TIMP1 Human, HEK

What is human TIMP1 and what is its basic structure?

Human TIMP1 is a 23 kDa glycoprotein consisting of 184 amino acids (positions 24-207 in the full sequence) that functions primarily as an endogenous inhibitor of matrix metalloproteinases (MMPs) . The protein contains multiple disulfide bonds that are essential for its tertiary structure and biological activity. Recombinant human TIMP1 produced in HEK293 cells typically has a purity of ≥95% and maintains the proper post-translational modifications required for biological activity . The amino acid sequence includes critical functional domains for MMP interaction, with the N-terminal domain primarily responsible for MMP inhibition while the C-terminal domain mediates additional protein-protein interactions .

What are the primary biological functions of TIMP1?

TIMP1 serves multiple biological functions beyond its classical role as an MMP inhibitor. Its primary functions include:

• Metalloproteinase inhibition: Forms 1:1 complexes with target MMPs and irreversibly inactivates them by binding to their catalytic zinc cofactor .

• Growth factor activity: Regulates cell differentiation, migration, and cell death independent of its MMP inhibitory function .

• Cellular signaling: Activates signaling cascades via CD63 and ITGB1 (integrin β1), playing a significant role in integrin signaling pathways .

• Erythropoiesis regulation: Mediates species-specific erythropoiesis in vitro, stimulating the growth and differentiation of human and murine erythroid progenitors .

• Extracellular matrix homeostasis: Maintains the balance between matrix destruction and formation, with imbalances potentially leading to excessive ECM deposition and fibrogenic processes .

How is recombinant human TIMP1 produced in HEK293 cells?

Recombinant human TIMP1 is typically produced in HEK293 cells through transfection with expression vectors containing the human TIMP1 gene sequence (amino acids 24-207) . HEK293 cells are preferred for recombinant TIMP1 production because they:

• Provide human-compatible post-translational modifications, particularly glycosylation patterns

• Ensure proper protein folding and disulfide bond formation

• Secrete functional protein into the cell culture medium

• Allow for scaled production with consistent protein quality

The production process generally involves stable transfection of HEK293 cells followed by selection, expansion, and protein purification through chromatographic techniques. The resulting recombinant protein undergoes quality control testing for purity (typically ≥95%), endotoxin levels (≤0.005 EU/μg), and functional activity through enzyme inhibition assays .

What methods are used to quantify TIMP1 in biological samples?

Several methodological approaches can be used to quantify TIMP1 in research samples:

• HTRF (Homogeneous Time-Resolved Fluorescence) immunoassay: Uses two specific anti-human TIMP1 antibodies labeled with donor and acceptor dyes to create a sandwich immunoassay. The signal intensity directly correlates with TIMP1 concentration in the sample .

• ELISA-based detection: Various commercial kits employ sandwich ELISA methodology using TIMP1-specific antibodies.

• Western blotting: For semi-quantitative detection and molecular weight confirmation.

• Mass spectrometry: For precise identification and absolute quantification in complex samples.

The HTRF-based protocol typically requires:

• 16 μL sample volume

• 384-well small volume white plates or low volume 96-well plates (20 μL final)

• HTRF donor and acceptor-labeled antibodies that can be pre-mixed

• Standard curves generated with purified TIMP1 protein

How does TIMP1 influence cancer biology and what are the appropriate experimental approaches?

TIMP1 has complex roles in cancer progression, often associated with poor prognosis in multiple cancer types including breast, gastric, colorectal, non-small cell lung carcinoma (NSCLC), and renal cell carcinoma . Key considerations for cancer-related TIMP1 research include:

• Effects on cell morphology and EMT: High TIMP1-expressing cancer cells (e.g., A549) display spindle-shaped, fibroblast-like morphology with increased cell scattering associated with EMT-like phenomena, while TIMP1 knockdown results in cobblestone-like epithelioid morphology .

• Relationship with miR-125a-5p: TIMP1 knockdown increases miR-125a-5p expression with concomitant increases in apoptosis. TIMP1 has been identified as a direct target of miR-125a-5p, and their interaction affects p53 expression .

• Experimental approaches:

  • TIMP1 knockdown studies using siRNA or CRISPR-Cas9

  • Luciferase reporter assays to confirm miRNA binding sites

  • Morphological assessment through microscopy

  • Protein expression analysis through western blotting for EMT markers (e.g., E-cadherin)

What is the role of TIMP1 in blood-brain barrier integrity and how can it be investigated?

TIMP1 protects tight junctions of brain endothelial cells against MMP-mediated disruption, particularly from MMP-3 and MMP-9 . Research methodologies to investigate this function include:

• In vitro BBB model generation using rat brain microvascular endothelial cells (RBMEC)

• Assessment of BBB integrity through:

  • Trans-endothelial electrical resistance (TEER) measurements

  • Permeability assays using fluorescent markers

  • Immunofluorescence microscopy of tight junction proteins

• Dose-dependent studies with MMPs (e.g., 1.5 μM MMP-3 or MMP-9 causes an over threefold increase in permeability)

• Inhibition studies using TIMP1-MMP complexes to demonstrate the protective effect of TIMP1

This experimental design allows for quantitative assessment of both the disruptive effects of MMPs and the protective function of TIMP1 in maintaining BBB integrity.

How can researchers differentiate between MMP-dependent and MMP-independent functions of TIMP1?

Differentiating between these functions requires specific experimental approaches:

• Site-directed mutagenesis: Generate TIMP1 mutants that lack MMP inhibitory activity but retain other functional domains

• Comparison studies: Use both wild-type TIMP1 and mutant versions in parallel experiments

• Competitive inhibition: Employ synthetic MMP inhibitors alongside TIMP1 to distinguish effects

• Receptor blocking: Use antibodies against CD63 or ITGB1 to block TIMP1 signaling while preserving MMP inhibition

• Downstream signaling analysis: Examine activation of pathways known to be MMP-independent (e.g., integrin signaling)

What is known about TIMP1-miRNA interactions and their experimental validation?

TIMP1 has been identified as a target of miR-125a-5p in lung adenocarcinoma models . The experimental validation of this interaction involved:

• Luciferase reporter constructs: The 3'UTR and 5'UTR regions of TIMP1 were cloned into a firefly luciferase reporter construct (psiCHECK) and transfected into HEK293 cells.

• Site-directed mutagenesis: The seed region in the 3'UTR was mutated using overlap extension PCR.

• Transfection studies: miR-125a-5p mimics repressed luciferase activity in wild-type 3'UTR constructs but not in the mutated 3'UTR constructs or 5'UTR constructs.

• Morphological assessment: Transfection of miR-125a-5p mimics into A549 cells induced morphological changes similar to those observed in TIMP1 knockdown cells, while antagomirs reversed these effects .

These methodologies conclusively demonstrated that TIMP1 is a direct target of miR-125a-5p, with significant functional consequences for cancer cell behavior.

What is the significance of TIMP1 in the balance of matrix remodeling, and how is this disrupted in pathological conditions?

TIMP1 plays a crucial role in maintaining extracellular matrix homeostasis through several mechanisms:

• MMP regulation: Forms 1:1 complexes with MMPs (particularly MMP-2 and MMP-9) to irreversibly inactivate them .

• Balance maintenance: TIMPs maintain the equilibrium between matrix destruction and formation, with the TIMP:MMP ratio being critical for tissue homeostasis .

• Pathological disruption: An imbalance between MMPs and TIMPs leads to excessive ECM deposition and fibrogenic processes in multiple conditions .

In various pathological states, this balance is disrupted:

• In cancer: Elevated TIMP1 levels are often associated with increased tumorigenesis and poor prognosis, potentially through anti-apoptotic effects and promotion of EMT-like changes .

• In fibrotic diseases: Excess TIMP1 can inhibit necessary matrix turnover, leading to pathological ECM accumulation.

• In inflammatory conditions: Dysregulated TIMP1/MMP ratios contribute to inappropriate tissue remodeling.

What are the optimal storage and handling conditions for recombinant human TIMP1?

Based on standard protocols for recombinant proteins and the specific nature of TIMP1:

• Storage temperature: Typically -80°C for long-term storage, with aliquoting recommended to avoid freeze-thaw cycles

• Buffer composition: Usually stored in a neutral pH buffer (pH 7.0-7.5) with minimal salt content

• Additives: May include stabilizers such as BSA (0.1-1%) or glycerol (10-50%)

• Handling precautions: Avoid repeated freeze-thaw cycles; maintain on ice when thawed; use low-binding tubes

• Working concentrations: Typically in the range of 1-100 ng/mL for cell culture experiments

What are the key considerations when designing TIMP1 functional assays?

When designing functional assays to study TIMP1 activity:

• MMP inhibition assays:

  • Use fluorogenic substrates specific for target MMPs

  • Include appropriate controls (EDTA as general MMP inhibitor)

  • Consider pre-incubation time between TIMP1 and MMPs

  • Account for the 1:1 stoichiometry of TIMP1-MMP interactions

• Cell-based assays:

  • Consider the expression of endogenous TIMP1 and MMPs in the chosen cell line

  • Use appropriate concentrations based on physiological ranges (typically 1-100 ng/mL)

  • Include controls for MMP-independent effects

  • Monitor multiple endpoints (proliferation, migration, apoptosis, morphology)

• In vivo models:

  • Consider species-specificity (TIMP1 shows species-specific effects in erythropoiesis)

  • Use appropriate delivery methods (recombinant protein vs. gene delivery)

  • Monitor both target inhibition and downstream physiological effects