Recombinant Human Plasminogen activator inhibitor 2 protein (SERPINB2) (Active)

What is SERPINB2 and what are its primary physiological functions?

SERPINB2 (Plasminogen Activator Inhibitor 2) is a member of the clade B or ovalbumin-like serine protease inhibitor (ov-serpin) subgroup of the serpin superfamily. It functions primarily as an inhibitor of urokinase plasminogen activator (uPA) and, to a lesser extent, tissue plasminogen activator (tPA) . These plasminogen activators convert plasminogen to the active protease plasmin, which has diverse physiological and pathological activities.

SERPINB2 exhibits high expression in blood, vasculature, and placenta, with upregulation during pregnancy and in activated monocytes/macrophages following exposure to various viral, bacterial, and parasitic agents . Functionally, SERPINB2 plays critical roles in:

• Regulating extracellular matrix degradation through inhibition of the plasminogen activation system

• Modulating inflammatory responses in macrophages

• Inhibiting cell migration

• Promoting cellular senescence

• Acting as a stress response protein in various toxic exposures

How does SERPINB2 expression vary across different cell types?

Expression is particularly high in tissues like placenta, blood vessels, and immune cells. Monocytes from HIV-1 infected patients show upregulated SERPINB2, suggesting it plays a role in modulating Th1/Th2 responses during infection . In stem cells, SERPINB2 expression increases dramatically in response to various toxic agents, indicating its role as a stress-response gene .

What is the molecular mechanism by which SERPINB2 inhibits its target proteases?

SERPINB2 inhibits serine proteases through the classical serpin inhibitory mechanism. The process involves:

• Initial binding of the target protease (primarily uPA) to the reactive center loop (RCL) of SERPINB2

• Cleavage of the RCL by the protease

• Rapid conformational change in SERPINB2 that distorts the protease active site

• Formation of a covalent complex between SERPINB2 and the protease

• Inactivation of the protease within this complex

The arginine at position 380 (R380) in SERPINB2 is critical for this inhibitory function. Mutation of this residue (R380A) produces a non-inhibitory variant that still binds the target protease but cannot complete the inhibitory mechanism . This mutation has been valuable in experimental designs to distinguish between SERPINB2's inhibitory and non-inhibitory functions.

How does SERPINB2 regulate cell migration, and what experimental models best demonstrate this function?

SERPINB2 serves as a negative regulator of cell migration through multiple mechanisms:

• Inhibition of uPA-mediated plasmin generation, which normally facilitates ECM degradation

• Association with cytoskeletal structures at focal adhesions and lamellipodia

• Counter-regulation of Gata6-regulated genes associated with migration

In experimental models, large peritoneal macrophages (LPM) from SerpinB2−/− and SerpinB2 R380A (active site mutant) mice demonstrate significantly faster migration on Matrigel compared to wild-type controls . Confocal microscopy reveals that SerpinB2 and F-actin staining overlap in focal adhesions and lamellipodia, suggesting direct interaction with migration machinery .

For researchers investigating this phenomenon, the following methodology is recommended:

• Compare migration rates of wild-type cells versus SERPINB2-knockout or R380A mutant cells

• Use Matrigel or similar ECM components as substrate

• Employ real-time cell tracking with time-lapse microscopy

• Conduct RNA-Seq analysis focusing on genes associated with migration and ECM interactions

• Perform gene set enrichment analyses (GSEA) to identify pathways regulated by SERPINB2

What is the role of SERPINB2 in cellular senescence, and how does it interact with the p21 pathway?

SERPINB2 functions as both a marker and mediator of cellular senescence. Its role in senescence involves:

• Direct binding to and stabilization of p21 in a proteasome-independent manner

• Acting as a downstream target of p53 activated by the DNA damage response pathway

• Induction of senescence when overexpressed in proliferating human fibroblasts

Notably, SERPINB2-induced senescence occurs through intracellular mechanisms rather than its extracellular function. This has been demonstrated through experiments showing that inhibition of SERPINB2 secretion, exogenous introduction of SERPINB2, or use of a SERPINB2 mutant that cannot bind uPA did not affect senescence induction .

To study this interaction experimentally:

• Use co-immunoprecipitation to detect SERPINB2-p21 binding

• Employ Western blotting to monitor p21 stability in the presence/absence of SERPINB2

• Utilize senescence markers such as SA-β-galactosidase staining

• Compare the effects of wild-type SERPINB2 versus secretion-deficient mutants

• Analyze p53 activation in relation to SERPINB2 expression

How can SERPINB2 expression patterns be utilized as a biomarker for stem cell toxicity?

SERPINB2 has emerged as a novel indicator of stem cell toxicity. Research has demonstrated that:

• SERPINB2 expression is significantly increased in response to various toxic agents in human stem cells both in vitro and in vivo

• There is a strong correlation between increased SERPINB2 expression and toxicity-related inflammatory diseases

• SERPINB2 expression negatively correlates with stem cell differentiation potential

• SERPINB2 can serve as a "universal" marker for predicting toxic responses to various types of toxic materials

For toxicity screening applications, researchers should:

• Monitor SERPINB2 mRNA expression via real-time PCR after exposure to test compounds

• Validate at the protein level using Western blotting

• Compare SERPINB2 induction across different concentrations of test compounds

• Correlate SERPINB2 expression with functional stem cell parameters (proliferation, differentiation, migration)

• Consider using SERPINB2 knockdown models as controls to confirm specificity

What are the optimal conditions for working with recombinant human SERPINB2 protein in functional assays?

When designing experiments with recombinant human SERPINB2:

Storage and Stability:

• Store lyophilized protein at -20°C to -80°C

• After reconstitution, aliquot and store at -80°C

• Avoid repeated freeze-thaw cycles

• Typical working concentrations range from 10-100 ng/mL

• For optimal activity, use within 6 months of reconstitution

Buffer Conditions:

• Reconstitute in PBS or similar physiological buffer

• Optimal pH range: 7.0-7.4

• Add 0.1% BSA as a carrier protein for dilute solutions

• For protease inhibition assays, physiological concentrations of calcium (2mM) may be required

Activity Considerations:

• Full inhibitory activity requires proper folding of the reactive center loop

• Consider using fluorogenic substrates for uPA/tPA to monitor inhibition kinetics

• Second-order rate constants for inhibition: uPA (105-106 M-1s-1), tPA (104-105 M-1s-1)

• Activity may be affected by oxidizing conditions

What are the recommended approaches for studying SERPINB2-mediated effects on cell migration?

When investigating SERPINB2's role in cell migration:

Experimental Design:

• Compare migration of cells with varying SERPINB2 expression levels:

  • Wild-type cells

  • SERPINB2 knockout cells

  • SERPINB2 R380A mutant (non-inhibitory) expressing cells

  • SERPINB2 overexpressing cells

• Migration assay options:

  • Transwell migration assays (8 μm pore size recommended)

  • Wound healing/scratch assays

  • Single-cell tracking via time-lapse microscopy

  • 3D invasion assays in Matrigel or collagen matrices

• Visualization techniques:

  • Immunofluorescence to co-localize SERPINB2 with F-actin and focal adhesion proteins

  • Live-cell imaging with fluorescently tagged SERPINB2

• Molecular analysis:

  • RNA-Seq or microarray analysis to identify co-regulated genes

  • Western blotting for key migration proteins (MMP-2, integrins, focal adhesion kinase)

  • Activity assays for uPA and plasmin to correlate with migration phenotypes

Research has shown that confocal microscopy reveals SERPINB2 and F-actin staining overlap in focal adhesions and lamellipodia, providing insight into its mechanism of action . RNA-Seq analysis of migrating resident peritoneal macrophages from wild-type and SERPINB2 R380A mice identifies genes associated with migration and extracellular matrix interactions .

What techniques are most effective for analyzing SERPINB2-p21 interactions in senescence studies?

To investigate SERPINB2's role in stabilizing p21 during senescence:

Protein-Protein Interaction Assays:

• Co-immunoprecipitation (Co-IP)

  • Pull down with anti-SERPINB2 antibody and probe for p21

  • Reverse Co-IP with anti-p21 antibody

  • Include appropriate controls (IgG, lysates from SERPINB2-knockout cells)

• Proximity ligation assay (PLA)

  • Enables visualization of protein interactions in situ

  • Provides spatial information on where interactions occur within cells

• FRET (Förster Resonance Energy Transfer)

  • Tag SERPINB2 and p21 with appropriate fluorophore pairs

  • Measure energy transfer as indicator of close proximity

Protein Stability Assays:

• Cycloheximide chase assay

  • Treat cells with cycloheximide to inhibit new protein synthesis

  • Monitor p21 degradation rate in presence/absence of SERPINB2

  • Western blot at multiple time points (0, 1, 2, 4, 8 hours)

• Proteasome inhibition studies

  • Compare p21 levels with/without proteasome inhibitors (MG132)

  • Determine if SERPINB2 protection is proteasome-dependent

Senescence Markers:

• SA-β-galactosidase staining

• Analysis of senescence-associated secretory phenotype (SASP)

• Cell cycle analysis (G1 arrest)

• DNA damage foci (γH2AX)

Research has shown that SERPINB2 binds to and stabilizes p21 in a proteasome-independent manner, suggesting a direct role in maintaining senescence .

How does SERPINB2 overexpression affect stem cell properties and what mechanisms underlie these effects?

SERPINB2 overexpression significantly impacts stem cell functions through multiple mechanisms:

Effects on Stem Cell Properties:

Molecular Mechanisms:

• Increased activation of apoptotic pathways

  • Elevated levels of cleaved caspase-3 and PARP

  • Enhanced DNA fragmentation

• Altered migration machinery

  • Significantly decreased MMP-2 expression

  • Possible effects on cytoskeletal reorganization

• Changes in differentiation pathways

  • Dysregulation of lineage-specific transcription factors

  • Altered response to differentiation stimuli

Research demonstrates that SERPINB2 knockdown produces opposite effects to overexpression, confirming its specific role in regulating these stem cell properties . These findings indicate that SERPINB2 functions as a negative regulator of stem cell self-renewal, migration, and differentiation potential.

What methodologies are recommended for using SERPINB2 as a biomarker for stem cell toxicity?

To utilize SERPINB2 as a toxicity biomarker in stem cell research:

Experimental Workflow:

• Exposure protocol:

  • Treat stem cells with test compounds at various concentrations

  • Include positive controls (known toxic compounds)

  • Maintain appropriate exposure duration (24-72 hours)

  • Consider both acute and chronic exposure models

• SERPINB2 expression analysis:

  • qRT-PCR for mRNA quantification (primary screening)

  • Western blotting for protein validation

  • Immunofluorescence for cellular localization

  • Flow cytometry for population-level analysis

• Correlation with functional parameters:

  • Cell viability/proliferation assays

  • Apoptosis markers (Annexin V, cleaved caspase-3)

  • Migration capability (transwell assays)

  • Differentiation potential (lineage-specific markers)

• In vivo validation:

  • Administer test compounds to animal models

  • Isolate tissue-specific stem cells (e.g., adipose-derived)

  • Analyze SERPINB2 expression ex vivo

  • Correlate with functional impairment in vivo

Research has validated that SERPINB2 expression is significantly increased in response to various toxic agents in human stem cells both in vitro and in vivo . Moreover, there is a strong correlation between increased SERPINB2 expression and toxicity-related inflammatory diseases or decreased differentiation potential, making it a valuable biomarker for toxicity prediction .

What are the most promising research directions for SERPINB2 in therapeutic applications?

Current evidence suggests several promising avenues for SERPINB2 research:

Cancer Research:

• SERPINB2 inhibition of uPA in tumors associates with favorable prognosis

• Potential for developing SERPINB2-based therapeutics targeting cancer cell migration

• Investigation of SERPINB2's role in modulating tumor microenvironment

Inflammation and Immunity:

• SERPINB2's role in modulating Th1/Th2 responses during infection

• Potential therapeutic applications in autoimmune diseases

• Development of anti-inflammatory strategies targeting SERPINB2 pathways

Senescence Modulation:

• Targeting SERPINB2-p21 interaction for senolytic approaches

• Exploring SERPINB2's role in age-related diseases

• Developing strategies to modulate cellular senescence through SERPINB2

Stem Cell Technologies:

• Using SERPINB2 as a biomarker for screening compound toxicity

• Developing methods to maintain stem cell function by modulating SERPINB2

• Exploring SERPINB2 knockdown approaches to enhance stem cell regenerative potential

Neurodevelopment:

• SERPINB2 colocalizes with CHL1 and Vitronectin and mediates neurite outgrowth during post-natal brain development

• Potential applications in neural regeneration and repair