TECPR1 Antibody

What is TECPR1 and what cellular functions does it regulate?

TECPR1 is a protein that plays a crucial role in noncanonical autophagy, specifically in a process called conjugation of ATG8 to single membranes (CASM). It forms E3 complexes that are critical mediators of CASM in cells treated with various pharmacological drugs, including nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents . TECPR1 functions primarily in response to membrane damage, particularly affecting endolysosomal membrane integrity, but does not respond to drugs that merely affect autophagy or endolysosomal proton gradients . Mechanistically, TECPR1 binds to the Atg12-Atg5 conjugate, with this interaction mediated by a region spanning amino acids 566-610 adjacent to its PH domain . This interaction is necessary and sufficient for promoting autophagosome maturation.

How does TECPR1 expression vary across different tissue and cell types?

TECPR1 expression varies significantly across different tissue types, with notably decreased expression in certain cancer tissues compared to normal tissues. Analysis using Gene Expression Profiling Interactive Analysis (GEPIA) has revealed that TECPR1 is low-expressed in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) when compared with normal tissues . This expression pattern has been consistently observed across multiple cell lines as well. For instance, TECPR1 expression is significantly lower in non-small cell lung carcinoma (NSCLC) cell lines (A549, NCI-H23, and NCI-H520) compared to normal human bronchial epithelial cells (16HBE) . It's worth noting that diverse cell lines display variations in their TECPR1 response when confronted with membrane damage, as evidenced by the varying degrees of TECPR1-dependent ATG8 lipidation in HEK293A, PC-3, and HeLa cells .

What structural domains characterize TECPR1 and how do they contribute to its function?

TECPR1 contains several distinct structural domains that are essential for its various functions:

• A pleckstrin homology (PH) domain - Involved in membrane interactions

• Nine beta-propeller repeats (TECPR domain) - Form the characteristic TECPR structure

• Two dysferlin domains - Contribute to membrane interaction capabilities

• An ATG5-interacting region (AIR) - Facilitates interaction with the ATG12-ATG5 complex

Additionally, TECPR1 possesses a LC3-interacting region (LIR) motif that enables binding to all human ATG8 proteins and an N-terminal beta-propeller (TR1) domain that selectively interacts with LC3C . TECPR1 also binds phosphatidylinositol-4-phosphate (PtdIns(4)P) both in vitro and in vivo, which is critical for its localization and function . This complex domain architecture explains how TECPR1 can simultaneously interact with multiple proteins and lipids to coordinate autophagosome maturation and protein aggregate clearance.

How does TECPR1 contribute to selective autophagy pathways?

TECPR1 plays a specialized role in selective autophagy, particularly in aggrephagy (the clearance of protein aggregates). Research demonstrates that TECPR1 selectively interacts with LC3C, unlike other human ATG8 proteins, and this interaction is critical for the degradation of LC3C-positive autophagosomes . When TECPR1 is deleted, there is a significant accumulation of LC3C puncta that colocalize with ubiquitin, the selective autophagy receptor p62, and the late autophagic marker STX17 .

The TECPR1-LC3C axis is particularly important in neural stem cells (NSCs), where overexpression of TECPR1 significantly reduces the accumulation of neurotoxic protein aggregates such as Huntingtin (Htt) in an autophagy-dependent manner . Conversely, depletion of LC3C augments protein aggregation. This mechanism appears to operate independently of the ATG12-ATG5 complex, as TECPR1 with a deleted ATG5-interacting region (AIR) still strongly colocalizes with LC3C . This suggests that TECPR1 employs multiple, potentially redundant mechanisms to promote selective autophagy.

What is the mechanism of TECPR1 activation in response to membrane damage?

TECPR1 activation is specifically triggered by damage to cellular membranes, particularly those of the endolysosomal system. Unlike ATG16L1-containing E3 complexes, which are activated by proton gradient loss, TECPR1-containing E3 complexes respond to a different type of membrane disruption . Research has shown that TECPR1 is activated when sphingomyelin (SM) is exposed due to membrane damage .

In vitro lipidation experiments have demonstrated that TECPR1 can facilitate the conjugation of all six ATG8 proteins (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2), but this process is specifically dependent on sphingomyelin . TECPR1 can also enable the use of phosphatidylserine (PS) as a substrate for ATG8 lipidation, albeit at lower rates, and there is a synergistic effect when both phosphatidylethanolamine (PE) and PS are present . This mechanism is distinct from that of ATG16L1, highlighting the complexity and redundancy in cellular responses to different types of membrane damage.

What is the role of TECPR1 in cancer cell apoptosis and its potential as a therapeutic target?

TECPR1 has emerged as a potential tumor suppressor in non-small cell lung carcinoma (NSCLC). Lower TECPR1 expression is associated with poorer outcomes in NSCLC patients, with Kaplan-Meier analysis revealing that lung cancer patients with low TECPR1 expression levels had a median survival time of 51.53 months, compared to 136.33 months for those with high TECPR1 expression .

Mechanistically, TECPR1 induces apoptosis in NSCLC cells through upregulation of ATG5, which promotes autophagy . Experimental evidence shows that overexpression of TECPR1 leads to decreased cell viability and enhanced apoptosis in NSCLC cell lines. This occurs through upregulation of Bax and LC3-II/LC3-I ratio, and downregulation of P62 and Bcl-2 . The formation of TECPR1-ATG5 complexes is critical for this process, as TECPR1 and ATG5 can regulate each other's expression levels .

These findings suggest that strategies to increase TECPR1 expression or activity could potentially be developed as therapeutic approaches for NSCLC and possibly other cancers where TECPR1 expression is reduced.

What are the optimal methods for detecting and analyzing TECPR1 expression?

For effective detection and analysis of TECPR1 expression, researchers should employ multiple complementary techniques:

• qRT-PCR: For quantitative analysis of TECPR1 mRNA expression. This method has been successfully used to compare TECPR1 expression between normal and cancer cells .

• Western blot: For protein-level detection, using validated TECPR1 antibodies. When analyzing TECPR1 function in autophagy, it's also advisable to examine the expression of autophagy markers such as LC3-II/LC3-I ratio and p62 .

• Immunofluorescence: For visualization of TECPR1 localization and co-localization with interaction partners. This approach is valuable for confirming the subcellular localization of TECPR1, particularly its association with lysosomal membranes .

• Co-immunoprecipitation: For validating TECPR1 interactions with proteins such as ATG5, ATG12, and LC3C. This method has been successfully employed to demonstrate that TECPR1 forms complexes with ATG12-ATG5 but not with ATG16 .

For comprehensive analysis, it's recommended to use cell lines with known TECPR1 expression patterns, such as HEK293A, HeLa, and PC-3 cells, which have been well-characterized in TECPR1 studies .

How can researchers effectively validate TECPR1 antibody specificity?

Validating TECPR1 antibody specificity is critical for reliable research outcomes. A systematic approach includes:

• Positive and negative controls: Use cell lines with known high (e.g., 16HBE) and low (e.g., NSCLC cell lines) TECPR1 expression .

• TECPR1 knockout validation: Generate TECPR1 knockout cell lines using CRISPR-Cas9 to confirm antibody specificity. The absence of signal in knockout cells provides strong evidence for antibody specificity .

• Overexpression validation: Compare antibody performance in cells with endogenous TECPR1 levels versus cells overexpressing tagged TECPR1 (e.g., EGFP-TECPR1) . This allows confirmation that the antibody recognizes both endogenous and overexpressed protein.

• Cross-reactivity testing: Examine potential cross-reactivity with related proteins, particularly those with beta-propeller repeat domains.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm that this blocks specific binding.

These validation steps should be performed across multiple experimental techniques (Western blot, immunofluorescence, immunoprecipitation) to ensure comprehensive validation of antibody performance in each application.

What experimental models are most suitable for studying TECPR1 function?

The choice of experimental model depends on the specific aspect of TECPR1 function being investigated:

• Cell lines:

  • HEK293A cells: Widely used for basic TECPR1 function studies

  • HeLa cells: Suitable for studying TECPR1 localization and trafficking

  • PC-3 cells: Show distinct TECPR1-dependent ATG8 lipidation patterns

  • Neural stem cells (NSCs): Ideal for studying TECPR1's role in clearing protein aggregates like Huntingtin

  • NSCLC cell lines (A549, NCI-H23, NCI-H520): Appropriate for investigating TECPR1's role in cancer

• Knockout models:

  • TECPR1-KO cells: Essential for determining TECPR1-specific effects

  • ATG16L1/TECPR1 double-knockout cells: Useful for distinguishing between ATG16L1 and TECPR1-dependent autophagy pathways

• Treatment conditions:

  • Membrane-damaging agents: Saponin, JetMESSENGER®, glycyl-L-phenylalanine 2-naphththylamide (GPN), astemizole

  • Lysosomal damage inducers: LLOMe

  • Autophagy inducers for comparison: Torin1, monensin, nigericin

The selection of an appropriate model should be guided by the specific research question, with consideration for cell type-specific variations in TECPR1 response that have been documented .

How can researchers distinguish between TECPR1-dependent and ATG16L1-dependent autophagy?

Distinguishing between TECPR1-dependent and ATG16L1-dependent autophagy requires a systematic approach:

• Use specific activators: TECPR1-dependent autophagy is activated by membrane-damaging agents like saponin, JetMESSENGER®, GPN, and astemizole, while ATG16L1 responds to drugs affecting the proton gradient like NH₄Cl, monensin, and nigericin . The table below summarizes key differences:

• Genetic approaches: Use TECPR1 knockout, ATG16L1 knockout, and double knockout cell lines, followed by rescue experiments with wild-type or mutant constructs . ATG16L1β expression rescues ATG8 lipidation in response to all treatments, while TECPR1 expression only rescues lipidation in response to membrane damage .

• Marker analysis: Monitor LC3C puncta accumulation, which is specifically associated with TECPR1 function . TECPR1 deletion leads to a selective accumulation of LC3C-positive autophagosomes that colocalize with Ub, p62, and STX17 .

• Lipidation substrate specificity: TECPR1-dependent ATG8 lipidation shows sphingomyelin specificity, while also facilitating phosphatidylserine as a substrate at lower rates . This differs from the typical phosphatidylethanolamine preference in conventional autophagy.

These approaches can provide clear evidence to distinguish between these two autophagy pathways and determine their relative contributions under different cellular conditions.

What are the common challenges in interpreting TECPR1 antibody staining patterns?

Interpreting TECPR1 antibody staining patterns presents several challenges that researchers should be aware of:

• Cell type-specific variation: Different cell lines display varying degrees of TECPR1-dependent ATG8 lipidation, as observed in HEK293A, PC-3, and HeLa cells . This can lead to inconsistent staining patterns across cell types.

• Subcellular localization complexity: TECPR1 localizes to lysosomes but can also be found on autophagosomes, particularly those containing LC3C . This dual localization can complicate interpretation of staining patterns.

• Membrane damage effects: Since TECPR1 is activated by membrane damage, fixation methods that affect membrane integrity can potentially influence TECPR1 distribution and lead to artifacts .

• Epitope accessibility: TECPR1's association with membranes and its complex domain structure may limit antibody accessibility to certain epitopes, potentially resulting in false negatives.

• Cross-reactivity with beta-propeller domains: TECPR1 contains nine beta-propeller repeats that share structural similarities with other proteins, potentially leading to cross-reactivity and false positives .

To address these challenges, researchers should use multiple antibodies targeting different TECPR1 epitopes, include appropriate controls (especially TECPR1 knockout cells), carefully select fixation methods that preserve membrane integrity, and validate findings using complementary approaches such as live-cell imaging with fluorescently tagged TECPR1.

How should researchers troubleshoot experiments when TECPR1 antibody detection is suboptimal?

When facing challenges with TECPR1 antibody detection, consider the following troubleshooting strategies:

• Fixation optimization: Test multiple fixation methods (paraformaldehyde, methanol, acetone) as TECPR1's membrane association may make certain epitopes inaccessible depending on fixation approach.

• Permeabilization adjustment: TECPR1's association with internal membranes may require stronger permeabilization (e.g., 0.1-0.5% Triton X-100 versus 0.1% saponin) for effective antibody access.

• Epitope retrieval: Implement antigen retrieval methods, particularly for formalin-fixed tissues or cells where epitopes may be masked.

• Blocking optimization: Test different blocking agents (BSA, normal serum, commercial blockers) to reduce background while preserving specific signal.

• Antibody concentration titration: Systematically test a range of primary antibody concentrations to determine the optimal signal-to-noise ratio.

• Signal amplification: Consider using signal amplification methods such as tyramide signal amplification or polymer-based detection systems for low abundance targets.

• Alternative detection methods: If immunostaining proves challenging, consider alternative approaches such as Western blotting following immunoprecipitation or proximity ligation assay to detect TECPR1 and its interaction partners.

For Western blot applications specifically, optimize protein extraction methods to ensure complete solubilization of membrane-associated TECPR1, potentially using detergents such as CHAPS or NP-40 that are effective for membrane proteins.

How can TECPR1 be targeted in neurodegenerative disease research?

TECPR1 shows significant promise as a target in neurodegenerative disease research due to its role in clearing protein aggregates:

• Aggregate clearance mechanism: TECPR1 selectively interacts with LC3C to promote the degradation of protein aggregates in neural stem cells . Overexpression of TECPR1 in NSCs diminishes the accumulation of neurotoxic protein aggregates such as Huntingtin (Htt) in an autophagy-dependent manner . This suggests that enhancing TECPR1 expression or activity could potentially reduce the burden of protein aggregates in neurodegenerative diseases.

• Experimental approaches:

  • Generate neuronal models expressing fluorescently tagged TECPR1 and disease-associated proteins (e.g., Htt, α-synuclein, tau)

  • Quantify aggregate clearance rates with TECPR1 modulation (overexpression, knockdown, pharmacological activation)

  • Investigate the interaction between TECPR1 and specific disease-associated proteins using co-immunoprecipitation and proximity ligation assays

  • Develop small molecules that enhance TECPR1 recruitment to protein aggregates or increase its activity

• Therapeutic strategies:

  • Screen for compounds that enhance TECPR1 expression or activity

  • Develop peptide-based approaches targeting the LC3C-TECPR1 interaction to enhance aggregate clearance

  • Explore gene therapy approaches to increase TECPR1 expression in affected neurons

  • Investigate the potential of TECPR1 as a biomarker for autophagy dysfunction in neurodegenerative diseases

The selective nature of TECPR1-mediated autophagy may provide advantages over general autophagy inducers, potentially offering greater specificity and reduced side effects for therapeutic applications.

What is the significance of TECPR1 in cancer research and potential therapeutic applications?

TECPR1 has emerged as a significant factor in cancer research, particularly in NSCLC:

• Prognostic value: Higher TECPR1 expression is associated with better outcomes in NSCLC patients, with a significant difference in median survival time (136.33 months for high expression vs. 51.53 months for low expression) . This suggests TECPR1 expression could serve as a prognostic biomarker.

• Tumor suppressive mechanism: TECPR1 induces apoptosis in NSCLC cells via ATG5 upregulation-induced autophagy promotion . This mechanism involves decreasing cell viability, promoting apoptosis, upregulating Bax and LC3-II/LC3-I, and downregulating P62 and Bcl-2 .

• Therapeutic strategies:

  • Develop approaches to increase TECPR1 expression in tumor cells

  • Screen for compounds that enhance the TECPR1-ATG5 interaction

  • Target the TECPR1-dependent autophagy pathway to promote cancer cell apoptosis

  • Investigate combination therapies that enhance TECPR1 function while inhibiting cancer cell survival mechanisms

• Research approaches:

  • Analyze TECPR1 expression across cancer types and correlate with patient outcomes

  • Investigate the mechanism of TECPR1 downregulation in cancer cells

  • Develop in vivo models to test TECPR1-enhancing strategies

  • Explore the relationship between TECPR1 expression and response to conventional cancer therapies

The dual role of TECPR1 in promoting both autophagy and apoptosis makes it a particularly interesting target for cancer research, potentially offering new strategies to overcome therapy resistance in tumors.

How does TECPR1 research contribute to understanding fundamental autophagy mechanisms?

TECPR1 research has significantly expanded our understanding of fundamental autophagy mechanisms in several key ways:

• Diversification of autophagy pathways: TECPR1 research has revealed that noncanonical autophagy (CASM) can be triggered by different cellular stresses and mediated by distinct E3 complexes . While ATG16L1-containing E3 complexes respond to proton gradient loss, TECPR1-containing E3 complexes are activated by membrane damage . This demonstrates that cells have evolved multiple, specialized autophagy mechanisms to respond to different types of cellular stress.

• Substrate specificity: TECPR1 research has uncovered unexpected complexity in the lipidation substrates used in autophagy. While conventional autophagy primarily uses phosphatidylethanolamine (PE) as the lipidation substrate, TECPR1 shows sphingomyelin specificity and can also facilitate the use of phosphatidylserine (PS) as a substrate . This expands our understanding of the lipid requirements for autophagosome formation.

• Selective autophagy mechanisms: The selective interaction between TECPR1 and LC3C provides insight into how cells achieve specificity in autophagy targeting . This mechanism helps explain how cells can selectively degrade protein aggregates while preserving essential cellular components.

• Integration of autophagy and cell death pathways: TECPR1's role in both autophagy promotion and apoptosis induction in cancer cells highlights the complex interplay between these two fundamental cellular processes . This challenges the simplistic view of autophagy as merely a cell survival mechanism and demonstrates how it can contribute to programmed cell death under certain conditions.

These insights have broad implications for understanding cellular homeostasis, stress responses, and the pathogenesis of various diseases, from neurodegeneration to cancer.