LAMTOR5 Antibody

What is LAMTOR5 and what cellular functions does it regulate?

LAMTOR5 is a component of the Ragulator complex that plays essential roles in several cellular functions. It serves as a late endosomal/lysosomal adaptor and activator for both MAPK and mTOR signaling pathways . LAMTOR5 physically associates with ATP6V1A, an essential subunit of vacuolar H⁺-ATPase (v-ATPase), and promotes the V0/V1 holoenzyme assembly to facilitate lysosome acidification . This protein is critical for immune homeostasis by integrating v-ATPase activity, lysosome function, and mTOR pathway regulation. Recent research has established that LAMTOR5 defects can lead to autoimmune conditions resembling systemic lupus erythematosus (SLE) . Additionally, LAMTOR5 has been identified as a key regulator in TLR4 signaling and inflammation control, as it associates with TLR4 and facilitates their colocalization at autolysosomes, affecting inflammatory response magnitudes .

What alternative names exist for LAMTOR5 in the scientific literature?

LAMTOR5 is known by several synonyms in scientific literature, which is important to recognize when conducting comprehensive literature searches:

• HBV X-interacting protein (HBXIP)

• HBX-interacting protein

• Hepatitis B virus X-interacting protein

• Ragulator complex protein LAMTOR5

• XIP

Understanding these alternative nomenclatures ensures thorough literature review and prevents researchers from missing relevant publications when investigating this protein.

What are the optimal applications for anti-LAMTOR5 antibodies in research?

Based on available data, anti-LAMTOR5 antibodies have been successfully validated for immunohistochemistry (IHC) applications . The antibody detects endogenous levels of total LAMTOR5 protein and shows specificity for human samples . While IHC is a well-validated application, researchers should consider that different antibodies may have varying specificities and optimal applications. When designing experiments, it's important to select antibodies that have been validated for your specific application and sample type. For optimal results in IHC applications, the recommended dilution is 1/100, as demonstrated in validation studies with human tonsil and thyroid cancer tissue samples .

What methodologies can be used to study LAMTOR5's role in lysosomal acidification?

To investigate LAMTOR5's role in lysosomal acidification, researchers can employ several complementary approaches:

• LysoSensor assays: LysoSensor Yellow/Blue DND-160, a dual-emission ratiometric probe, can be used to measure lysosomal pH in LAMTOR5 wildtype versus knockout/knockdown cells .

• Co-immunoprecipitation studies: These can detect physical interaction between LAMTOR5 and ATP6V1A (v-ATPase subunit) to understand the molecular basis for LAMTOR5's effect on acidification .

• Confocal microscopy: This technique allows visualization of lysosomal markers and acidification in live cells under different conditions.

• Autophagic flux assessment: Using tandem mCherry-EGFP-LC3B reporter systems can help visualize acidified autophagic structures, which appear as red puncta (since GFP is more sensitive to acidic conditions while mCherry remains stable) .

• Biochemical assays: Measuring the ratio of LC3-II/I and p62 levels can indicate autophagosome formation and maturation, which are affected by lysosomal acidification and function .

What controls should be included when using LAMTOR5 antibodies for immunohistochemistry?

When using LAMTOR5 antibodies for immunohistochemistry, several controls should be included to ensure specificity and validity of results:

• Negative controls:

  • Omission of primary antibody

  • Isotype control (non-specific IgG from the same species)

  • Tissues known not to express LAMTOR5

• Positive controls:

  • Human tonsil tissue, which has been validated to show LAMTOR5 expression

  • Human thyroid cancer tissue, which also demonstrates LAMTOR5 expression

• Blocking controls:

  • Pre-incubation with fusion protein (as shown in validation data where fusion protein treatment blocks the antibody signal)

• Dilution optimization:

  • Test different antibody dilutions (starting with manufacturer's recommendation of 1/100)

• Secondary antibody controls:

  • Suitable secondary antibodies include: Goat Anti-Rabbit IgG H&L with various conjugates (AP, Biotin, FITC, or HRP) depending on detection method

How does LAMTOR5 deficiency impact macrophage phenotype and function?

LAMTOR5 deficiency profoundly alters macrophage phenotype and function, shifting them toward a hyperactivated, inflammatory state. Experimental data shows that LAMTOR5 knockout macrophages exhibit:

• Upregulated surface markers: Increased expression of MHCII and costimulatory molecules (CD80, CD86, and CD40)

• Enhanced phagocytic function: Significantly increased phagocytic capacity compared to control cells

• Altered cytokine profile:

  • Increased production of proinflammatory cytokines and M1 marker genes

  • Repressed expression of immunosuppressive cytokines and M2-featured genes

• Dysregulated gene expression: Differential expression of 42 lysosome-associated genes, including Rilpl2, Atp6v0d2, TMEM150a, and IFITM3

• Impaired lysosomal acidification: Elevated lysosomal pH compared to wildtype macrophages

• Altered autophagy: Reduced LC3-II/I ratio (indicating impaired autophagosome formation) and elevated p62 levels (suggesting decreased autophagic degradation)

This shift toward an inflammatory phenotype in LAMTOR5-deficient macrophages contributes to the development of autoimmune-like conditions observed in experimental models and potentially in human disease conditions like SLE.

What is the relationship between LAMTOR5 and TLR4 signaling in inflammatory responses?

LAMTOR5 plays a crucial regulatory role in TLR4 signaling and inflammatory response control through several mechanisms:

• Direct TLR4 interaction: LAMTOR5 associates with TLR4 via their LZ/TIR domains, forming a physical complex

• Subcellular colocalization: LAMTOR5 facilitates the colocalization of TLR4 at autolysosomes following LPS stimulation

• mTORC1 regulation: LAMTOR5 prevents lysosomal tethering and activation of mTORC1 upon LPS stimulation

• TFEB derepression: By modulating mTORC1 activity, LAMTOR5 derepresses TFEB (Transcription Factor EB), promoting autophagic degradation of TLR4

• Inflammatory resolution: LAMTOR5 contributes to the timely degradation of TLR4, helping to resolve inflammatory responses after pathogen recognition

In LAMTOR5-deficient conditions, TLR4 degradation is delayed, leading to sustained inflammatory signaling and increased susceptibility to endotoxic shock. Notably, Lamtor5 haploinsufficient mice showed increased mortality during endotoxic shock, demonstrating the physiological importance of this regulatory pathway .

How does LAMTOR5 integrate nutrient sensing with immune regulation?

LAMTOR5 serves as a critical molecular bridge between metabolic sensing and immune regulation through several interconnected mechanisms:

• Nutrient-dependent immune modulation: Nutrient deprivation, particularly leucine deprivation, blunts inflammatory signaling and provides protection during endotoxic shock, an effect that is largely abrogated upon LAMTOR5 deletion

• Amino acid sensing machinery component: As part of the Ragulator complex, LAMTOR5 contributes to amino acid sensing and subsequent mTORC1 activation or inhibition

• GEF function for RAG GTPase: LAMTOR5, along with other Lamtor proteins, functions as a guanine exchange factor for RAG GTPase to transduce nutrient-sensing signals

• Autolysosomal pathway regulation: Through modulation of TFEB activity, LAMTOR5 influences autophagy processes that are essential for both metabolic adaptation and immune response resolution

This integration of pathogenic signals and nutrient availability through LAMTOR5 represents a homeostatic mechanism that optimizes inflammatory responses based on metabolic status. This function has significant implications for understanding and potentially treating TLR4-associated inflammatory and metabolic disorders .

What are common challenges when working with LAMTOR5 antibodies and how can they be addressed?

When working with LAMTOR5 antibodies, researchers may encounter several challenges:

• Cross-reactivity concerns:

  • Solution: Validate antibody specificity using positive and negative controls, including fusion protein blocking experiments as demonstrated in validation studies

  • Approach: Compare results using multiple antibodies targeting different epitopes of LAMTOR5

• Variable expression levels:

  • Solution: Optimize sample preparation and antibody concentration

  • Approach: For IHC applications, the recommended starting dilution is 1/100, but titration may be necessary for specific tissues

• Storage and stability issues:

  • Solution: Store antibody at -20°C as recommended

  • Approach: Avoid repeated freeze-thaw cycles and prepare working aliquots

• Detection sensitivity:

  • Solution: Choose appropriate secondary antibodies based on application

  • Approach: For LAMTOR5 rabbit polyclonal antibodies, suitable secondaries include Goat Anti-Rabbit IgG H&L with various conjugates (AP, Biotin, FITC, or HRP)

• Background signal:

  • Solution: Optimize blocking conditions and washing steps

  • Approach: Include appropriate negative controls in each experiment

How can researchers effectively study the relationship between LAMTOR5 and lysosomal function?

To effectively study the relationship between LAMTOR5 and lysosomal function, researchers should consider a multi-faceted approach:

• Genetic manipulation:

  • Generate LAMTOR5 knockout/knockdown models using CRISPR-Cas9 or shRNA

  • Create cell-specific conditional knockout models (e.g., myeloid-specific Lamtor5 knockout mice as described in the literature)

• Lysosomal pH measurement:

  • Use LysoSensor Yellow/Blue DND-160 for dual-emission ratiometric pH measurement

  • Compare pH in LAMTOR5-sufficient and LAMTOR5-deficient cells

• Protein interaction studies:

  • Perform co-immunoprecipitation to detect LAMTOR5 interaction with ATP6V1A and other v-ATPase components

  • Use proximity ligation assays to visualize protein interactions in situ

• Autophagy assessment:

  • Monitor LC3-II/I ratios and p62 levels by Western blotting

  • Use mCherry-EGFP-LC3B reporter systems to visualize autophagic flux

• Transcriptional profiling:

  • Conduct RNA-seq to identify differentially expressed lysosome-associated genes

  • Perform GO analysis to identify enriched pathways related to lysosomal function

• Functional assays:

  • Assess lysosomal enzyme activity

  • Measure degradation rates of known lysosomal substrates

What are promising therapeutic applications based on LAMTOR5 biology?

Based on current understanding of LAMTOR5 biology, several promising therapeutic applications could be developed:

• Autoimmune disease treatment: Since LAMTOR5 deficiency leads to SLE-like autoimmunity, strategies to restore or enhance LAMTOR5 function could potentially treat autoimmune conditions. The research indicates that enforced expression of LAMTOR5 resumed lysosomal acidity in PBMCs from SLE patients .

• Anti-inflammatory approaches: Targeting the LAMTOR5-TLR4 interaction could provide novel ways to modulate inflammatory responses in conditions like sepsis. This is supported by findings that LAMTOR5 haploinsufficient mice showed increased mortality during endotoxic shock .

• Metabolic inflammation targeting: The unique position of LAMTOR5 at the intersection of nutrient sensing and immune regulation makes it a potential target for treating metabolic disorders with inflammatory components. The finding that leucine deprivation blunts inflammatory signaling through LAMTOR5-dependent mechanisms suggests nutritional or pharmacological approaches .

• Cancer therapy: Given LAMTOR5's associations with oncogenic proteins like c-FOS, c-Myc, Survivin, and p53, as well as its role in promoting proliferation in cancer cells, targeting LAMTOR5 might represent a novel approach in cancer treatment .

• Lysosomal storage disease applications: LAMTOR5's role in lysosomal acidification suggests potential applications in lysosomal storage diseases, where lysosomal function is compromised.

What are key unresolved questions regarding LAMTOR5 function?

Despite recent advances, several key questions about LAMTOR5 remain unresolved:

• Tissue-specific functions: How does LAMTOR5 function differ across various tissue and cell types beyond macrophages and immune cells?

• Regulatory mechanisms: What regulates LAMTOR5 expression and activity under different physiological and pathological conditions?

• Structural biology: What are the precise structural determinants of LAMTOR5's interactions with TLR4, v-ATPase, and Rag GTPase?

• Systems biology perspective: How does LAMTOR5 coordinate its multiple functions in nutrient sensing, immune regulation, and lysosomal function in an integrated cellular context?

• Evolutionary significance: How conserved is LAMTOR5 function across species, and what does this reveal about the co-evolution of metabolic and immune systems?

• Disease relevance beyond autoimmunity: What roles might LAMTOR5 play in neurodegeneration, metabolic diseases, or other conditions where lysosomal function is implicated?

• Interaction with other Ragulator components: How do the different components of the Ragulator complex (Lamtor1-5) coordinate their functions, and are there redundancies?

What methodological advances might improve LAMTOR5 research?

Future advances in LAMTOR5 research could benefit from several methodological improvements:

• Live-cell imaging techniques: Development of specific fluorescent tags for LAMTOR5 that maintain protein function would allow real-time visualization of its dynamics and interactions.

• Tissue-specific conditional knockout models: Generation of additional tissue-specific LAMTOR5 knockout models would help delineate its function in different physiological contexts.

• High-throughput screening approaches: Development of assays suitable for identifying small molecule modulators of LAMTOR5 function could accelerate therapeutic development.

• Improved antibodies and detection methods: Development of highly specific monoclonal antibodies against different epitopes of LAMTOR5 would enhance research capabilities.

• Proteomics approaches: Comprehensive interactome studies would help identify the full range of LAMTOR5 binding partners across different cellular conditions.

• Single-cell analysis: Application of single-cell technologies could reveal cell-to-cell variation in LAMTOR5 function and its consequences.

• Structural biology techniques: Cryo-EM or X-ray crystallography studies of LAMTOR5 in complex with its binding partners would provide mechanistic insights at the atomic level.