Recombinant Human Insulin-like growth factor-binding protein 5 protein (IGFBP5) (Active)

What is the structural organization of IGFBP5 and how does it relate to its function?

IGFBP5 consists of three distinct structural domains, each with specific functional properties:

N-terminal domain (N-domain): A highly conserved globular structure containing 12 cysteine residues and a hydrophobic patch critical for IGF binding. This domain forms part of the high-affinity IGF-binding site and can independently bind IGF with substantial affinity .

Central linker domain (L-domain): A non-conserved flexible region containing multiple proteolytic cleavage sites, phosphorylation sites, and O-glycosylation sites that regulate IGFBP5 activity. This domain undergoes post-translational modifications that can significantly alter IGFBP5 function .

C-terminal domain (C-domain): A conserved globular domain containing six cysteine residues and an arginine-lysine rich (RK-rich) sequence that contributes to IGF binding, nuclear localization, and interaction with acid-labile subunit (ALS). This domain also contains binding sites for extracellular matrix (ECM) components and heparin .

The functional integrity of IGFBP5 depends on the coordinated interaction between these domains. Specific mutations or truncations can significantly alter its biological activities, as demonstrated by studies showing that fragments of IGFBP5 retain certain bioactivities while losing others .

How does IGFBP5 regulate IGF signaling in cellular systems?

IGFBP5 regulates IGF signaling through multiple mechanisms that either inhibit or potentiate IGF activities:

Inhibitory actions:

• Forms high-affinity complexes with IGFs in circulation and tissues, sequestering them from interaction with IGF1R

• Creates binary complexes with IGF or ternary complexes with IGF and acid-labile subunit (ALS) in the bloodstream to extend IGF half-life

• Acts as a major mediator of mTORC1-dependent feedback inhibition of IGF1 signaling

Potentiating actions:

• Releases bound IGF upon interaction with ECM components or cell surface molecules

• Undergoes proteolytic cleavage that lowers its affinity for IGF, increasing local IGF bioavailability

• Stores IGF in tissues for controlled release upon appropriate stimuli

Importantly, the IGFBP5:IGF ratio is a critical determinant of whether IGFBP5 inhibits or potentiates IGF action. At high IGFBP5:IGF ratios, inhibitory effects predominate, while at lower ratios or in specific tissue contexts, potentiating effects may occur .

What are the known IGF-independent functions of IGFBP5?

IGFBP5 exhibits several important IGF-independent functions through multiple mechanisms:

Cell surface interactions:

• Binds to putative membrane receptors to initiate intracellular signaling cascades

• Interacts with tumor necrosis factor receptor 1 (TNF1R), inhibiting TNFα-dependent NFκB activation in lymphoma cells but supporting TNFα activity in MDA-MB-231 breast cancer cells

• Binds α2/β1 integrins to enhance cell-ECM interactions through activation of GTPase Cdc42 and AKT, while inactivating p38 stress-response kinase

Nuclear functions:

• Translocates to the nucleus through its nuclear localization signal in the C-domain

• Interacts with nuclear co-factors to regulate transcription and other nuclear processes

ECM interactions:

• Binds to ECM components including collagen types 3 and 4, laminin, fibronectin, thrombospondin, and osteopontin

• The C-terminal heparin-binding domain (HBD) stimulates mesangial cell migration in an IGF-independent manner

Cell signaling pathway modulation:

• Regulates ERK1/2 and p38 MAPK pathways in human intestinal smooth muscle cells

• Decreases VEGF-A and MMP-9 expression, inhibits Akt and ERK phosphorylation, and reduces NF-kB activity in ovarian cancer models

• Acts as a growth factor that regulates bone formation independently of IGFs

These IGF-independent functions demonstrate IGFBP5's role as a multifunctional signaling molecule beyond its canonical IGF-binding activities .

What are the optimal methods for producing and purifying active recombinant IGFBP5?

Production and purification of active recombinant IGFBP5 involves several key methodological considerations:

Expression systems:

• Escherichia coli is the most common expression system for IGFBP5 production, yielding protein with >95% purity suitable for various applications including SDS-PAGE, functional studies, Western blotting, and inhibition assays

• Mammalian expression systems can be used when post-translational modifications are critical for the specific application

Purification strategy:

• Initial isolation via affinity chromatography (often using His-tag or other fusion tags)

• Secondary purification steps typically include ion-exchange chromatography

• Final polishing via size exclusion chromatography to ensure homogeneity

Quality control measures:

• Verification of molecular weight (approximately 28.6 kDa for the full-length 253 amino acid human protein)

• Confirmation of purity via SDS-PAGE under reducing and non-reducing conditions

• Functional validation through IGF binding assays

• Assessment of secondary structure integrity via circular dichroism spectroscopy

Storage considerations:

• For long-term stability, lyophilized protein should be stored at -20°C to -80°C

• Reconstituted protein should be used promptly or aliquoted and stored at appropriate temperatures according to the specific Certificate of Analysis

When designing recombinant IGFBP5 constructs, researchers should carefully consider which domains are necessary for their specific application, as truncation mutants containing specific domains may retain certain biological functions while losing others .

How can researchers effectively measure IGFBP5-IGF interactions in experimental settings?

Several complementary methodologies can be employed to measure IGFBP5-IGF interactions with varying levels of sensitivity and informational output:

Surface plasmon resonance (SPR):

• Provides real-time, label-free measurements of binding kinetics (kon and koff rates)

• Enables determination of binding affinity (KD values)

• Can detect conformational changes upon binding

• Requires immobilization of either IGFBP5 or IGF on a sensor chip

Isothermal titration calorimetry (ITC):

• Measures thermodynamic parameters of binding (ΔH, ΔS, ΔG)

• Provides stoichiometry information

• Works in solution without requiring protein modification

Biosensor real-time analysis:

• Particularly useful when combined with heparin ligand blotting to study how proteolytic cleavage modifies heparin-binding domain interactions

IGF binding assays with radiolabeled ligands:

• Traditional approach using 125I-labeled IGFs

• Competitive binding assays can determine relative affinities

• Can be performed with full-length IGFBP5 or domain fragments to map binding regions

Functional cellular assays:

• Measuring IGF-dependent cellular responses (e.g., proliferation, AKT phosphorylation) in the presence of IGFBP5

• Comparing wild-type IGFBP5 with mutants deficient in IGF binding

When interpreting results, researchers should consider that binding dynamics can be significantly affected by:

• Post-translational modifications of IGFBP5

• The presence of ECM components or heparin (which can reduce IGF binding affinity by up to 17-fold)

• Proteolytic processing of IGFBP5

• The specific experimental conditions including pH, temperature, and ionic strength

What in vitro and in vivo models are most appropriate for studying IGFBP5 functions?

Researchers can utilize various models to study IGFBP5 functions, each with specific advantages for investigating different aspects of IGFBP5 biology:

In vitro cellular models:

In vivo models:

• Transgenic mouse models:

  • Heart-specific IGFBP5 knockdown mice show inhibited myocardial apoptosis and increased cardiomyocyte proliferation after myocardial infarction

  • Global IGFBP5 overexpression in mice leads to severe body growth reduction, neonatal mortality, reduced fertility, and decreased skeletal muscle weight

  • IGFBP5 knockout mice show no obvious growth phenotype, suggesting functional compensation by other IGFBPs

• Xenograft models:

  • Ovarian cancer xenografts demonstrate that the C-terminal region of IGFBP5 significantly decreases tumor growth

  • Patient-derived xenografts provide a clinically relevant system to study IGFBP5 effects on tumor angiogenesis

• Ex vivo systems:

  • Aortic ring assays for studying IGFBP5 effects on angiogenesis

  • Isolated organ cultures for tissue-specific responses

• Disease models:

  • Myocardial infarction models reveal IGFBP5's role in cardiac repair and remodeling

  • Ischemic and hypoxic injury models demonstrate increased IGFBP5 expression

When selecting a model system, researchers should consider the specific aspect of IGFBP5 biology they aim to investigate, whether it's tissue-specific functions, IGF-dependent vs. independent actions, or disease-relevant processes.

How does IGFBP5 contribute to cardiac repair following myocardial infarction?

IGFBP5 plays complex roles in cardiac repair following myocardial infarction (MI), affecting multiple aspects of the repair process:

Expression pattern in cardiac injury:

• IGFBP5 shows high expression in multiple models of ischemic and hypoxic cardiac injury

• Expression changes during different phases of the repair process, suggesting stage-specific functions

Dual roles in cardiomyocyte survival and death:

• IGFBP5 affects proliferation of neonatal rat cardiomyocytes (NRCMs)

• Modulates cardiomyocyte apoptosis induced by oxygen-glucose deprivation (OGD)

• Heart-specific IGFBP5 knockdown inhibits myocardial apoptosis and increases cardiomyocyte proliferation in mice with MI

Mechanistic pathway:

• IGFBP5 regulates cardiomyocyte survival through the insulin-like growth factor 1 receptor (IGF1R)/protein kinase B (PKB/AKT) pathway

• During chronic remodeling stages, heart-specific regulation of IGFBP5 ameliorates pathological cardiac remodeling and dysfunction

Therapeutic implications:

• IGFBP5 represents a potential therapeutic target for myocardial ischemic injury

• Modulation of IGFBP5 during specific repair phases may improve cardiac outcomes

• The timing of intervention appears critical, as IGFBP5 functions may differ between acute and chronic phases

These findings indicate that targeted manipulation of IGFBP5 expression or activity could be a promising approach for improving cardiac repair following MI, but the complexity of its functions requires careful consideration of the timing and extent of intervention .

What is the role of IGFBP5 in cancer progression and how does it vary across different cancer types?

IGFBP5 exhibits context-dependent roles in cancer progression that vary significantly between cancer types and even within the same cancer type under different conditions:

Tumor suppressor functions:

• Ovarian cancer: IGFBP5 is markedly downregulated in ovarian cancer tissue; the C-terminal region of IGFBP5 decreases tumor growth in ovarian cancer xenografts by inhibiting angiogenesis via regulation of the Akt/ERK and NF-kB–VEGF/MMP-9 signaling pathway

• Melanoma: Functions as a tumor suppressor in melanoma tumorigenicity and metastasis, though expression patterns vary among melanoma cell lines (high in A2058 and UACC903, low in A375)

• Breast cancer: Shows pro-apoptotic activity in triple-negative breast cancer cell lines (MDA-MB-231 and Hs578T)

Tumor promoting functions:

• Breast cancer: In some contexts, can switch from pro-apoptotic to anti-apoptotic in the presence of ceramide by promoting protein kinase C-dependent conversion of ceramide to anti-apoptotic sphingosine-1-phosphate

• Luminal A breast cancer: High IGFBP5 expression correlates with poor survival

Dual roles:

• Estrogen receptor-positive breast cancer: Contradictory findings in MCF-7 cells where IGFBP5 has been reported to both promote and inhibit apoptosis depending on experimental conditions and IGFBP5 dosage

Drug response modulation:

IGFBP5 can either sensitize or desensitize cancer cells to therapeutic agents:

The variable effects of IGFBP5 in cancer suggest it may function as a molecular switch that can either promote or suppress tumor growth depending on the cellular context, genetic background, and tumor microenvironment .

How does IGFBP5 influence cellular senescence and aging processes?

IGFBP5 plays significant roles in cellular senescence and aging processes through multiple mechanisms:

Expression changes with aging:

• IGFBP5 levels are markedly reduced in senescent mouse embryonic fibroblasts (MEFs) compared to early passage cells

• IGFBP5 levels in serum, bone, and skeletal muscle are lower in aged individuals compared to young adults

• Decreased serum IGFBP5 levels are observed in patients with age-related conditions including type 1 and type 2 diabetes and hip fractures

Mechanistic involvement in senescence:

• During replicative senescence in MEFs, IGFBP5 mRNA levels are significantly reduced as cells undergo growth arrest and display senescence markers (SA-β-GAL staining, p16/p19 upregulation)

• IGFBP5 can modulate IGF signaling pathways that influence cellular aging processes, including the IGF1R/AKT pathway that regulates metabolism and cellular lifespan

Tissue-specific effects in aging:

• In muscle tissue, age-related IGFBP5 reduction may contribute to sarcopenia (age-related muscle loss)

• In bone, decreased IGFBP5 may affect bone formation and repair capacity in older individuals

Relationship to other aging-associated pathways:

• IGFBP5 downregulation may contribute to the dysregulation of IGF bioavailability that occurs with aging

• The connection between IGFBP5 and aging pathways suggests potential interventions targeting IGFBP5 might influence aging processes

Understanding IGFBP5's role in senescence and aging could provide insights into age-related diseases and potential interventions to modulate age-associated decline in tissue function .

How do post-translational modifications of IGFBP5 alter its biological activities?

Post-translational modifications (PTMs) of IGFBP5 serve as critical regulatory mechanisms that dynamically modulate its diverse functions:

Proteolytic processing:

• Multiple proteases cleave IGFBP5 at specific sites in the L-domain

• Proteolysis reduces IGF binding affinity, potentially releasing IGFs to interact with their receptors

• Proteolytic fragments retain distinct biological activities; for example, the N-terminal fragment maintains significant IGF binding capability

• C-terminal fragments may have IGF-independent activities, as demonstrated by the anti-tumorigenic activity of the C-terminal region in ovarian cancer models

Phosphorylation:

• IGFBP5 contains multiple serine/threonine phosphorylation sites in the L-domain

• Phosphorylation can alter IGF binding affinity and interactions with other proteins

• May regulate nuclear localization and intracellular signaling functions

Glycosylation:

• O-glycosylation sites in the L-domain affect protein stability and function

• Glycosylation patterns may vary in different tissues and pathological states

Heparin binding and ECM interactions:

• The RK-rich sequence in the C-domain interacts with heparin and ECM components

• Binding of this region to heparin reduces IGF affinity by 17-fold, promoting release of bound IGF

• These interactions can localize IGFBP5 within tissues and affect its bioavailability

Experimental approaches to study PTMs:

• Site-directed mutagenesis to create IGFBP5 variants lacking specific modification sites

• Mass spectrometry to identify and quantify PTMs

• Phospho-specific or glyco-specific antibodies to detect modified forms

• In vitro modification systems to generate specifically modified IGFBP5

• Cell-based assays comparing wild-type and modification-resistant IGFBP5 variants

Understanding the complex interplay of these modifications is essential for fully elucidating IGFBP5's physiological and pathological roles and for developing targeted therapeutic approaches .

What are the molecular mechanisms underlying IGFBP5's dual role as both an inhibitor and enhancer of IGF signaling?

IGFBP5's ability to function as both an inhibitor and enhancer of IGF signaling depends on complex molecular mechanisms that are context-dependent:

Inhibitory mechanisms:

• Sequestration of IGFs:

  • IGFBP5 binds IGFs with higher affinity than the IGF1R (KD in sub-nanomolar range)

  • Both N- and C-domains participate in IGF binding, creating a high-affinity binding pocket

  • Formation of binary IGFBP5-IGF complexes or ternary complexes with ALS prevents IGF interaction with receptors

• Regulatory feedback:

  • Once secreted, IGFBP5 acts as a major mediator of mTORC1-dependent feedback inhibition of IGF1 signaling

  • This creates a homeostatic mechanism to prevent excessive IGF signaling

Enhancing mechanisms:

• Controlled release of IGFs:

  • IGFBP5 proteolysis by specific proteases reduces its affinity for IGFs, releasing them to activate receptors

  • The IGFBP5-IGF complex can act as a reservoir, providing sustained release of IGFs over time

  • Sudden proteolytic release of IGF from IGFBP5 can create high local IGF concentrations, potentially enhancing receptor activation

• ECM and cell surface interactions:

  • Binding of IGFBP5 to ECM components through its C-terminal heparin-binding domain reduces its affinity for IGFs by up to 17-fold

  • This mechanism allows IGFBP5 to deliver IGFs to the cell surface where they can activate receptors

  • IGFBP5-ECM interactions can concentrate IGFs near their receptors, enhancing signaling efficiency

• Indirect enhancement:

  • In human intestinal smooth muscle cells, IGFBP5 stimulates IGF1 secretion in an IGF-independent manner by activating ERK1/2 and p38 kinase pathways

  • In turn, IGF1 promotes IGFBP5 expression, creating a positive feedback loop

Determinants of inhibitory versus enhancing effects:

• IGFBP5:IGF ratio: At high ratios, inhibitory effects predominate; at lower ratios, enhancing effects may occur

• Tissue context: Different cell types express varying proteases and ECM components that influence IGFBP5 functions

• Post-translational modifications: Proteolysis, phosphorylation, and glycosylation alter IGFBP5's binding properties and interactions

• Developmental stage: IGFBP5 functions may differ during prenatal versus postnatal development, as evidenced by transgenic mouse studies

This dual functionality makes IGFBP5 a sophisticated regulator that can fine-tune IGF signaling depending on physiological context and cellular needs .

What novel therapeutic approaches are being developed based on IGFBP5 biology?

Several innovative therapeutic approaches leveraging IGFBP5 biology are in development, targeting various disease states:

Peptide-based therapeutics:

• The BP5-C peptide derived from the C-terminus of IGFBP5 has shown significant anti-angiogenic and anti-tumorigenic effects in ovarian cancer models by decreasing VEGF-A and MMP-9 expression, inhibiting Akt and ERK phosphorylation, and reducing NF-kB activity

• This peptide significantly decreased tumor weight and angiogenesis in both ovarian cancer orthotopic xenograft and patient-derived xenograft mice

• Structure-activity relationship studies have helped identify the minimal amino acid motif that retains anti-tumorigenic activity

Targeting IGFBP5 in cardiovascular disease:

• Heart-specific modulation of IGFBP5 shows promise for improving cardiac repair following myocardial infarction

• IGFBP5 regulates cardiomyocyte survival through the IGF1R/AKT pathway, offering a potential therapeutic target for myocardial ischemic injury

• Stage-specific IGFBP5 modulation may ameliorate pathological cardiac remodeling and dysfunction during chronic remodeling stages

Cancer therapy approaches:

• Sensitization to anti-estrogen therapy: IGFBP5 can enhance responsiveness to tamoxifen and fulvestrant in certain breast cancer contexts by inhibiting Bcl-3 expression or preventing ERα phosphorylation

• Combination therapies targeting both IGFBP5 and specific oncogenic pathways may overcome treatment resistance

• Context-specific manipulation of IGFBP5 could exploit its dual roles in cancer progression

Aging and senescence intervention:

• IGFBP5's involvement in cellular senescence suggests potential interventions to modulate age-related processes

• Restoring youthful IGFBP5 levels might impact age-associated conditions, particularly in tissues where IGFBP5 levels decline with age (muscle, bone)

Delivery challenges and solutions:

• Recombinant IGFBP5 proteins, domains, or peptides require appropriate delivery systems to reach target tissues

• Tissue-specific targeting approaches using nanoparticles, liposomes, or viral vectors

• Gene therapy approaches to modulate IGFBP5 expression in specific tissues

Experimental considerations for therapeutic development:

• The complex and sometimes contradictory functions of IGFBP5 necessitate careful context-specific evaluation

• Potential compensation by other IGFBP family members must be considered, as observed in IGFBP5 knockout mice

• Therapeutic window determination is critical, as complete inhibition or excessive activation of IGFBP5 pathways may have unintended consequences

These emerging therapeutic approaches highlight IGFBP5's potential as a target for intervention in multiple disease states, though translational challenges remain due to its multifunctional nature and context-dependent effects .

What are the key considerations when designing experiments to distinguish between IGF-dependent and IGF-independent IGFBP5 functions?

Distinguishing between IGF-dependent and IGF-independent functions of IGFBP5 requires careful experimental design:

Molecular approach strategies:

• Domain-specific mutants and fragments:

  • Use IGFBP5 mutants with disrupted IGF binding (typically mutations in the N-domain hydrophobic patch or C-domain RK-rich sequence)

  • Compare full-length IGFBP5 with specific domain fragments that retain or lack IGF binding capacity

  • The mini-IGFBP5 (residues 40-92) can be used as it binds IGFs with reduced affinity (10-fold reduced for IGF-I, 80-fold for IGF-II)

• Specific peptides:

  • The C-terminal BP5-C peptide has demonstrated IGF-independent anti-angiogenic effects

  • Compare responses to IGFBP5 domains that lack IGF binding capacity

Cellular model strategies:

• IGF1R manipulation:

  • Use IGF1R knockout cells or IGF1R inhibition (via specific inhibitors or dominant-negative constructs)

  • If IGFBP5 effects persist in the absence of IGF1R signaling, this suggests IGF-independence

• Cell line selection:

  • Study IGFBP5 effects in cell lines lacking functional IGF survival pathways (e.g., MDA-MB-231, Hs578T breast cancer cells)

  • Compare with cells where IGF signaling is active and important for survival

• Growth factor deprivation:

  • Conduct experiments in serum-free or IGF-depleted conditions

  • Add exogenous IGFs to determine if they rescue or alter IGFBP5 effects

Signaling pathway analysis:

• Monitor IGF1R-independent pathways:

  • Assess non-canonical pathways activated by IGFBP5 (e.g., interactions with TNF1R, α2/β1 integrins)

  • Examine nuclear localization and potential nuclear functions of IGFBP5

• Temporal signaling dynamics:

  • Compare immediate versus delayed signaling events

  • IGF-dependent effects typically require IGF1R activation and show characteristic signaling kinetics

Controls and validation approaches:

• Parallel IGF neutralization:

  • Use IGF neutralizing antibodies alongside IGFBP5 treatments

  • If neutralizing antibodies don't block IGFBP5 effects, this suggests IGF-independence

• Gene expression analysis:

  • Compare transcriptional profiles induced by IGFBP5 versus IGFs

  • Identify IGFBP5-specific gene signatures distinct from IGF responses

• In vivo validation:

  • Study IGFBP5 effects in IGF1 or IGF2 knockout backgrounds

  • Look for IGFBP5 phenotypes that diverge from those displayed by IGF null animals

By systematically employing these strategies, researchers can more definitively attribute observed IGFBP5 effects to either IGF-dependent or IGF-independent mechanisms .

How can researchers address the problem of functional redundancy among IGFBP family members in experimental systems?

Functional redundancy among IGFBP family members poses a significant challenge for researchers studying IGFBP5. The following strategies can help address this issue:

Combinatorial genetic approaches:

• Multiple IGFBP knockout/knockdown:

  • Create cell lines or animal models with simultaneous depletion of multiple IGFBPs

  • Mice lacking IGFBP-3, -4, and -5 show more pronounced phenotypes than single knockouts, including reduced growth, metabolic changes, and significant reduction in circulating and bioactive IGF-1 levels

  • Use inducible systems to avoid developmental compensation

• Domain-specific targeting:

  • Target unique domains or regions of IGFBP5 that are distinct from other family members

  • Focus on the L-domain, which has the least conservation among IGFBPs

Biochemical and expression-based strategies:

• Expression pattern analysis:

  • Study tissues or conditions where IGFBP5 is the predominant IGFBP expressed

  • Exploit developmental windows when IGFBP5 function is most critical and less redundant, such as the immediate postnatal period when tissue programming occurs

• Isoform-specific antibodies and inhibitors:

  • Develop and validate highly specific antibodies against IGFBP5 epitopes not shared with other IGFBPs

  • Design peptide inhibitors that selectively disrupt IGFBP5 functions

Functional discrimination approaches:

• Exploit unique binding partners:

  • Target IGFBP5-specific protein interactions (e.g., with TNF1R or specific ECM components)

  • Focus on cellular processes where IGFBP5 has unique functions compared to other IGFBPs

• Cellular assays with differential IGFBP responses:

  • Identify cell types or processes where IGFBPs have distinct or even opposing effects

  • For example, IGFBP5 overexpression increased fractional brain weight, while IGFBP1 or IGFBP3 overexpression decreased it, and IGFBP2 had little effect

Advanced genetic strategies:

• CRISPR-based approaches:

  • Use precise genome editing to modify IGFBP5-specific regulatory elements rather than the coding sequence

  • Introduce specific mutations that alter post-translational modifications unique to IGFBP5

• Cell-type specific modulation:

  • Use tissue-specific promoters to drive IGFBP5 overexpression or depletion

  • Heart-specific IGFBP5 knockdown, for example, revealed specific roles in cardiac repair

Experimental design considerations:

• Compensatory response monitoring:

  • Systematically measure expression changes in other IGFBPs following IGFBP5 manipulation

  • Account for these changes when interpreting phenotypes

• Acute versus chronic manipulation:

  • Use rapid induction systems to minimize time for compensatory mechanisms

  • Compare acute versus chronic IGFBP5 depletion to identify compensation-masked phenotypes

By implementing these strategies, researchers can better isolate IGFBP5-specific functions despite the functional redundancy that has historically complicated IGFBP biology research .

How should researchers interpret contradictory findings about IGFBP5 functions in the literature?

The literature contains numerous contradictory findings regarding IGFBP5 functions, presenting significant challenges for researchers. A systematic approach to interpreting these contradictions includes:

Context-dependent analysis framework:

• Experimental system differences:

  • Cellular context: IGFBP5 shows opposite effects in epithelial versus mesenchymal cells. In NMuMG cells, IGFBP5 increased adhesion of epithelial cells to ECM but decreased adhesion of mesenchymal variants

  • Species variations: Though IGFBP5 is highly conserved, species-specific differences may exist in regulatory mechanisms

  • In vitro versus in vivo: IGFBP5 effects observed in cell culture may not translate directly to complex tissue environments

• Methodological variations:

  • Protein concentration effects: IGFBP5 can switch from anti-apoptotic to pro-apoptotic as concentration increases

  • Full-length versus fragments: A 22 kDa fragment of IGFBP5 shows different activities than the complete protein

  • Expression level considerations: Global overexpression of IGFBP5 in mice caused growth inhibition, while localized or moderate expression may yield different outcomes

Mechanistic reconciliation strategies:

• Dual functionality reconciliation:

  • IGFBP5 has established dual roles as both an inhibitor and enhancer of IGF activity depending on context

  • In breast cancer, IGFBP5 shows both pro-apoptotic and anti-apoptotic effects depending on cellular context and presence of other factors like ceramide

• Temporal dynamics consideration:

  • Different effects may occur at different time points after IGFBP5 exposure

  • Developmental timing: IGFBP5 has maximal growth inhibition effects postnatally before the onset of growth hormone-dependent growth

• Signaling network integration:

  • Consider the status of interconnected pathways (e.g., PI3K/AKT, ERK1/2, p38 MAPK)

  • The presence of specific binding partners can redirect IGFBP5 functions

Practical approach to literature evaluation:

When evaluating contradictory findings, researchers should systematically assess these parameters and recognize that apparent contradictions may actually reflect the genuine multifunctionality of IGFBP5 rather than experimental inconsistencies .

What are the limitations of current research models for studying IGFBP5 functions?

Current research models for studying IGFBP5 have several important limitations that researchers should consider when designing experiments and interpreting results:

Genetic model limitations:

• Compensation in knockout models:

  • IGFBP5 knockout mice show minimal phenotypes due to functional compensation by other IGFBPs, masking its physiological roles

  • This compensation makes it difficult to identify IGFBP5-specific functions in conventional knockout models

• Overexpression artifacts:

  • Global overexpression can cause non-physiological effects due to abnormal IGFBP5 levels or inappropriate tissue expression

  • In transgenic mice ubiquitously overexpressing IGFBP5, circulating IGFBP5 concentrations increased only 4-fold despite pronounced phenotypic effects, suggesting complex relationships between expression levels and biological outcomes

In vitro system limitations:

• ECM component absence:

  • Many cell culture systems lack the complex ECM environment that modulates IGFBP5 functions in vivo

  • IGFBP5 interactions with ECM components are critical for its localization and IGF-binding properties

• Post-translational modification differences:

  • Cell lines may process IGFBP5 differently than primary tissues

  • Proteolytic processing that occurs in vivo may be absent or altered in vitro

• Concentration considerations:

  • Local tissue concentrations of IGFBP5 are difficult to replicate in vitro

  • Dose-response relationships may be non-linear, with different effects at varying concentrations

Technical challenges:

• Detection specificity issues:

  • Antibody cross-reactivity with other IGFBP family members can confound results

  • Proteolytic fragments of IGFBP5 may not be detected by antibodies targeting specific epitopes

• Protein stability concerns:

  • Recombinant IGFBP5 can have variable stability in experimental systems

  • Storage and handling conditions can affect bioactivity

Translational limitations:

• Species differences:

  • While IGFBP5 is highly conserved, regulatory mechanisms and interacting partners may differ between species

  • Mouse models may not fully replicate human IGFBP5 biology

• Disease model fidelity:

  • Cancer cell lines may not recapitulate the heterogeneity of primary tumors

  • Acute injury models may not reflect chronic disease processes

Future model improvements:

• Tissue-specific and inducible systems:

  • Develop more refined spatial and temporal control of IGFBP5 expression

  • Heart-specific IGFBP5 knockdown has already revealed cardiac-specific functions

• Domain-specific approaches:

  • Create models targeting specific domains or functions rather than the entire protein

  • The C-terminal region approach in ovarian cancer has shown promising results

• Humanized models:

  • Generate models with human IGFBP5 in physiologically relevant contexts

  • Patient-derived xenografts provide more clinical relevance

• Multi-IGFBP models:

  • Develop systems addressing redundancy by targeting multiple IGFBPs simultaneously

  • Conditional approaches to avoid developmental lethality

Recognizing these limitations is essential for designing robust experiments and appropriately interpreting results in IGFBP5 research .

What are the most promising emerging areas of IGFBP5 research?

Several promising research directions are emerging in the IGFBP5 field that have potential for significant scientific and therapeutic advances:

IGFBP5 in tissue regeneration and repair:

• Recent findings on IGFBP5's role in cardiac repair following myocardial infarction suggest broader applications in regenerative medicine

• Exploring IGFBP5's function in other regenerative contexts, such as skeletal muscle, neuronal, and skin repair

• Developing targeted delivery systems to modulate IGFBP5 activity in damaged tissues

Precision targeting of IGFBP5 domains:

• The C-terminal region of IGFBP5 has shown anti-angiogenic and anti-tumorigenic effects in ovarian cancer models

• Further refinement of domain-specific peptides with therapeutic potential

• Structure-function studies to design optimized IGFBP5-derived therapeutics with improved stability and efficacy

IGFBP5 in aging and longevity:

• Investigating the mechanisms underlying IGFBP5 downregulation in senescent cells and aged tissues

• Exploring interventions to restore youthful IGFBP5 expression patterns

• Understanding IGFBP5's role in age-related diseases such as osteoporosis, sarcopenia, and neurodegeneration

IGFBP5 interaction networks:

• Comprehensive mapping of IGFBP5 protein-protein interactions beyond IGFs

• Identification of novel binding partners that mediate IGF-independent actions

• Characterization of tissue-specific interactomes that explain context-dependent functions

Post-translational modification regulation:

• Systematic study of how specific PTMs alter IGFBP5 function

• Identification of the enzymes responsible for IGFBP5 modifications

• Development of tools to detect and quantify specific modified forms of IGFBP5

IGFBP5 in metabolic regulation:

• Investigating IGFBP5's role in glucose homeostasis and insulin sensitivity

• Exploring connections between IGFBP5 and obesity, diabetes, and metabolic syndrome

• Potential therapeutic applications in metabolic disorders

Advanced in vivo models:

• Development of humanized IGFBP5 models to better translate findings to human physiology

• Tissue-specific and temporally controlled genetic systems

• Patient-derived organoids and xenografts to study IGFBP5 in human disease contexts

IGFBP5 in personalized medicine approaches:

• Exploration of IGFBP5 as a biomarker for disease progression or therapeutic response

• Development of personalized treatment strategies based on IGFBP5 expression or modification patterns

• IGFBP5-targeted therapies for specific patient subgroups, particularly in cancer treatment

These emerging research areas hold promise for advancing our understanding of IGFBP5 biology and developing novel therapeutic approaches for various diseases .

What methodological advances would most benefit IGFBP5 research?

Several methodological advances would significantly enhance IGFBP5 research and overcome existing limitations:

Advanced protein analysis techniques:

• High-resolution structural studies:

  • Cryo-electron microscopy of full-length IGFBP5 and its complexes with IGFs and other binding partners

  • Structural determination of IGFBP5 bound to cell surface receptors

  • NMR studies of dynamic IGFBP5 conformational changes upon binding to various partners

• Post-translational modification profiling:

  • Mass spectrometry-based techniques for comprehensive mapping of IGFBP5 modifications

  • Development of modification-specific antibodies and biosensors

  • In situ visualization of modified IGFBP5 forms in tissues

• Protein-protein interaction analysis:

  • Proximity labeling approaches (BioID, APEX) to identify IGFBP5 interacting partners in living cells

  • Single-molecule techniques to study IGFBP5 binding dynamics

  • Protein complementation assays optimized for secreted protein interactions

Genetic and cell-based innovations:

• Precision genome editing:

  • CRISPR-Cas9 approaches to introduce specific IGFBP5 mutations

  • Base editing to modify regulatory elements controlling IGFBP5 expression

  • Knock-in models expressing tagged IGFBP5 for improved tracking

• Advanced cellular models:

  • Organoid systems recapitulating tissue-specific IGFBP5 functions

  • Microfluidic organ-on-chip models incorporating multiple cell types

  • Co-culture systems to study IGFBP5 in complex cellular environments

• Single-cell approaches:

  • Single-cell transcriptomics to identify cell populations responsive to IGFBP5

  • Spatial transcriptomics to map IGFBP5 expression in complex tissues

  • Single-cell proteomics to detect IGFBP5-induced signaling changes

In vivo methodological improvements:

• Enhanced genetic models:

  • Tissue-specific and temporally controlled IGFBP5 expression/deletion

  • Humanized IGFBP5 models to improve clinical relevance

  • Reporter mice for real-time visualization of IGFBP5 expression

• Improved delivery systems:

  • Nanoparticle formulations for targeted delivery of IGFBP5 or its fragments

  • Cell-penetrating peptide conjugates for intracellular delivery

  • Sustained release formulations for chronic IGFBP5 modulation

• In vivo imaging:

  • PET and SPECT imaging with IGFBP5-targeted tracers

  • Optical imaging of IGFBP5 distribution and activity

  • Multiplexed imaging of IGFBP5 and its binding partners

Computational and data analysis approaches:

• Systems biology integration:

  • Network analysis tools to place IGFBP5 in broader signaling contexts

  • Mathematical modeling of IGFBP5-IGF interactions and signaling

  • Multi-omics data integration approaches

• AI and machine learning applications:

  • Prediction of IGFBP5 interaction sites and functional domains

  • Identifying patterns in contradictory IGFBP5 literature

  • Drug design targeting specific IGFBP5 functions

• Improved bioinformatics resources:

  • Databases cataloging IGFBP5 variants, modifications, and interactions

  • Standardized data reporting for IGFBP5 research

  • Predictive tools for IGFBP5 function in various contexts

These methodological advances would address current research limitations and accelerate progress in understanding IGFBP5's complex biology and therapeutic potential.

What are the optimal storage and handling conditions for recombinant IGFBP5 to maintain its activity?

Proper storage and handling of recombinant IGFBP5 is crucial for maintaining its biological activity in research applications:

Storage recommendations:

• Lyophilized protein:

  • Store at -20°C to -80°C for long-term stability

  • Protect from light and moisture

  • Avoid frequent temperature fluctuations

  • Typical shelf life: 12-24 months when properly stored

• Reconstituted protein:

  • For short-term use (1-2 weeks): Store at 4°C

  • For longer-term storage: Prepare single-use aliquots and store at -20°C to -80°C

  • Avoid repeated freeze-thaw cycles (limit to ≤3 cycles)

  • Add carrier protein (0.1-1% BSA or HSA) to prevent surface adsorption and improve stability

Reconstitution guidelines:

• Recommended buffers:

  • Phosphate-buffered saline (PBS, pH 7.2-7.4)

  • Tris-buffered saline (TBS, pH 7.4-7.6)

  • 25 mM HEPES buffer (pH 7.0-7.5)

  • Buffer should be sterile and preferably endotoxin-free

• Reconstitution process:

  • Allow the vial to reach room temperature before opening

  • Reconstitute by gently adding buffer along the sides of the vial

  • Allow to stand for 5-10 minutes

  • Gently swirl or rotate the vial until completely dissolved (avoid vigorous shaking or vortexing)

  • Filter sterilize using a 0.22 μm filter if required for cell culture applications

Stability considerations:

• Factors affecting stability:

  • pH: Maintain between 6.5-7.8 for optimal stability

  • Temperature: Higher temperatures accelerate degradation

  • Protein concentration: Very dilute solutions (<10 μg/ml) may lose activity more rapidly

  • Presence of proteases: Use protease inhibitors when working with complex biological samples

  • Metal ions: Some metal ions can promote oxidation; consider adding EDTA (0.1-1 mM)

• Quality control measures:

  • Verify protein integrity via SDS-PAGE before critical experiments

  • Confirm bioactivity using IGF binding assays or functional cell-based assays

  • Monitor for proteolytic degradation

  • Check for protein aggregation via dynamic light scattering if available

Working solution preparation:

• Dilution recommendations:

  • Prepare fresh working solutions immediately before use when possible

  • Use polypropylene tubes to minimize protein adsorption

  • Include carrier protein (0.1-1% BSA) in dilution buffer for very dilute solutions

  • Maintain sterility for cell culture applications

• Application-specific considerations:

  • For cell culture: Use endotoxin-tested, cell culture-grade preparations

  • For animal studies: Ensure appropriate formulation for the delivery route

  • For biochemical assays: Consider buffer compatibility with downstream applications

Always refer to the Certificate of Analysis or product-specific recommendations from the manufacturer, as optimal conditions may vary between different recombinant IGFBP5 preparations .

How can researchers effectively validate antibody specificity for IGFBP5 detection in their experimental systems?

Validating antibody specificity for IGFBP5 is critical for obtaining reliable results, especially considering the significant homology among IGFBP family members:

Comprehensive validation workflow:

• Initial selection criteria:

  • Choose antibodies raised against unique epitopes of IGFBP5 not conserved in other IGFBPs

  • Review literature for previously validated antibodies with demonstrated specificity

  • Consider monoclonal antibodies for greater epitope specificity

• Positive and negative controls:

  • Positive controls:

    • Recombinant human IGFBP5 protein as standard

    • Cell lines with confirmed high IGFBP5 expression (e.g., A2058 and UACC903 melanoma cells)

    • Tissues known to express IGFBP5 (e.g., bone, muscle, lung)

  • Negative controls:

    • IGFBP5 knockout or knockdown samples

    • Cell lines with confirmed low IGFBP5 expression (e.g., A375 melanoma cells)

    • Antibody pre-absorption with recombinant IGFBP5

Technical validation approaches:

• Western blot validation:

  • Run recombinant IGFBP5 alongside other recombinant IGFBP family members

  • Include multiple positive and negative control cell/tissue lysates

  • Verify correct molecular weight (28.6 kDa for full-length human IGFBP5)

  • Check for absence of non-specific bands or cross-reactivity with other IGFBPs

  • Validate antibody performance in both reducing and non-reducing conditions

• Immunoprecipitation validation:

  • Perform IP followed by mass spectrometry to confirm protein identity

  • Conduct reciprocal IP with different IGFBP5 antibodies recognizing distinct epitopes

  • Include isotype control antibodies as negative controls

• Immunohistochemistry/Immunofluorescence validation:

  • Compare staining patterns with mRNA expression (ISH or public databases)

  • Include IGFBP5 knockdown samples as negative controls

  • Perform peptide competition assays to confirm specificity

  • Compare multiple antibodies against different IGFBP5 epitopes

• ELISA/quantitative assay validation:

  • Generate standard curves with recombinant IGFBP5

  • Test for cross-reactivity with other IGFBPs and relevant proteins

  • Assess recovery by spiking known amounts of IGFBP5 into samples

  • Validate linearity of dilution and dynamic range

Genetic manipulation validation:

• Expression modulation:

  • Verify antibody signal decrease following IGFBP5 knockdown or knockout

  • Confirm signal increase following IGFBP5 overexpression

  • Use inducible systems to demonstrate dynamic changes in signal

• Tagged protein approach:

  • Express epitope-tagged IGFBP5 and compare antibody staining with epitope tag antibodies

  • Useful for antibody validation in challenging samples

Addressing common challenges:

• Proteolytic fragments:

  • Test antibody detection of known IGFBP5 fragments

  • Use domain-specific antibodies to distinguish intact protein from fragments

• Post-translational modifications:

  • Determine if antibody recognition is affected by phosphorylation, glycosylation, or other modifications

  • Compare detection in samples with different modification states

• Matrix effects:

  • Validate antibody performance in different sample types (serum, tissue lysates, cell media)

  • Optimize extraction methods to maximize IGFBP5 recovery