TRAIL Human (114-281 a.a.)

What is TRAIL Human (114-281 a.a.) and how does it function in cellular pathways?

TRAIL Human (114-281 a.a.) refers to the extracellular domain of human TRAIL protein that mediates activation of the extrinsic apoptosis pathway. TRAIL is expressed as a homotrimeric type II transmembrane or soluble protein that binds to and activates its death receptors DR4 and DR5 . The binding induces receptor trimerization, which is essential for initiating the apoptotic signaling pathway . The extracellular domain (amino acids 114-281) retains the binding capacity to these receptors and can be produced recombinantly for research purposes.

The interaction between TRAIL and its receptors creates a cascade where the cytoplasmic domains of DR4 and DR5 serve as docking sites for adapter proteins that ultimately activate the caspase cascade leading to controlled cell death . Notably, TRAIL also interacts with three decoy receptors (DcR1, DcR2, and OPG), which can modulate its apoptotic activity .

Why does TRAIL (114-281 a.a.) preferentially induce apoptosis in cancer cells while sparing normal cells?

TRAIL exhibits remarkable tumor specificity, selectively inducing apoptosis in malignant cells while generally sparing normal tissues . This selective activity is attributed to several factors:

• Differential receptor expression: Cancer cells often upregulate death receptors DR4 and DR5 compared to normal cells

• Decoy receptor distribution: Normal cells may express higher levels of decoy receptors (DcR1, DcR2, OPG) that bind TRAIL without triggering apoptosis

• Intracellular anti-apoptotic mechanisms: Cancer cells frequently have alterations in apoptotic machinery that paradoxically make them more susceptible to death receptor activation

Research has demonstrated that this selective toxicity profile makes TRAIL an attractive candidate for cancer therapy compared to other TNF family members like FasL and TNF-α, which cause significant systemic toxicity when administered .

What expression systems are optimal for producing functional recombinant TRAIL (114-281 a.a.)?

Several expression systems have been successfully employed to produce recombinant TRAIL (114-281 a.a.), each with specific advantages:

Human recombinant TRAIL (hrTRAIL) and metabolically labeled 15N-rhTRAIL have been successfully expressed in E. coli and purified using established protocols . For applications requiring more native-like protein structure, CHO cell expression systems have been employed, particularly when producing fusion proteins like TRAIL-Trimer, which contains disulfide bond-linked homotrimers .

When selecting an expression system, researchers should consider whether post-translational modifications are critical for their specific experimental design and whether protein yield or structural authenticity is the priority.

What methodological approaches can enhance TRAIL stability and activity for in vivo applications?

A significant challenge with native TRAIL (114-281 a.a.) is its relatively rapid clearance in vivo, which limits its therapeutic potential. Several methodological approaches have been developed to address this limitation:

• Trimer-Tag fusion: In-frame fusion of human C-propeptide of α1(I) collagen (Trimer-Tag) to the C-terminus of mature human TRAIL creates a disulfide bond-linked homotrimer with significantly improved pharmacokinetic profiles

• Receptor-selective variants: Engineering TRAIL variants with preferential binding to either DR4 or DR5 based on the cancer type being targeted (some cancers preferentially signal through one receptor over the other)

The TRAIL-Trimer approach has demonstrated particular promise, retaining similar bioactivity and receptor binding kinetics as native TRAIL in vitro while showing 4–5 orders of magnitude superior activity compared to dimeric TRAIL-Fc constructs . Most importantly, TRAIL-Trimer manifests more favorable pharmacokinetic and antitumor pharmacodynamic profiles in vivo than native TRAIL, with direct evidence that antitumor efficacy correlates with systemic drug exposure .

What are the recommended approaches for quantifying TRAIL (114-281 a.a.) in experimental samples?

Accurate quantification of TRAIL is essential for consistent experimental results. Several complementary analytical methods are available:

• LC-MS/MS analysis: Antibody-free liquid chromatography-tandem mass spectrometry provides highly specific protein analysis of TRAIL with potential for absolute quantification

• Bioactivity assays: Functional testing using TRAIL-sensitive cell lines to determine biological activity rather than just protein concentration

• Receptor binding kinetics: Surface plasmon resonance (SPR) or similar techniques to measure binding affinity to DR4/DR5 receptors

For the most comprehensive characterization, researchers should employ multiple orthogonal methods. A particular advantage of LC-MS/MS approaches is the ability to perform antibody-free analysis, which can be especially valuable when antibody availability or specificity is limiting .

How should researchers evaluate receptor selectivity when working with TRAIL (114-281 a.a.)?

Understanding receptor selectivity is crucial when studying TRAIL mechanisms. While it was initially assumed that TRAIL-R2 (DR5) played a dominant role in initiating apoptosis, more recent evidence suggests that receptor preference varies by cancer type . Some methodological approaches to evaluate receptor selectivity include:

• Comparative receptor expression analysis: Quantify DR4, DR5, and decoy receptor levels on target cells

• Receptor-specific blocking antibodies: Selectively inhibit either DR4 or DR5 to determine their relative contribution to apoptosis

• Receptor knockdown/knockout: Use RNAi or CRISPR-based approaches to specifically reduce receptor expression

• Receptor-selective TRAIL variants: Compare the activity of TRAIL mutants engineered for preferential binding to either DR4 or DR5

Research has demonstrated that some cancer types, such as chronic lymphocytic leukemia (CLL), predominantly signal to cell death via TRAIL-R1 (DR4), challenging the previous assumption of TRAIL-R2 dominance . Importantly, the relationship between receptor expression levels and response to TRAIL-R1 or TRAIL-R2 activating compounds is not straightforward and requires careful experimental design to elucidate .

What molecular mechanisms contribute to TRAIL resistance in cancer cells?

Despite TRAIL's promise as a cancer therapeutic, many tumors exhibit intrinsic or acquired resistance. Several key resistance mechanisms have been identified:

• Inhibitor of Apoptosis Proteins (IAPs): Many cancer types, including pancreatic carcinoma, overexpress IAPs such as X-linked inhibitor of apoptosis (XIAP), which prevents apoptosis by binding to and inhibiting activated caspase-3 and caspase-9

• Decoy receptor expression: Increased expression of non-apoptotic TRAIL receptors (DcR1, DcR2, OPG) can sequester TRAIL without triggering cell death

• Death receptor downregulation: Reduced expression or internalization of DR4/DR5 receptors limits TRAIL sensitivity

• Defects in apoptotic signaling: Alterations in downstream components of the apoptotic machinery can prevent effective signal transduction

XIAP represents a particularly important resistance factor as it acts at the core of the apoptotic machinery, blocking cell death at the effector phase . This makes therapeutic modulation of XIAP a potentially valuable strategy to overcome TRAIL resistance in cancer treatment protocols .

What experimental approaches can overcome TRAIL resistance in research models?

Several methodological strategies have shown promise in addressing TRAIL resistance:

• Combination treatments: Many standard chemotherapeutics can sensitize resistant cells to TRAIL through various mechanisms, including upregulation of death receptors or downregulation of anti-apoptotic proteins

• IAP antagonists: Small molecule inhibitors that target XIAP and other IAPs can restore TRAIL sensitivity in resistant cancer cells by removing the block at the effector phase of apoptosis

• Epigenetic modifiers: HDAC inhibitors and DNA methyltransferase inhibitors can increase death receptor expression in some cancer types

• Improved TRAIL formulations: Novel delivery systems or engineered TRAIL variants with enhanced stability and receptor binding properties

The selection of sensitizing agents should be guided by the specific resistance mechanism present in the experimental model. For instance, IAP antagonists would be most appropriate when dealing with XIAP-mediated resistance, whereas epigenetic modifiers might be more suitable for cases with epigenetically silenced death receptors.

What factors might explain the limited efficacy of TRAIL therapies in clinical trials despite promising preclinical results?

Despite encouraging preclinical data, TRAIL-based therapies have shown modest efficacy in clinical trials . Several factors may explain this disconnect:

• Pharmacokinetic limitations: Evidence suggests that the clinical failures may be due to rapid systemic clearance of native TRAIL, limiting effective drug exposure at tumor sites

• Receptor agonist potency: Dimeric agonist monoclonal antibodies targeting DR4/DR5 have shown poor apoptosis-inducing potency despite their long serum half-lives

• Tumor heterogeneity: Variability in receptor expression and resistance mechanisms within and between tumors may limit consistent responses

• Decoy receptor interference: The presence of decoy receptors in the tumor microenvironment may sequester therapeutic TRAIL

Research has provided direct evidence that in vivo antitumor efficacy of TRAIL correlates with systemic drug exposure, suggesting that approaches to extend half-life while maintaining or enhancing receptor activation could improve clinical outcomes .

What methodological approaches align with current human subjects research regulations when designing TRAIL-based clinical studies?

When designing clinical studies involving TRAIL (114-281 a.a.), researchers must navigate complex regulatory requirements for human subjects research:

• Clinical trial definition: Studies must determine whether they meet the NIH definition of a clinical trial, which encompasses "a research study in which one or more human subjects are prospectively assigned to one or more interventions to evaluate the effects of those interventions on health-related biomedical or behavioral outcomes"

• Regulatory compliance: Research must follow institutional and national guidelines for human subjects research, including IRB approval and informed consent procedures

• Trial registration: Clinical trials involving TRAIL must be registered on ClinicalTrials.gov with appropriate documentation of protocol and results reporting

• Does the study involve human participants?

• Are the participants prospectively assigned to an intervention?

• Is the study designed to evaluate the effect of the intervention on the participants?

• Is the effect being evaluated a health-related biomedical or behavioral outcome?

How can researchers employ LC-MS/MS for antibody-free analysis of TRAIL (114-281 a.a.) in complex biological matrices?

LC-MS/MS offers powerful capabilities for TRAIL analysis without relying on antibodies, which can be particularly valuable when antibody availability, specificity, or cross-reactivity is a concern:

• Sample preparation: For complex matrices like serum or tissue lysates, protein precipitation, immunodepletion of abundant proteins, or solid-phase extraction may be necessary

• Peptide selection: Identify unique peptides (signature peptides) from TRAIL that can serve as quantitative surrogates for the intact protein

• Isotope dilution: Use of metabolically labeled 15N-rhTRAIL as an internal standard enables accurate quantification

• Sensitivity enhancement: Techniques such as multiple reaction monitoring (MRM) can improve detection limits for TRAIL peptides in complex backgrounds

LC-MS/MS approaches have been successfully applied to quantify other proteins in human serum with lower limits of quantification (LLOQ) in the nanomolar range, suggesting this methodology could be adapted for TRAIL quantification in clinical samples .

What biomarkers can help predict TRAIL sensitivity in experimental cancer models?

Identifying predictive biomarkers for TRAIL sensitivity remains an active area of research. Several potential markers have emerged:

• Death receptor expression: While not a perfect predictor, DR4/DR5 expression levels provide baseline information about potential TRAIL responsiveness

• Decoy receptor levels: The ratio of death receptors to decoy receptors may offer better predictive value than absolute expression of either alone

• XIAP and other IAP expression: Higher levels typically correlate with resistance

• C-reactive protein (CRP): Some studies have investigated relationships between inflammatory markers like CRP and therapeutic responses, though direct links to TRAIL sensitivity require further investigation

Researchers should consider using a panel of biomarkers rather than relying on a single predictor, given the complex and multifactorial nature of TRAIL response mechanisms in cancer cells.

How does the TRAIL pathway interact with immune checkpoint proteins in cancer immunotherapy?

An exciting frontier in TRAIL research involves its intersection with cancer immunotherapy:

• TRAIL-immune checkpoint interactions: Emerging evidence indicates that the TRAIL pathway may interact with immune checkpoint proteins, including programmed death-ligand 1 (PD-L1), potentially modulating PD-L1-based tumor immunotherapies

• TRAIL in immune surveillance: Beyond direct apoptosis induction, TRAIL expressed by immune cells plays a critical role in tumor surveillance

• Combination approaches: TRAIL-based therapies may complement immune checkpoint inhibitors by targeting cancer cells through orthogonal mechanisms

These interactions suggest potential synergies between TRAIL-targeted therapies and immune checkpoint blockade strategies, opening new avenues for combination treatments that simultaneously activate apoptotic pathways and reinvigorate anti-tumor immune responses .

What emerging technologies might address the pharmacokinetic limitations of TRAIL (114-281 a.a.) in future research?

Several innovative approaches are being explored to overcome the pharmacokinetic limitations of native TRAIL:

• Engineered fusion proteins: The TRAIL-Trimer approach using C-propeptide of α1(I) collagen has already demonstrated improved pharmacokinetics while maintaining receptor binding efficiency

• Nanoparticle delivery systems: Encapsulation or surface presentation of TRAIL on various nanoparticles may improve stability and tumor targeting

• Cell-based delivery: Engineered cells expressing TRAIL could provide sustained local delivery in the tumor microenvironment

• Gene therapy approaches: Viral vectors encoding TRAIL could enable long-term expression within tumors

The promising results with TRAIL-Trimer, which showed significantly improved pharmacokinetic and antitumor pharmacodynamic profiles compared to native TRAIL, provide proof-of-concept that addressing the rapid clearance issue can enhance therapeutic efficacy .

What strategies can address batch-to-batch variability in recombinant TRAIL (114-281 a.a.) preparations?

Consistency in TRAIL preparations is crucial for reproducible research. Several approaches can minimize variability:

• Standardized expression protocols: Maintain consistent culture conditions, induction parameters, and harvest timing

• Quality control metrics: Implement multiple orthogonal QC assays including:

  • SDS-PAGE for purity assessment

  • Size exclusion chromatography to confirm trimeric structure

  • Bioactivity assays on reference cell lines

  • Mass spectrometry for identity confirmation

• Reference standards: Maintain well-characterized internal reference standards to calibrate new batches

• Storage optimization: Investigate and standardize buffer compositions and storage conditions that maximize stability

Researchers should document batch characteristics comprehensively and include appropriate controls when comparing results across different TRAIL preparations.

How can researchers distinguish between TRAIL-specific and non-specific cell death in experimental systems?

Differentiating TRAIL-specific apoptosis from other cell death mechanisms requires careful experimental design:

• Blocking controls: Use TRAIL-neutralizing antibodies or soluble decoy receptors to confirm specificity

• Receptor dependence: Employ receptor-specific blocking antibodies or receptor knockdown to verify the involvement of DR4/DR5

• Apoptosis markers: Assess hallmarks of apoptosis including caspase activation, PARP cleavage, and phosphatidylserine externalization

• Caspase inhibition: Determine whether pan-caspase inhibitors (e.g., z-VAD-fmk) prevent the observed cell death

• Alternative death pathways: Evaluate markers of necroptosis, pyroptosis, or other death mechanisms to rule out non-apoptotic death