TRIAP1 Human

What is TRIAP1 and what is its evolutionary significance?

TRIAP1 (TP53-regulated inhibitor of apoptosis 1, also known as p53CSV for p53-inducible cell survival factor) is the human homolog of yeast Mdm35, a well-characterized chaperone protein. It represents an evolutionarily conserved protein family extending from yeast to humans . The conservation of this protein throughout evolutionary history indicates its fundamental importance in cellular function. TRIAP1 is part of a unique heterodimeric lipid transfer system that has been maintained throughout eukaryotic evolution, suggesting its critical role in mitochondrial function and cellular homeostasis .

What is the basic structure of TRIAP1?

Structural analyses using X-ray crystallography and NMR spectroscopy have revealed that TRIAP1 adopts a helical coiled-coil structure with twin CX₉C motifs. The N-terminal region forms a structured domain while the C-terminal region (after residue K53) exhibits significant flexibility and is highly disordered, as evidenced by NMR data showing elevated transverse ¹⁵N relaxation times and narrow linewidths characteristic of dynamic regions . Crystal structures of TRIAP1 have been solved at a resolution of 2.12 Å in space group P2₁ with two molecules per asymmetric unit . This structural information provides crucial insights into how TRIAP1 functions as a chaperone in mitochondrial lipid transfer.

What are the primary cellular functions of TRIAP1?

TRIAP1 functions primarily as a chaperone that interacts with members of the PRELI family (PRELID1, PRELID3A, and PRELID3B) to facilitate the intramitochondrial transfer of phospholipids, particularly phosphatidic acid (PA) and phosphatidylserine (PS) . This lipid transfer activity is essential for the synthesis of cardiolipin (CL) and phosphatidylethanolamine in the inner mitochondrial membrane. TRIAP1 contributes significantly to maintaining cardiolipin levels, which in turn helps sequester cytochrome c within the mitochondria . Beyond its mitochondrial lipid trafficking role, TRIAP1 has been identified as a p53-responsive antiapoptotic protein that is induced in response to sublethal genotoxic stress and can function as a p53 antagonist by inhibiting the expression of the cell-cycle regulator p21 .

How does TRIAP1 interact with its binding partners at the molecular level?

The crystal structure of the TRIAP1-SLMO1 complex, determined at 3.58 Å resolution, reveals an intimate interaction between a hydrophobic stripe on the TRIAP1 chaperone and the PRELI-like domain of SLMO1 . The TRIAP1-binding region on SLMO1 spans residues P13 to L39, encompassing the edge β2 strand, an ordered proline-rich loop, and the α1-helix. Key interactions include:

• A cluster of three phenylalanine residues (F19, F23, and F28) at the tip of the TRIAP1 coiled-coil structure interacts with a hydrophobic cluster formed by M22, V38, and L49 on SLMO1.

• A second hydrophobic patch comprises V33, L34, and V36 from SLMO1 together with F41, Y44, V48, and I52 from the other end of the hydrophobic strip on TRIAP1 .

Site-directed mutagenesis and pull-down assays have confirmed the importance of these residues. Mutations of V36 or L49 in SLMO1 to alanine prevented complex formation, while mutations of neighboring V38 and a double mutant of V33A/L34A did not disrupt the complex. The double mutant TRIAP1-F41A/SLMO1-V38A abrogated complex formation, highlighting the critical nature of these hydrophobic interactions in stabilizing the complex .

What are the crystallographic parameters of TRIAP1 and its complexes?

The following table summarizes the crystallographic parameters of TRIAP1 and the TRIAP1-SLMO1 complex:

This crystallographic data has been crucial for understanding the structural basis of TRIAP1 function and interactions .

How does the structure of TRIAP1 relate to other lipid transfer proteins?

The PRELI-like domain of SLMO1, with which TRIAP1 interacts, exhibits structural resemblance to mammalian phosphatidylinositol transfer proteins (PITPs). This structural similarity suggests they share comparable lipid transfer mechanisms . In both cases, access to a buried phospholipid-binding cavity appears to be regulated by conformationally adaptable loops. The 3D models of the Ups1-Mdm35 (yeast homologs) and TRIAP1-PRELID1 complexes have been constructed using protein structure threading programs and refined independently, providing insights into the evolutionary conservation of their lipid transfer mechanisms . This structural homology underscores the fundamental importance of these protein families in maintaining proper lipid distribution within cellular compartments.

How does TRIAP1 contribute to cancer cell proliferation?

Studies in colorectal cancer cell models have demonstrated that TRIAP1 supports cancer cell proliferation and tumorigenesis . Experimental evidence shows that:

• Knockdown of TRIAP1 in HCT116 colorectal cancer cells increased their doubling time and compromised their capacity to clone at low density .

• TRIAP1 depletion decreased the proliferation rate of cancer cells, with the negative effect on cell proliferation being most visible at low cell density .

• While TRIAP1 depletion did not cause cell death, it significantly affected growth parameters, suggesting its importance in sustaining the rapid proliferation characteristic of cancer cells .

These findings indicate that TRIAP1 provides metabolic and proliferative advantages to cancer cells, potentially through its role in mitochondrial lipid trafficking and its interaction with p53-regulated pathways .

What is the relationship between TRIAP1 and p53 signaling in cancer?

TRIAP1 has a complex relationship with p53 signaling that appears to be significant in cancer:

• TRIAP1 was initially described as a p53-responsive antiapoptotic protein that is induced in response to sublethal genotoxic stress .

• It also functions as a p53 antagonist by inhibiting the expression of the cell-cycle regulator p21 .

• High levels of TRIAP1 have been reported in various types of cancer, suggesting that its overexpression may provide a survival advantage to cancer cells by modulating p53-mediated surveillance pathways .

The TRIAP1/p53 axis appears to be interconnected with mitochondrial lipid trafficking activity, though the exact mechanisms linking these functions remain an active area of investigation . Understanding this relationship is crucial for deciphering the advantage that TRIAP1 expression provides to cancer cells and may offer new therapeutic opportunities.

How does TRIAP1 affect mitochondrial structure and function in cancer cells?

Research has shown that depletion of TRIAP1 perturbs mitochondrial ultrastructure in cancer cells, though without a major impact on cardiolipin levels and mitochondrial activity . In HeLa cells, knockdown of either TRIAP1 or PRELID1 has been reported to partially reduce cardiolipin levels, favor the release of cytochrome c, and increase the vulnerability of the cells to apoptosis .

Quantitative analysis of mitochondrial parameters following TRIAP1 silencing has been performed using transmission electron microscopy (TEM) at ×2,900 magnification. Parameters evaluated include mitochondria number, size, perimeter, mitochondria-to-cytoplasm area ratio, and aspect factor (major axis/minor axis ratio), which reflects the length-to-width ratio of mitochondria . These studies provide important insights into how TRIAP1 influences mitochondrial morphology and potentially contributes to the altered mitochondrial dynamics observed in cancer cells.

What are the recommended techniques for studying TRIAP1-protein interactions?

Several complementary techniques have proven valuable for investigating TRIAP1 interactions:

• X-ray Crystallography: This technique has been successfully employed to solve the structures of free TRIAP1 and the TRIAP1-SLMO1 complex, providing atomic-level insight into their interaction . Data collection at synchrotron facilities (e.g., Diamond Light Source) followed by processing with software packages such as XDS, SCALA, and PHASER has yielded high-quality structural information .

• NMR Spectroscopy: Full-length TRIAP1 (300 μl) in 50 mM sodium phosphate pH 6.5, 50 mM NaCl, and 10% D₂O has been used to record standard triple resonance experiments (HNCACB, CBCACONH, HNCO, HN(CA)CO) on a Bruker 600 spectrometer equipped with TXI cryoprobe at 303 K . This approach has achieved approximately 95% backbone assignments and provided valuable information on protein dynamics through ¹⁵N T₂ relaxation measurements .

• Pull-down Assays: These assays have been effectively used to assess the formation of TRIAP1-SLMO1 complexes, particularly when combined with site-directed mutagenesis to evaluate the contribution of specific residues to the interaction. In these experiments, the complex can be retrieved via a His-tag on SLMO1 while TRIAP1 remains untagged .

• Protein Structure Threading: Programs such as PHYRE have been used to construct 3D models of protein complexes, which can then be refined with tools like ModRefiner. This approach has been applied to model the Ups1-Mdm35 and TRIAP1-PRELID1 complexes .

What methods are most effective for studying TRIAP1 in cancer cell models?

Research on TRIAP1 in cancer cell models has employed several effective methodologies:

• RNA Interference: Knockdown of TRIAP1 using distinct interfering RNAs (shRNAs) has been instrumental in assessing its functional significance in cancer cells. This approach has revealed effects on cell doubling time, clonogenic capacity, and proliferation rate .

• Proliferation Assays: Assessing the impact of TRIAP1 depletion on cancer cell growth at different cell densities has provided insights into its role in proliferation. These assays have shown that the negative effect of TRIAP1 depletion on cell proliferation is most evident at low cell density .

• Transmission Electron Microscopy (TEM): TEM at ×2,900 magnification has been used to analyze the impact of TRIAP1 silencing on mitochondrial morphology. Manual segmentation of plasma membranes, mitochondria, and nuclei using software like IMOD enables the extraction of quantitative parameters such as mitochondria number, size, and perimeter .

• Western Blotting: For whole-extract analysis, cells can be washed with PBS, lysed with 1% SDS, boiled, sonicated, and stored at -80°C. Proteins in the lysates are quantified using assays such as the DC Protein Assay (Bio-Rad), resolved by SDS-PAGE, and subjected to immunoblot analyses .

• Statistical Analysis: Non-parametric Mann-Whitney tests have been employed for statistical comparisons of mitochondrial parameters, with p < 0.05 considered statistically significant .

How can researchers effectively express and purify TRIAP1 for structural studies?

Based on published methodologies, the following approach is recommended for expressing and purifying TRIAP1 for structural studies:

• Construct Design: Design expression constructs that include appropriate tags for purification. For instance, MBP-TRIAP1 fusions have been successfully used for crystallization studies .

• Expression Systems: Express the protein in suitable bacterial systems, optimizing conditions for high yield and proper folding.

• Purification Strategy: Implement a multi-step purification protocol that may include affinity chromatography, ion exchange chromatography, and size exclusion chromatography to obtain highly pure protein.

• Complex Formation: For studies of TRIAP1 in complex with its binding partners (e.g., SLMO1 or PRELID proteins), co-express or mix the purified proteins and isolate the complex using appropriate techniques.

• Quality Control: Verify the purity and integrity of the protein using SDS-PAGE, mass spectrometry, and other analytical techniques before proceeding with structural studies.

• Crystallization: For crystallography, screen various conditions to identify those that yield diffraction-quality crystals. For the TRIAP1-SLMO1 complex, crystals have been obtained in 100 mM sodium formate pH 7.0, 15% (w/v) PEG 3350, and 25% (w/v) PEG 200 .

• NMR Sample Preparation: For NMR studies, prepare isotopically labeled samples (¹⁵N, ¹³C) in suitable buffer conditions, such as 50 mM sodium phosphate pH 6.5, 50 mM NaCl, and 10% D₂O .

What are the current limitations in understanding TRIAP1 function?

Despite significant advances, several challenges remain in fully understanding TRIAP1 function:

• Physiological Relevance: While structural analyses and in vitro assays have demonstrated TRIAP1's interaction with PRELI family members and its lipid transfer capabilities, understanding the regulation and physiological relevance of its mitochondrial action in human cells is still in its infancy .

• Cancer Mechanism: The precise molecular mechanisms by which TRIAP1 overexpression contributes to cancer progression remain incompletely understood. It is particularly important to determine whether the advantage provided to cancer cells is primarily related to TRIAP1's mitochondrial lipid trafficking activity or to its interaction with p53-regulated surveillance pathways .

• Integrated Function: The integration of TRIAP1's various roles—as a mitochondrial lipid transfer chaperone, an antiapoptotic factor, and a p53 antagonist—into a cohesive functional model remains a challenge.

• Therapeutic Targeting: Given TRIAP1's involvement in cancer, developing strategies to target it therapeutically represents both an opportunity and a challenge that requires deeper understanding of its structure-function relationships.

What are the most promising directions for future TRIAP1 research?

Based on current knowledge, several promising research directions emerge:

• Detailed Mechanism of Lipid Transfer: Further investigation into the precise mechanisms by which TRIAP1-PRELI complexes facilitate lipid transfer across mitochondrial membranes, possibly utilizing advanced biophysical techniques and real-time imaging of lipid movement.

• Tissue-Specific Functions: Exploration of potential tissue-specific functions of TRIAP1 and its differential expression or regulation across various cell types and tissues.

• Post-translational Modifications: Investigation of post-translational modifications that might regulate TRIAP1 function and their potential dysregulation in disease states.

• Therapeutic Development: Given its role in cancer cell proliferation, research aimed at developing small molecule inhibitors or peptide mimetics that could disrupt TRIAP1's pro-survival functions selectively in cancer cells.

• Systems Biology Approach: Integration of TRIAP1 function into broader cellular networks, particularly those involving mitochondrial dynamics, lipid metabolism, and stress response pathways.

• Genetic Models: Development of genetic models (e.g., conditional knockout mice) to better understand the systemic consequences of TRIAP1 modulation in vivo.