lyrm4 Antibody

What is LYRM4 and why is it important in research?

LYRM4 (also known as ISD11, CGI-203, or C6orf149) functions as a stabilizing factor of the core iron-sulfur cluster (ISC) assembly complex. It regulates, in association with NDUFAB1, the stability and cysteine desulfurase activity of NFS1 and participates in the [2Fe-2S] clusters assembly on the scaffolding protein ISCU . The core ISC assembly complex is involved in the de novo synthesis of [2Fe-2S] clusters, which represents the first step of mitochondrial iron-sulfur protein biogenesis. This process begins with the cysteine desulfurase complex (NFS1:LYRM4:NDUFAB1) producing persulfide that is delivered to the scaffold protein ISCU in a FXN-dependent manner . LYRM4 is essential for maintaining the stability and activity of the human cysteine desulfurase complex NFS1-LYRM4-ACP, making it a critical component for cellular function and potential disease research .

What applications are LYRM4 antibodies suitable for in research settings?

LYRM4 antibodies have been validated for multiple research applications:

• Immunohistochemistry-paraffin (IHC-P): For detecting LYRM4 in fixed, paraffin-embedded tissue sections.

• Western blotting (WB): For protein quantification and molecular weight determination.

• Immunocytochemistry/Immunofluorescence (ICC/IF): For cellular localization studies .

When designing experiments, researchers should consider that commercial LYRM4 antibodies typically react with human samples and are generated using immunogens corresponding to recombinant fragments within human LYRM4 amino acid 1 to C-terminus .

How can specificity of LYRM4 antibodies be validated for experimental use?

Validation of LYRM4 antibody specificity should follow these methodological approaches:

• Positive and negative controls: Use tissues/cells known to express LYRM4 (e.g., liver tissues) as positive controls, and LYRM4-knockout or knockdown samples as negative controls.

• Western blot analysis: Confirm a single band at the expected molecular weight (~12 kDa for human LYRM4).

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining.

• Multiple antibody validation: Use antibodies from different sources or raised against different epitopes to confirm specificity.

• Correlation with mRNA expression: Compare protein detection with RT-PCR or RNA-seq data for LYRM4 expression .

What is the recommended protocol for using LYRM4 antibodies in Western blotting?

For optimal Western blot results with LYRM4 antibodies:

• Sample preparation:

  • Extract proteins from tissues or cells using RIPA buffer containing protease inhibitors

  • Include both cytosolic and mitochondrial fractions since LYRM4 localizes primarily to mitochondria

• Gel electrophoresis:

  • Use 15-18% SDS-PAGE gels due to LYRM4's small size (~12 kDa)

  • Load 20-30 μg of total protein per lane

• Transfer and blocking:

  • Transfer to PVDF membrane (0.2 μm pore size recommended for small proteins)

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • Dilute primary LYRM4 antibody 1:500-1:1000 in blocking buffer

  • Incubate overnight at 4°C

  • Wash 3× with TBST

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) reagents

  • Exposure times typically range from 30 seconds to 5 minutes

How can LYRM4 antibodies be used to investigate its role in cancer?

Recent research has identified LYRM4 as a potential biomarker in liver hepatocellular carcinoma (LIHC). When designing studies to investigate LYRM4's role in cancer using antibodies:

• Expression profiling:

  • Perform IHC on tissue microarrays (TMAs) containing tumor and adjacent normal tissues

  • Quantify expression using H-score or similar methods

  • Correlate with clinical parameters (tumor stage, grade, patient survival)

• Mechanistic investigations:

  • Use Western blotting to compare LYRM4 levels across cancer cell lines

  • Combine with co-immunoprecipitation to identify cancer-specific interaction partners

  • Perform cellular fractionation to determine subcellular localization changes in cancer cells

• Prognostic evaluation:

  • According to studies, LYRM4 mRNA expression correlates with clinical stratifications, prognosis, and survival of LIHC patients

  • Immunohistochemistry results confirmed high expression in LIHC tissues, with significant correlation to age, serum low-density lipoprotein (LDL), and triglyceride content

  • LYRM4 expression in LIHC also shows significant positive correlation with infiltrating levels of six immune cell types

The comprehensive analysis should include evaluating both the expression patterns and functional implications, as LYRM4 overexpression appears to lead to ISC-dependent metabolic reprogramming in cancer cells .

What approaches are recommended for studying LYRM4 mutations and their impact on iron-sulfur cluster assembly?

To investigate LYRM4 mutations and their effects on iron-sulfur cluster assembly:

• Mutation identification and characterization:

  • Use massively parallel sequencing methods (e.g., MitoExome sequencing) to identify mutations in patient samples

  • Confirm mutations with Sanger sequencing

  • Perform in silico analysis to predict impact on protein structure and function

• Functional validation in cell models:

  • Generate cell lines expressing mutant LYRM4 using CRISPR/Cas9 or site-directed mutagenesis

  • Measure activities of iron-sulfur proteins (e.g., complexes I, II, III of OXPHOS, aconitase, ferrochelatase)

  • Assess mitochondrial function through oxygen consumption rate measurements

• Biochemical characterization:

  • Purify recombinant wild-type and mutant LYRM4 proteins

  • Perform binding assays with NFS1 and other components of the ISC machinery

  • Assess stability differences between wild-type and mutant proteins

• Yeast complementation studies:

  • Test the ability of mutant LYRM4 to complement ISD11 deletion in yeast

  • Measure growth rates and respiratory chain enzyme activities

  • Assess iron-sulfur protein activities in complemented yeast strains

Research indicates that mutations in LYRM4 (such as c.203G>T, p.R68L) can lead to combined OXPHOS deficiency affecting complexes I, II, and III, which all contain iron-sulfur clusters. These mutations can also impact other Fe-S proteins including aconitases and ferrochelatase .

What techniques can be combined with LYRM4 antibodies to study protein-protein interactions in the iron-sulfur cluster assembly complex?

For comprehensive analysis of LYRM4's interactions within the ISC assembly complex:

• Co-immunoprecipitation (Co-IP):

  • Use LYRM4 antibodies to pull down protein complexes

  • Identify interaction partners by mass spectrometry

  • Confirm key interactions by Western blotting with specific antibodies for NFS1, NDUFAB1, ISCU, and FXN

• Proximity ligation assay (PLA):

  • Utilize LYRM4 antibody in combination with antibodies against suspected interaction partners

  • Visualize protein-protein interactions in situ with subcellular resolution

  • Quantify interaction signals under different cellular conditions

• FRET/BRET approaches:

  • Generate fluorescent/bioluminescent protein fusions with LYRM4 and potential partners

  • Measure energy transfer as indicator of protein proximity

  • Analyze interaction dynamics in living cells

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map interaction interfaces between LYRM4 and its partners

  • Identify structural changes upon complex formation

  • Characterize effects of mutations on protein-protein interactions

• Crosslinking mass spectrometry (XL-MS):

  • Covalently link interacting proteins in physiological conditions

  • Digest and analyze by mass spectrometry to identify crosslinked peptides

  • Determine spatial constraints within protein complexes

Research has confirmed that LYRM4 forms a complex with NFS1 and is essential for both ISC biogenesis and maintenance of normal cellular iron homeostasis .

How can LYRM4 antibodies be used to study the relationship between iron-sulfur cluster assembly and mitochondrial disease?

To investigate the connection between LYRM4, iron-sulfur cluster assembly, and mitochondrial diseases:

• Patient tissue analysis:

  • Perform IHC and Western blot analysis on patient-derived tissues

  • Compare LYRM4 expression and localization between control and disease samples

  • Correlate with clinical parameters and disease severity

• Biochemical assays for iron-sulfur protein function:

  • Measure activities of mitochondrial complex I and aconitase hydratase (ACO2)

  • Assess changes in the citric acid cycle and oxidative phosphorylation

  • Analyze iron homeostasis markers in patient samples

• Cellular models of disease:

  • Create patient-specific iPSCs and differentiate to affected cell types

  • Knockdown/knockout LYRM4 in relevant cell lines

  • Perform rescue experiments with wild-type LYRM4

• Mitochondrial function assessment:

  • Combine LYRM4 immunostaining with mitochondrial markers

  • Measure mitochondrial membrane potential and ROS production

  • Assess changes in mitochondrial morphology and distribution

• Therapeutic screening:

  • Test compounds that might bypass defects in iron-sulfur cluster assembly

  • Evaluate approaches to enhance cysteine availability, as limited sulfur donor availability may affect clinical outcomes

Studies have shown that mutations in LYRM4 can lead to deficiency of OXPHOS complexes containing iron-sulfur clusters (complexes I, II, and III) as well as other mitochondrial and cytosolic Fe-S proteins, resulting in severe clinical phenotypes including neonatal lactic acidosis .

What considerations are important when using LYRM4 antibodies for analyzing subcellular localization?

For accurate subcellular localization studies with LYRM4 antibodies:

• Sample preparation optimization:

  • Test different fixation methods (4% PFA, methanol, etc.)

  • Optimize permeabilization conditions to ensure antibody access to all cellular compartments

  • Consider antigen retrieval methods for fixed tissues

• Co-localization studies:

  • Use established markers for mitochondria (e.g., TOMM20, MitoTracker)

  • Include markers for other relevant compartments (cytosol, nucleus)

  • Employ high-resolution microscopy techniques (confocal, STED, SIM)

• Fractionation controls:

  • Perform subcellular fractionation to separate mitochondrial and cytosolic components

  • Use Western blotting to confirm localization patterns observed by microscopy

  • Include controls for fraction purity (e.g., VDAC for mitochondria, GAPDH for cytosol)

• Special considerations for LYRM4:

  • Account for potential dual localization in mitochondria and cytoplasm

  • Consider that LYRM4 may interact with both mitochondrial and cytoplasmic forms of NFS1

  • Be aware that subtle changes in localization may occur under stress conditions

Research suggests that LYRM4 may participate in iron-sulfur protein biogenesis not only in mitochondria but also in the cytoplasm through interaction with the cytoplasmic form of NFS1 .

What are the optimal conditions for immunohistochemistry using LYRM4 antibodies?

For successful immunohistochemistry with LYRM4 antibodies:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section at 4-5 μm thickness onto positively charged slides

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval methods:

    • Citrate buffer (pH 6.0) for 20 minutes

    • EDTA buffer (pH 9.0) for 20 minutes

  • Allow slides to cool to room temperature for 20 minutes after retrieval

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5% normal serum for 1 hour

  • Incubate with primary LYRM4 antibody (typically 1:100-1:200 dilution) overnight at 4°C

  • Use appropriate detection system (e.g., HRP-polymer and DAB)

• Controls and counterstaining:

  • Include positive control tissue (liver is recommended based on LYRM4 expression data)

  • Use isotype control antibodies as negative controls

  • Counterstain with hematoxylin and mount with permanent mounting medium

• Scoring and analysis:

  • Evaluate both intensity and percentage of positive cells

  • Consider both cytoplasmic and nuclear staining patterns

  • Use digital image analysis for quantification when possible

How can LYRM4 antibodies be used to analyze changes in protein expression under different experimental conditions?

To effectively analyze LYRM4 expression changes:

• Experimental design considerations:

  • Include appropriate time course analyses (early and late timepoints)

  • Use multiple biological and technical replicates

  • Include positive controls (known inducers of ISC biogenesis stress)

• Quantitative Western blotting:

  • Use fluorescently-labeled secondary antibodies for wider dynamic range

  • Include loading controls appropriate for subcellular fraction (β-actin for total lysate, VDAC for mitochondria)

  • Normalize LYRM4 expression to these controls

  • Use digital image acquisition to avoid saturation

• Proteomics approaches:

  • Consider targeted proteomics (PRM/MRM) for absolute quantification

  • Use SILAC or TMT labeling for relative quantification across conditions

  • Analyze post-translational modifications that might affect function

• Transcriptional analysis correlation:

  • Compare protein-level changes with mRNA expression (qRT-PCR or RNA-seq)

  • Analyze relationship between transcriptional and translational regulation

  • Identify potential discrepancies indicating post-transcriptional regulation

Research has shown that LYRM4 expression can be significantly altered in pathological conditions such as hepatocellular carcinoma, where both mRNA and protein levels are upregulated .

What new methodologies are being developed to study LYRM4 and iron-sulfur cluster biogenesis?

Emerging techniques for LYRM4 and ISC biogenesis research include:

• Cryo-electron microscopy:

  • High-resolution structural analysis of the entire ISC assembly complex

  • Visualization of conformational changes during cluster assembly

  • Structural basis for disease-causing mutations

• Live-cell imaging of ISC assembly:

  • Development of fluorescent sensors for iron-sulfur cluster formation

  • Real-time monitoring of assembly complex dynamics

  • Single-molecule tracking of components including LYRM4

• Systems biology approaches:

  • Integration of proteomics, transcriptomics, and metabolomics data

  • Network analysis of ISC biogenesis and related pathways

  • Machine learning for predicting functional consequences of mutations

• Therapeutic targeting strategies:

  • High-throughput screening for compounds that enhance ISC biogenesis

  • Development of mitochondria-targeted peptides to stabilize the assembly complex

  • Gene therapy approaches for LYRM4-related disorders

How can researchers effectively study the relationship between LYRM4 and immune cell infiltration in cancer?

Based on findings showing correlation between LYRM4 expression and immune infiltration:

• Multiplex immunofluorescence:

  • Co-stain for LYRM4 and immune cell markers (CD4, CD8, etc.)

  • Analyze spatial relationships between LYRM4-expressing cells and immune cells

  • Quantify immune cell densities in relation to LYRM4 expression levels

• Single-cell analysis approaches:

  • Perform single-cell RNA-seq on tumor samples

  • Identify cell populations based on LYRM4 expression

  • Correlate with immune cell signatures and activation states

• Functional assays:

  • Co-culture LYRM4-manipulated cancer cells with immune cells

  • Assess changes in immune cell activation and function

  • Measure cytokine production and immune checkpoint expression

• In vivo models:

  • Generate LYRM4-overexpressing or knockout tumor models

  • Analyze immune infiltration patterns

  • Test immunotherapy responses in relation to LYRM4 status

Research indicates that LYRM4 expression in liver hepatocellular carcinoma is significantly positively correlated with the infiltrating levels of six immune cell types, and both factors are strongly associated with prognosis .

What are the methodological challenges in detecting post-translational modifications of LYRM4?

Investigating post-translational modifications (PTMs) of LYRM4 presents several challenges:

• PTM-specific antibody development:

  • Generate and validate antibodies against predicted modification sites

  • Confirm specificity through peptide competition assays

  • Consider using a combination of modification-specific and total LYRM4 antibodies

• Mass spectrometry approaches:

  • Enrich for LYRM4 through immunoprecipitation before MS analysis

  • Use multiple proteases to increase sequence coverage

  • Apply enrichment strategies for specific modifications (e.g., TiO2 for phosphopeptides)

  • Consider targeted MS methods for low-abundance modified peptides

• Functional impact assessment:

  • Generate site-specific mutants (phosphomimetic, non-phosphorylatable)

  • Assess effects on NFS1 binding and activity

  • Determine impact on ISC assembly efficiency

  • Evaluate changes in protein stability and localization

• Regulation analysis:

  • Identify kinases, phosphatases, or other enzymes regulating LYRM4 modifications

  • Determine physiological conditions affecting modification status

  • Investigate cross-talk between different modification types

By addressing these methodological challenges, researchers can gain deeper insights into how post-translational regulation affects LYRM4 function in iron-sulfur cluster biogenesis.