P4HB Human, Active

What is P4HB and what are its primary cellular functions?

P4HB (also known as PDI, PDIA1, PHD, PO4DB, PO4HB, ERBA2L) is a multifunctional and highly abundant enzyme that belongs to the protein disulfide isomerase family . The protein contains two thioredoxin domains that catalyze the formation, breakage, and rearrangement of disulfide bonds, which is crucial for proper protein folding . When present as a tetramer consisting of two alpha and two beta subunits, P4HB participates in the hydroxylation of prolyl residues in preprocollagen .

At the cellular level, P4HB has distinct functions depending on its location. Inside the cell, it primarily forms and rearranges disulfide bonds of nascent proteins, while at the cell surface, it acts as a reductase that cleaves disulfide bonds of proteins attached to the cell . Interestingly, at high concentrations and following phosphorylation by FAM20C, P4HB functions as a chaperone that inhibits aggregation of misfolded proteins, whereas at low concentrations, it may facilitate aggregation (anti-chaperone activity) .

What are optimal storage and handling conditions for active P4HB protein?

For recombinant P4HB research applications, proper storage and handling are crucial to maintain enzymatic activity. The protein should be stored at 4°C if the entire vial will be used within 2-4 weeks. For longer periods, storage at -20°C is recommended .

To maximize stability during long-term storage, researchers should consider adding a carrier protein (0.1% HSA or BSA) to the P4HB solution . Multiple freeze-thaw cycles should be strictly avoided as they can significantly compromise the protein's activity and integrity .

Most commercially available recombinant P4HB is supplied in a formulation containing buffer components such as 20mM Tris-HCl pH-8 and 10% glycerol, which help maintain protein stability . When designing experiments, researchers should account for these buffer components to prevent experimental artifacts.

How can researchers verify P4HB enzymatic activity in experimental settings?

The specific activity of P4HB can be assessed using established enzymatic assays. A standard method involves measuring the aggregation of insulin in the presence of DTT, with properly functioning P4HB showing specific activity > 100 A650/cm/min/mg .

Researchers should also verify protein purity using SDS-PAGE, with commercial preparations typically showing greater than 90% purity . When conducting functional studies, it's important to confirm that the recombinant protein retains its disulfide isomerase activity by using oxidized RNase A refolding assays or reduction assays with di-eosin-GSSG as an alternative approach.

What evidence supports P4HB's role in cancer development and progression?

Recent comprehensive studies have demonstrated P4HB's oncogenic role in tumorigenesis and cancer development across multiple cancer types . Pan-cancer analysis has revealed that P4HB expression significantly correlates with prognosis in several cancer types, including:

• Urinary tumors (adrenocortical carcinoma, kidney renal papillary cell carcinoma, kidney renal clear cell carcinoma, kidney chromophobe, bladder urothelial carcinoma, and prostate adenocarcinoma)

• Lung cancers (lung adenocarcinoma and lung squamous cell carcinoma)

• Gliomas (glioblastoma multiforme, brain lower grade glioma)

• Cervical squamous cell carcinoma and endocervical adenocarcinoma

• Uveal melanoma

In these cancers, differential expression between tumor and normal samples suggests P4HB could serve as a potential biomarker . For example, in breast cancer (BRCA), P4HB expression levels may predict patient prognosis and response to neoadjuvant chemotherapy (NCT) .

What are the key molecular mechanisms through which P4HB contributes to cancer progression?

Mechanistic studies have identified several pathways through which P4HB promotes cancer progression:

• Direct protein interactions: Co-immunoprecipitation (Co-IP) assays have demonstrated that P4HB can directly interact with collagen type X alpha 1 (COL10A1) . In breast cancer, high expression of COL10A1 amplifies the proliferation and metastatic capabilities of cancer cells, while P4HB downregulation suppresses these effects .

• Role in cachexia: In esophageal squamous cell carcinoma (ESCC), extracellular vesicles containing P4HB induce apoptosis in myoblasts by upregulating the expression of cleaved PARP, caspase-3, and caspase-8 . P4HB directly interacts with phosphoglycerate dehydrogenase (PHGDH), with the first domain of P4HB (amino acids 1-138) retaining the interaction capability .

• Drug resistance mechanisms: P4HB overexpression contributes to temozolomide (TMZ) resistance in glioblastoma multiforme (GBM) . Inhibition of P4HB disrupts its protective function and enhances the sensitivity of glioma cells to TMZ by activating the protein kinase R-like endoplasmic reticulum kinase (PERK) arm of the ER stress response .

What approaches can researchers use to effectively manipulate P4HB expression?

To study P4HB function through expression modulation, researchers can employ several strategies:

• siRNA/shRNA knockdown: RNA interference has been successfully used to reduce P4HB expression in various cancer cell lines. Studies have shown that knockdown of P4HB increases apoptosis in human HT29 cells .

• CRISPR-Cas9 genome editing: For complete knockout or targeted modifications of the P4HB gene, CRISPR-Cas9 approaches provide precise editing capabilities.

• Overexpression systems: Plasmid-based overexpression of wild-type or mutant P4HB can be used to study gain-of-function effects. Evidence shows that upregulation of P4HB enhances breast cancer cell proliferation, clone-forming capacity, migration, and invasion .

• Domain-specific constructs: Researchers can express specific domains of P4HB (e.g., the first domain spanning amino acids 1-138) to study domain-specific functions, such as interaction with PHGDH .

When implementing these approaches, researchers should validate manipulation efficiency through Western blotting, qRT-PCR, and functional assays specific to P4HB activity.

How can researchers investigate P4HB interactions with other proteins?

To study protein-protein interactions involving P4HB, researchers can employ multiple complementary techniques:

• Co-immunoprecipitation (Co-IP): Successfully used to demonstrate direct interactions between P4HB and proteins such as COL10A1 and PHGDH .

• Glutathione S-transferase (GST) affinity isolation: This approach has been used alongside Co-IP to confirm direct protein interactions with P4HB .

• Domain mapping: Expression of truncated P4HB constructs allows identification of specific interaction domains, as demonstrated in studies showing that the redox-active domain a (amino acids 1-138) facilitates interaction with PHGDH .

• Proximity labeling: Techniques such as BioID or APEX2 can be used to identify proteins in close proximity to P4HB in living cells.

• Surface plasmon resonance (SPR): For quantitative measurement of binding affinities between P4HB and potential interacting partners.

What is the evidence for P4HB as a therapeutic target in cancer?

Emerging evidence supports P4HB as a promising therapeutic target, particularly in cancer treatment:

• Drug resistance modulation: Inhibition or knockdown of P4HB can sensitize previously resistant cancer cells to therapies. In glioblastoma, P4HB inhibition enhanced sensitivity to temozolomide (TMZ) by activating the PERK arm of the ER stress response .

• MicroRNA regulation: miRNA-210 has been shown to downregulate P4HB expression, resulting in reduced TMZ resistance in glioma cells . This suggests microRNA-based approaches could be developed to modulate P4HB expression therapeutically.

• Drug discovery potential: Computational prediction models have identified potential drugs targeting P4HB, which could lead to new therapeutic opportunities for cancer patients .

What methodologies are available for screening P4HB inhibitors?

When developing or screening for P4HB inhibitors, researchers can employ several approaches:

• Enzymatic activity assays: High-throughput screening using insulin aggregation assays in the presence of DTT, with activity measured spectrophotometrically (A650) .

• Cellular assays: Evaluation of compounds in cell-based systems that report on disulfide bond formation or ER stress responses.

• Drug sensitivity analysis: Assessment of how P4HB expression levels correlate with sensitivity to various compounds across cancer cell lines.

• In silico screening: Computational approaches to identify compounds that may bind to and inhibit P4HB based on its structure.

• Target engagement assays: Cellular thermal shift assays (CETSA) or related methods to confirm direct binding of compounds to P4HB in cells.