BIRC7 Human, (1-280 a.a.)

What is the molecular architecture of BIRC7 Human protein and how does it relate to its function?

BIRC7 (Baculoviral IAP repeat-containing protein 7, also known as Livin) is a member of the Inhibitor of Apoptosis Protein (IAP) family. The protein contains a single baculovirus IAP repeat (BIR) domain and a RING-type zinc finger domain. The BIR domain is necessary for inhibitory activity and interacts with caspases, while the RING finger domain possesses E3 ubiquitin ligase activity .

When examining the functional interactions, BIRC7 directly contacts several caspases including caspase-3, caspase-7, and caspase-9. Mechanistically, it inhibits the activation of caspase-9 induced by Apaf-1, cytochrome c, and dATP . The protein also binds to SMAC/DIABLO via the BIR domain to target the degradation of these molecules and active caspases .

For experimental work with recombinant BIRC7 Human (1-280 a.a.), the protein is typically produced in E. coli as a single, non-glycosylated polypeptide chain containing 304 amino acids (the 1-280 a.a. sequence fused to a 24 amino acid His-tag at the N-terminus) with a molecular mass of 33.4kDa .

How do the alternative splice variants of BIRC7 differ in structure and function?

BIRC7 encodes two splice variants termed Livin-α and Livin-β. These isoforms are identical with each other except for a 54 bp truncation at exon 6 in the Livin-β variant . This structural difference manifests in distinct functional characteristics:

Research has demonstrated that these isoforms can have differential effects on cell survival pathways, highlighting the importance of specifying which variant is being studied in experimental contexts .

What experimental approaches can be used to study BIRC7-caspase interactions?

Several complementary methodologies have proven effective for investigating BIRC7-caspase interactions:

• Structural studies: X-ray crystallography has been successfully used to determine the structure of the human dimeric RING domain from BIRC7 in complex with the E2 UbcH5B covalently linked to Ub at its active site (UbcH5B~Ub) at 2.18 Å resolution . NMR analyses demonstrate that BIRC7 binding to UbcH5B~Ub induces peak shift perturbations in the donor Ub consistent with the crystallographically-observed BIRC7-Ub interactions .

• Biochemical assays:

  • Single turnover lysine discharge assays to exclusively monitor the effects on donor Ub transfer

  • Di-Ub formation assays (though these may be complicated by BIRC7's preference for autoubiquitination)

  • BIRC7 autoubiquitination and SMAC ubiquitination assays

• Protein-protein interaction assays:

  • Binding affinity measurements using techniques like surface plasmon resonance

  • Pull-down assays with recombinant proteins

  • Mutational studies targeting specific residues in the BIR domain

When designing such experiments, researchers should consider using both wild-type BIRC7 and mutants that disrupt specific interactions, as mutations in non-covalent interaction sites or RING dimerization regions have been shown to reduce UbcH5B~Ub binding affinity and ubiquitination activity .

What are the optimal storage and handling conditions for BIRC7 Human recombinant protein?

To maintain the stability and activity of BIRC7 Human recombinant protein, researchers should follow these evidence-based protocols:

• Short-term storage: Store at 4°C if the entire vial will be used within 2-4 weeks

• Long-term storage: Store frozen at -20°C

  • For extended periods, it is recommended to add a carrier protein (0.1% HSA or BSA)

  • Avoid multiple freeze-thaw cycles

• Formulation: BIRC7 protein solution (1mg/ml) typically contains 20mM Tris pH-8, 1mM DTT, 0.1M NaCl, 2mM EDTA, and 20% glycerol

• Reconstitution: For lyophilized BIRC7, add 0.2ml of distilled water to yield a concentration of 500μg/ml

These conditions are critical for maintaining protein integrity during experimental use and ensuring reproducible results.

How does BIRC7 expression correlate with tumor progression and clinical outcomes?

BIRC7 expression has significant implications for tumor progression and patient prognosis across multiple cancer types:

• Expression patterns: While BIRC7 is generally not detectable in normal differentiated adult tissues (except placenta, spleen, lymph nodes, and developing embryonic tissues), it is overexpressed in various tumor types, including melanoma, non-small cell lung cancer, malignant pleural mesothelioma, gallbladder cancer, breast cancer, and prostate cancer .

• Clinical significance in prostate cancer: High BIRC7 expression might serve as an independent prognostic biomarker. Research has established that BIRC7 expression is positively correlated with the pathological grading of prostate cancer . Importantly, BIRC7 upregulation might serve as a valuable biomarker of increased recurrence risk in advanced T stages and medium-grade prostate cancer .

• Cutaneous melanoma: BIRC7 expression in skin cutaneous melanoma (SKCM, n = 461) is significantly higher than in normal samples (n = 558), as shown by GEPIA data . This overexpression is associated with melanoma progression.

• Prognostic value across ethnicities: Using TCGA data analyzed through the UALCAN domain, researchers have studied the expression pattern of BIRC7/Livin based on patient race (African-American, Whites, and Asian) and its effect on colorectal cancer survival, revealing potential ethnic differences in prognostic significance .

These correlations highlight BIRC7's potential value as both a biomarker and therapeutic target.

What is the relationship between BIRC7 expression and hypoxia in tumor microenvironments?

The interplay between BIRC7 and hypoxia represents a significant aspect of tumor biology:

• Transcriptional regulation: BIRC7 is a downstream factor of HIF-1α, which acts as a transcription factor for BIRC7 and promotes its transcription under hypoxic conditions . This relationship has been demonstrated through:

  • RNA-seq analysis showing BIRC7 among the most significantly upregulated genes in hypoxic melanoma cells

  • Luciferase assays confirming HIF-1α binding to the BIRC7 promoter

  • The inhibitory effect of HIF-1α inhibitors on hypoxia-induced BIRC7 protein expression

• Functional consequences in melanoma cells: In A875 and M14 melanoma cell lines, hypoxia:

  • Promotes cell proliferation

  • Inhibits apoptosis

  • Enhances cell invasion

Notably, knockdown of BIRC7 under hypoxic conditions blocks these hypoxia-induced effects, demonstrating BIRC7's essential role in mediating the cellular response to hypoxia .

• Quantitative findings: RT-qPCR and Western blot analyses have confirmed significant upregulation of BIRC7 at both mRNA and protein levels after hypoxia treatment in melanoma cells .

This hypoxia-BIRC7 axis has important implications for therapeutic approaches targeting tumor hypoxia and resistance mechanisms.

What is the structural basis for BIRC7's E3 ubiquitin ligase activity?

The structural basis for BIRC7's E3 ubiquitin ligase activity is revealed through crystallographic studies of its RING domain interacting with E2~Ub conjugates:

• Dimeric RING domain structure: The human dimeric RING domain from BIRC7 was crystallized in complex with the E2 UbcH5B covalently linked to Ub at its active site (UbcH5B~Ub) at 2.18 Å resolution. Key crystallographic parameters include:

• Key interaction interfaces: The structure reveals:

  • The known E2-RING contacts

  • Extensive non-covalent donor Ub interactions with UbcH5B

  • Interactions with both subunits of the RING domain dimer

• Functional validation: Mutations that disrupt the non-covalent interactions or RING dimerization reduce UbcH5B~Ub binding affinity and ubiquitination activity .

• Mechanistic implications: BIRC7 promotes ubiquitination activity by stabilizing an optimal E2~donor Ub arrangement for Ub transfer. This represents the first detailed structural evidence supporting this mechanistic hypothesis .

These structural insights provide a foundation for understanding how BIRC7 functions as an E3 ubiquitin ligase and potentially for designing inhibitors targeting these interactions.

How do SMAC mimetics antagonize BIRC7 function at the molecular level?

SMAC (Second Mitochondria-derived Activator of Caspases) is a key antagonist of IAPs including BIRC7. The molecular basis of this antagonism involves:

• Multi-site engagement: SMAC engages BIRC7 at multiple sites resulting in a sub-nanomolar affinity, enabling SMAC to competitively displace caspases, thus antagonizing BIRC7's anti-apoptotic function .

• Binding interfaces:

  • N-terminal residues of SMAC bind to the BIR domain via a conserved Asp342-containing pocket

  • Additional contacts involve the SMAC helical bundle interacting with a unique loop extending from the DOC domain tip of BIRC7

• Binding affinities:

  • BIRC7 binding to monomeric SMAC: Kd 12.1 ± 1.5 nM

  • BIRC7 binding to isolated SMAC peptide: Kd 35.6 ± 3.0 nM

  • Loss of Asp342 abrogates binding

• Structural contributions: The positively charged DOC domain tip (residues 3189-3193, HRRaR) contributes to binding affinity, as mutation to all Ala or all Asp reduces the binding affinity to that measured between BIRC7 and SMAC N-terminal peptide .

Understanding these molecular interactions provides a foundation for developing SMAC mimetics as potential therapeutic agents targeting BIRC7 in cancer contexts.

What are the most effective methods for modulating BIRC7 expression in experimental models?

Several approaches have been validated for modulating BIRC7 expression in experimental models:

• RNA interference:

  • siRNA transfection has been successfully used to down-regulate BIRC7 expression in melanoma cell lines (A875 and M14 cells)

  • This approach has demonstrated significant effects on cell proliferation, apoptosis, and invasion in both normoxic and hypoxic conditions

• Transcriptional regulation:

  • HIF-1α inhibitors can reduce hypoxia-induced BIRC7 expression

  • This approach targets the transcriptional upregulation pathway rather than directly targeting BIRC7

• Protein-based approaches:

  • SMAC mimetics can functionally antagonize BIRC7 activity without altering expression levels

  • These compounds compete with caspases for binding to BIRC7's BIR domain

When designing experiments to study BIRC7 function, researchers should consider the following experimental controls:

• Comparison of normal vs. hypoxic conditions

• Use of HIF-1α inhibitors as positive controls for BIRC7 expression modulation

• Appropriate vector controls for siRNA experiments

• Dose-dependent studies to establish optimal concentrations

What functional assays are most informative for evaluating BIRC7's roles in cancer cell biology?

A comprehensive panel of functional assays has been validated for investigating BIRC7's roles in cancer biology:

• Cell viability and proliferation:

  • CCK-8 (Cell Counting Kit-8) assay has been used to measure the effects of BIRC7 knockdown on melanoma cell proliferation under both normoxic and hypoxic conditions

  • Results show that si-BIRC7 inhibits the proliferation and viability of A875 and M14 cells under normoxia, and blocks the promoting effect of hypoxia on cell proliferation

• Apoptosis quantification:

  • Flow cytometry with appropriate staining (e.g., Annexin V/PI) can detect apoptotic cells

  • Studies show si-BIRC7 increases the percentage of apoptosis in melanoma cells under normoxia, and hinders the inhibitory effect of hypoxia on apoptosis

• Cell invasion assays:

  • Transwell invasion assays demonstrate that BIRC7 knockdown decreases the number of invaded cells, while hypoxia treatment increases invasion

  • Knockdown of BIRC7 under hypoxia blocks the promotion of cell invasion by hypoxia

• Biochemical assays:

  • Single turnover lysine discharge assays to monitor donor Ub transfer

  • BIRC7 autoubiquitination and SMAC ubiquitination assays to assess E3 ligase activity

• Gene expression analysis:

  • RNA-seq to identify downstream targets and pathways affected by BIRC7 modulation

  • qRT-PCR for validation of key target genes

When designing these assays, researchers should ensure appropriate statistical analysis and consider potential cell line-specific effects.

How might BIRC7 serve as a therapeutic target in cancer treatment strategies?

BIRC7's roles in tumor progression and therapy resistance position it as a promising therapeutic target:

• Rationale for targeting BIRC7:

  • Overexpression in multiple cancer types but limited expression in normal tissues

  • Association with malignancy and chemoresistance

  • Key role in inhibiting apoptosis, a hallmark of cancer

  • Involvement in hypoxia response pathways

• Potential targeting approaches:

  • Small molecule inhibitors targeting the BIR domain to disrupt caspase binding

  • SMAC mimetics that competitively bind to BIRC7

  • siRNA or antisense oligonucleotides to reduce BIRC7 expression

  • Inhibition of the RING domain's E3 ligase activity

• Combination therapy potential:

  • Targeting BIRC7 in glioblastoma has been shown to enhance the efficacy of radiotherapy and chemotherapy

  • Potential synergy with HIF-1α inhibitors in hypoxic tumors

• Cancer-specific considerations:

  • In melanoma: BIRC7 knockdown reverses hypoxia-induced proliferation and invasion, suggesting particular value in highly hypoxic melanomas

  • In prostate cancer: BIRC7 upregulation might serve as a biomarker of increased recurrence risk in advanced T stages and medium-grade prostate cancer

These approaches represent promising avenues for translational research targeting BIRC7 in cancer therapy.

What are the key considerations when using BIRC7 expression as a biomarker in clinical research?

When evaluating BIRC7 as a biomarker in clinical research, several methodological considerations are critical:

• Detection methodologies:

  • Immunohistochemistry using anti-BIRC7 antibodies (0.5-1μg/ml concentration is typically used for human tissues)

  • qRT-PCR for mRNA expression analysis (reference genes and primer sequences must be carefully selected)

  • Western blot analysis (0.1-0.5μg/ml antibody concentration for human and rat samples)

• Isoform specificity:

  • Consider whether to measure total BIRC7 or distinguish between α and β isoforms

  • The two splice variants may have different prognostic implications in specific cancer types

• Data interpretation factors:

  • Expression thresholds: Determined based on Youden index for stratifying patients in survival analyses

  • Correlation with clinicopathological parameters: Assessed using χ² tests

  • Survival analysis: Difference between recurrence-free survival curves compared using log-rank test

  • Multivariate analysis: Cox regression models to evaluate independent prognostic value

• Regulatory mechanisms to consider:

  • BIRC7 upregulation is regulated by both copy number alteration and DNA methylation

  • HIF-1α-mediated transcriptional activation in hypoxic environments

• Ethnic considerations:

  • Expression patterns and prognostic significance may vary across different ethnic groups (African-American, White, Asian)

  • Clinical studies should account for potential ethnic variation in biomarker performance