzymes

• RT-qPCR and western blotting to measure expression of glycolytic genes

• Metabolite profiling using mass spectrometry or NMR spectroscopy

• Seahorse XF analysis to measure glycolytic capacity and mitochondrial respiration

What experimental approaches have been successful in developing and testing LCMT1 inhibitors?

Several experimental approaches have proven successful in developing and testing LCMT1 inhibitors:

• Structural analysis and molecular docking: Using x-ray crystallographic structures of human LCMT-1 obtained from the RCSB Protein Data Bank (e.g., https://doi.org/10.2210/pdb3O7W/pdb) for molecular docking studies . Software tools such as AutoDock Vina have been utilized to analyze interactions between LCMT-1 and potential inhibitor compounds when SwissDock couldn't be used due to the size of LCMT-1 .

• In vitro methyltransferase assays: Testing potential inhibitors using recombinant human LCMT-1 and its substrate PP2Ac in controlled reaction conditions. For example, incubating rhLCMT-1 with test compounds at 50 μM final concentration for 10 minutes at room temperature before adding the substrate and methyl donor .

• Cell-based assays: Evaluating the effects of LCMT1 inhibitors on cell viability and proliferation in both normal and cancer cell lines. These experiments often include LCMT1-overexpressing cells to determine if overexpression can rescue the inhibitory effects .

• Visualization techniques: Converting docking outputs to Protein Data Bank file format with OpenBabel software and visualizing them with UCSF ChimeraX software to understand inhibitor-protein interactions at the molecular level .

When developing LCMT1 inhibitors, researchers should consider comparative studies with known methyltransferase inhibitors (such as sinefungin) and evaluate specificity by testing against other methyltransferases with similar catalytic domains.

What are the latest techniques for analyzing LCMT1-mediated post-translational modifications?

Analyzing LCMT1-mediated post-translational modifications requires sophisticated techniques:

• Mass spectrometry-based approaches: Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify and quantify methylated residues on target proteins. This can be enhanced with stable isotope labeling with amino acids in cell culture (SILAC) for comparative studies.

• Antibody-based detection: Using specific antibodies that recognize methylated versus unmethylated forms of target proteins, such as the antibody directed to demethylated PP2Ac used in dot immunoblot assays .

• Genetic code expansion: Incorporating methyl-lysine analogs site-specifically into proteins using amber suppression technology to study the functional consequences of methylation at specific sites.

• Proximity labeling proteomics: Employing BioID or APEX2 fused to LCMT1 to identify proximal proteins that may be substrates for methylation.

• Functional readouts: Assessing the downstream consequences of LCMT1-mediated methylation, such as changes in protein-protein interactions, enzyme activity, or cellular localization.

These techniques should be combined with LCMT1 manipulation through overexpression, silencing, or inhibition to establish causality between LCMT1 activity and observed post-translational modifications.

How does LCMT1 expression correlate with cancer progression and patient survival?

LCMT1 expression has significant implications for cancer progression and patient outcomes, particularly in hepatocellular carcinoma (HCC). Analysis of clinical data reveals:

The following table summarizes LCMT1 expression across different GEO datasets:

For researchers investigating LCMT1's clinical significance, methodological approaches should include:

• Multivariate Cox regression analysis to adjust for confounding factors

• Meta-analysis of multiple independent cohorts

• Correlation analyses with established prognostic markers

• Stratified analyses based on tumor stage, grade, and molecular subtype

How can LCMT1 be targeted therapeutically, and what are the challenges in developing LCMT1-specific inhibitors?

Targeting LCMT1 therapeutically presents several opportunities and challenges:

Methodological approaches for developing LCMT1-targeted therapeutics should include:

• Structure-based drug design utilizing crystallographic data

• Medicinal chemistry optimization of lead compounds

• Selectivity profiling against other methyltransferases

• Combination therapy strategies to address potential resistance mechanisms

• Biomarker development to identify patients most likely to respond to LCMT1 inhibition

What are the recommended experimental models for studying LCMT1 function in different cellular contexts?

Researchers have successfully employed several experimental models to study LCMT1 function across different cellular contexts:

• Cell line models:

  • HCC cell lines (HepG2, Huh7, Hep3B) have been used to study LCMT1's role in liver cancer metabolism and proliferation

  • HEK-293 cells serve as a versatile model for overexpression studies and inhibitor testing due to their high transfection efficiency

  • Breast cancer cell lines (MDA-MB-231, MCF7) have been employed to study LCMT1's role in different cancer subtypes

• Genetic manipulation approaches:

  • Transient transfection with plasmid vectors (e.g., pCMV6-LCMT1) using reagents optimized for specific cell types (PolyJet for HEK-293, Lipofectamine 3000 for MDA-MB-231)

  • Stable transfection with selection using G418 sulfate (700 ng/mL) for long-term studies

  • RNA interference using siRNA or shRNA to silence LCMT1 expression

• Functional readouts:

  • Cell viability assays using calcein-AM fluorescence measurement

  • Metabolic assays measuring glucose uptake, lactate production, and enzymatic activities

  • Gene expression analysis using RT-PCR for key downstream targets

When selecting experimental models, researchers should consider:

• Baseline LCMT1 expression levels in different cell types

• The specific cellular process being studied (proliferation, metabolism, etc.)

• The need for acute versus chronic manipulation of LCMT1 levels

• Appropriate positive and negative controls for each experimental system

How can researchers best analyze and interpret contradictory data regarding LCMT1's role in different experimental settings?

Analyzing contradictory data regarding LCMT1 function requires a systematic approach: