LZTR1 Antibody

What is LZTR1 and why is it important for research?

LZTR1 is a BTB-Kelch family protein that acts as a substrate adaptor for the CUL3-based ubiquitin E3 ligase complex. It plays a crucial role in regulating proteostasis of the RAS subfamily, including RIT1, MRAS, HRAS, KRAS, and NRAS through ubiquitin-dependent degradation . The significance of LZTR1 extends to several pathological conditions as mutations in LZTR1 have been identified in patients with RAS/MAPK signaling pathway-dependent congenital malformation syndrome (RASopathies), glioblastoma, and various cancers . Furthermore, LZTR1 has recently been implicated in tumor metastasis through novel mechanisms beyond RAS regulation, making it an important target for cancer research .

What are the typical applications of LZTR1 antibodies in research?

LZTR1 antibodies are employed in multiple experimental applications including:

• Western blotting to detect LZTR1 protein levels and modifications

• Immunoprecipitation for protein-protein interaction studies with RAS subfamily members and other potential binding partners such as KLHL12

• Immunohistochemistry to examine tissue expression patterns, as demonstrated in studies showing LZTR1 expression in distinct cell types of the cortex, amygdala, hippocampus, and white matter in the telencephalon

• Immunofluorescence to determine subcellular localization and co-localization with interacting proteins

• Chromatin immunoprecipitation (ChIP) when investigating potential transcriptional regulation roles

How should researchers validate LZTR1 antibody specificity?

Validation of LZTR1 antibodies should include:

• Western blot analysis comparing wild-type and LZTR1 knockout samples, as demonstrated in studies using LZTR1 knockout A549 cell lines

• Peptide competition assays to confirm epitope-specific binding

• RNA interference experiments showing corresponding reduction in antibody signal with LZTR1 knockdown

• Testing across multiple techniques (Western blot, immunoprecipitation, immunohistochemistry) to confirm consistent specificity

• Cross-validation with multiple antibodies targeting different epitopes of LZTR1

How can researchers optimize LZTR1 antibodies for studying RAS ubiquitination?

Optimizing LZTR1 antibodies for studying RAS ubiquitination requires:

• Selection of antibodies targeting the Kelch domain of LZTR1, as this region is crucial for substrate recognition including RAS proteins

• Validation using in vivo ubiquitination assays similar to those described in previous studies

• Designing experiments with proteasome inhibitors (e.g., MG132) to allow accumulation of ubiquitinated species

• Creating appropriate positive controls by overexpressing LZTR1 with ubiquitin and RAS proteins

• Implementation of denaturing conditions during immunoprecipitation to disrupt protein-protein interactions that might interfere with detection

A typical protocol would include treatment of cells with proteasome inhibitors, followed by lysis under denaturing conditions (1% SDS, 5 mM N-ethylmaleimide), immunoprecipitation of either LZTR1 or RAS proteins, and western blotting for ubiquitin, LZTR1, and RAS proteins.

What are the critical controls needed when using LZTR1 antibodies in studies of disease-associated mutations?

When studying disease-associated LZTR1 mutations, researchers should implement:

• Wild-type LZTR1 expression as a primary control

• LZTR1 knockout/knockdown samples as negative controls

• Multiple mutant constructs covering different functional domains, particularly focusing on mutations like G248R and R283Q (involved in RIT1 recognition) and R412C (potential allosteric mutation)

• Controls for substrate specificity by testing interaction with multiple RAS subfamily members

• Verification of antibody binding to mutant forms, as mutations might affect epitope recognition

A structured experimental approach should include both loss-of-function and gain-of-function studies to comprehensively characterize mutation effects.

How can LZTR1 antibodies help distinguish between its roles in RAS regulation versus newly discovered functions in ECM remodeling?

To differentiate between LZTR1's dual functions:

• Combine LZTR1 antibodies with co-immunoprecipitation studies targeting both RAS proteins and KLHL12-SEC31A complexes

• Implement sequential immunoprecipitation to isolate distinct LZTR1-containing complexes

• Use proximity ligation assays to visualize and quantify interactions between LZTR1 and its different binding partners in situ

• Perform subcellular fractionation followed by immunoblotting to determine compartment-specific associations

• Design rescue experiments with domain-specific mutants of LZTR1 that selectively disrupt either RAS binding or KLHL12 interaction

Research has shown that LZTR1-RIT1 and LZTR1-KLHL12 function independently regarding molecular interactions and do not directly interfere with each other , suggesting these pathways can be studied separately with appropriate controls.

What fixation and antigen retrieval methods are optimal for LZTR1 immunohistochemistry in different tissue types?

Based on published protocols:

• For brain tissue analysis, 4% paraformaldehyde fixation followed by citrate buffer (pH 6.0) heat-induced epitope retrieval has been effective for detecting LZTR1 in neuronal and glial cells

• For xenograft tumor samples, both 10% neutral buffered formalin and 4% paraformaldehyde have been used successfully

• For lung tissue, especially when examining metastatic lesions, proteinase K-based antigen retrieval may be required to penetrate the collagen-rich ECM

Optimization experiments should test multiple fixation durations (4-24 hours) and antigen retrieval methods to determine ideal conditions for specific tissue types.

What are the technical considerations when using LZTR1 antibodies for detecting substrate ubiquitination?

When studying LZTR1-mediated ubiquitination:

• Include protease inhibitors and deubiquitinase inhibitors (N-ethylmaleimide) in lysis buffers

• Consider using tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins

• Perform sequential immunoprecipitation: first pull down the substrate (e.g., RAS), then immunoblot for ubiquitin

• Include controls with overexpression of catalytically inactive ubiquitin ligase components

• Verify results using mass spectrometry to identify specific ubiquitination sites

The following table summarizes key experimental considerations for ubiquitination studies:

How can researchers address false negative results when attempting to detect LZTR1-mediated ubiquitination of RAS proteins?

When troubleshooting negative results:

• Verify LZTR1 antibody functionality through immunoprecipitation of overexpressed tagged LZTR1

• Ensure proteasome inhibition is effective by monitoring accumulation of other ubiquitinated proteins

• Test multiple lysis conditions, as LZTR1-RAS interactions may be sensitive to detergent types and concentrations

• Consider that ubiquitination may be transient or have low stoichiometry; increase sample concentration or use TUBEs for enrichment

• Ensure the specific RAS isoform being studied is a substrate for LZTR1, as different isoforms may have different requirements. For example, research has shown that the conservation of K170 across RAS isoforms does not necessarily mean all isoforms are ubiquitinated by LZTR1 at this residue

What are the potential pitfalls when interpreting LZTR1 expression data across different cellular contexts?

Researchers should be aware of:

• Cell type-specific expression patterns, as LZTR1 is enriched in distinct cell types including those in the cortex, amygdala, hippocampus, and white matter

• Variability in LZTR1 function between tissue/cancer types, as reports indicate different roles in various contexts

• Potential antibody cross-reactivity with other BTB-Kelch family proteins

• Changes in LZTR1 expression or localization in response to cellular stress or treatment

• Post-translational modifications that may mask antibody epitopes

When interpreting inconsistent results, consider that LZTR1 may have different roles in each tissue or cancer type, and unknown molecules may be involved in LZTR1-related processes .

How can LZTR1 antibodies be utilized to investigate its role in metastasis beyond RAS regulation?

Recent findings indicate LZTR1 deficiency promotes metastasis through mechanisms including:

• Enhanced sensitivity to EMT induction

• Promotion of collagen secretion through regulation of KLHL12-SEC31A complexes

Researchers can:

• Use LZTR1 antibodies in conjunction with antibodies against EMT markers (N-Cadherin) in immunofluorescence studies of cells treated with TGF-β1

• Perform co-immunoprecipitation with LZTR1 antibodies followed by mass spectrometry to identify novel interaction partners

• Use ascorbate chase analyses with LZTR1 antibodies to monitor collagen secretion dynamics in response to LZTR1 manipulation

• Implement proximity ligation assays to visualize LZTR1-KLHL12 interactions in situ

What are the recent advances in understanding structural interactions between LZTR1 and its substrates that may inform antibody selection?

Computational studies have revealed:

• The interaction model between LZTR1 and RIT1 is stabilized by an electrostatic bond network between protein surfaces

• Specific mutations (G248R, R283Q) are directly involved in RIT1 recognition

• R412C mutation may function as an allosteric mutation affecting communication between the Kelch and C-terminal BTB domains

When selecting antibodies, researchers should consider:

• Targeting epitopes outside predicted substrate-binding regions to avoid interference with protein interactions

• Using antibodies specific to different domains to distinguish various functional states

• Selecting antibodies that can recognize LZTR1 in complex with its substrates