MMP14 Human, His

What is the basic domain structure of human MMP14 and how does a His-tag affect its structure?

Human MMP14 consists of several domains including a signal peptide, prodomain, catalytic domain, hinge region, hemopexin-like domain, transmembrane domain, and cytoplasmic tail. The full protein spans 582 amino acids, with the catalytic domain (where most enzymatic activity occurs) residing approximately between amino acids 112-292.

When working with His-tagged MMP14, researchers should note that tag placement can affect protein folding and function. N-terminal His-tags are typically placed after the signal peptide (which is removed upon ER insertion) but before or after the prodomain, depending on whether you want to study the activation process . C-terminal His-tags may interfere with membrane insertion if the protein is to be expressed in its full transmembrane form.

For most structural and enzymatic studies, researchers use truncated versions (e.g., AA 110-539 or AA 112-541) lacking the transmembrane and cytoplasmic domains to improve solubility while maintaining catalytic function .

How do specific MMP14 mutations affect protein function and processing in research models?

MMP14 mutations significantly impact protein processing and function in various ways:

• The p.T17R mutation affects the signal peptide, disrupting proper ER targeting and subsequent processing, resulting in severe loss of function .

• The p.R92C mutation impacts prodomain cleavage and cell surface localization, severely compromising MMP14 function .

• The p.R111H mutation (located in the catalytic domain) represents a hypomorphic allele that partially impairs proteolytic activity while maintaining normal trafficking to the cell membrane, resulting in residual enzymatic activity .

• The p.S466P mutation in the hemopexin domain affects substrate specificity and protein-protein interactions but may maintain some proteolytic function .

These mutations provide valuable research tools for understanding structure-function relationships in MMP14 and can be reproduced in recombinant His-tagged versions for comparative studies.

What expression systems are optimal for producing functional His-tagged human MMP14?

The choice of expression system for His-tagged human MMP14 depends on your specific research requirements:

• Bacterial systems (E. coli): Suitable for producing the catalytic domain alone (AA 112-284) with His-tags. Advantages include high yield and low cost, but proteins often lack post-translational modifications and may form inclusion bodies requiring refolding protocols.

• Yeast systems: Provide better protein folding and some post-translational modifications. Successfully used for producing MMP14 fragments (AA 110-539) with His-tags .

• Mammalian cell systems (HEK293, CHO): Optimal for full-length or transmembrane-containing MMP14 constructs that require proper glycosylation and disulfide bond formation. These systems best recapitulate the natural processing of MMP14 including signal peptide removal and prodomain cleavage .

• Insect cell/baculovirus systems: Offer a balance between proper folding and higher yields compared to mammalian cells.

When studying MMP14 activation and processing pathways, mammalian expression systems are strongly recommended as they correctly process the signal peptide upon ER insertion and contain the proprotein convertases necessary for prodomain removal .

What purification strategies maximize yield and activity of His-tagged MMP14?

For optimal purification of His-tagged MMP14 while preserving enzymatic activity:

• Lysis buffer composition: Use buffers containing 50 mM Tris-HCl pH 7.5, 150-300 mM NaCl, 10% glycerol, and 0.1% non-ionic detergent (for transmembrane versions). Include zinc (1-5 μM ZnCl₂) to stabilize the catalytic domain.

• IMAC purification: Use Ni-NTA or Co²⁺ resins with gradient elution (20-250 mM imidazole) rather than step elution to separate differentially processed forms of MMP14.

• Buffer exchange: Immediately after elution, exchange into a storage buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 0.5-1 μM ZnCl₂, and 5 mM CaCl₂ to maintain enzymatic activity.

• Activity preservation: Add 1 mM APMA (aminophenylmercuric acetate) to activate pro-forms if immediate activity is desired, or maintain the prodomain intact by including protease inhibitors (excluding metalloprotease inhibitors) if studying activation mechanisms.

• Storage: Store at -80°C in single-use aliquots with 10% glycerol. Avoid repeated freeze-thaw cycles which significantly reduce activity.

For research requiring the separation of different processed forms (proenzyme vs. active enzyme), size exclusion chromatography following IMAC purification is recommended.

How can researchers accurately measure the enzymatic activity of purified His-tagged MMP14?

Several complementary approaches can be used to assess MMP14 activity:

Gelatin Zymography (Indirect Method):

• This assay measures MMP14's ability to activate pro-MMP2 rather than direct MMP14 activity

• Co-incubate purified His-tagged MMP14 with pro-MMP2, then perform gelatin zymography

• Active MMP14 will convert pro-MMP2 to active MMP2, visible as cleared bands at lower molecular weight

• This method is useful for comparing relative activities of different MMP14 constructs or mutants

Fluorogenic Peptide Substrates (Direct Method):

• Use peptides with quenched fluorescent groups that become fluorescent upon cleavage

• Common substrates include MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂

• Monitor increase in fluorescence over time to calculate reaction kinetics

• Allows determination of enzymatic parameters (K_m, k_cat) under various conditions

Gelatin Degradation Assay (Cell-Based):

• For transmembrane MMP14 expressed in cells, plate cells on fluorescently labeled gelatin

• Areas of degradation appear as dark spots against the fluorescent background

• Can be quantified by image analysis to compare wild-type vs. mutant MMP14 activity

Native Substrate Cleavage:

• For mechanistic studies, monitor cleavage of physiological substrates like collagen I, fibronectin, or CD44

• Use western blotting to detect appearance of cleavage fragments

• More biologically relevant but less quantitative than synthetic substrate assays

What are the most effective methods for studying MMP14's role in epithelial-mesenchymal transition (EMT)?

MMP14 has been implicated as a key regulator of EMT, particularly in cancer progression. For studying this role:

Gene Expression Modulation:

• Create stable cell lines with inducible MMP14 expression or knockdown using shRNA/CRISPR

• Express wild-type or catalytically inactive His-tagged MMP14 to distinguish between proteolytic and non-proteolytic functions

• Monitor changes in EMT markers (E-cadherin, vimentin, ZEB1, Twist) by qRT-PCR and western blotting

Cell-Based Functional Assays:

• Migration assays (wound healing, transwell)

• 3D invasion assays using matrices such as Matrigel or collagen I

• Cell morphology assessment using phase-contrast microscopy and cytoskeletal staining

• These assays can reveal how MMP14 expression levels correlate with invasive behavior

Protein Interaction Studies:

• Use His-tagged MMP14 for pull-down assays to identify interaction partners during EMT

• Perform co-immunoprecipitation with EMT transcription factors to investigate non-catalytic signaling functions

• Crosslinking followed by mass spectrometry can identify novel interaction partners

In vivo Models:

• Xenograft models with cells expressing wild-type or mutant MMP14

• Tissue analysis for EMT markers and correlation with MMP14 expression

• Primary cell isolation from these models for ex vivo analysis

Research has shown that MMP14 not only facilitates invasion through ECM degradation but also directly regulates the expression of EMT-associated genes including E-cadherin, vimentin, Twist, and ZEB1, making it a central player in cancer progression .

How do MMP14 mutations contribute to Winchester syndrome pathogenesis?

Winchester syndrome is a rare inherited disorder characterized by skeletal abnormalities, short stature, osteoporosis, and distinctive facial features. Research has revealed that MMP14 mutations are central to its pathogenesis:

Mutation Spectrum and Functional Consequences:

• The p.T17R mutation affects the signal peptide, preventing proper ER targeting and resulting in complete loss of function

• The p.R111H mutation represents a hypomorphic allele with partial impairment of catalytic activity while maintaining normal trafficking, resulting in a milder phenotype

• Other mutations like p.R92C affect prodomain processing and trafficking

Pathophysiological Mechanisms:

• Defective extracellular matrix remodeling, particularly collagen turnover

• Impaired podosome formation affecting cell migration and tissue morphogenesis

• Disrupted activation of other MMPs, creating a cascade effect on matrix remodeling

• Abnormal cell-matrix adhesion affecting skeletal development

Genotype-Phenotype Correlations:

• Complete loss-of-function mutations (p.T17R) cause severe phenotypes

• Hypomorphic mutations with residual activity (p.R111H) result in mitigated forms of the syndrome

• Murine models with similar mutations (p.R92C, p.S466P) recapitulate many aspects of the human phenotype

This research highlights how different MMP14 mutations affect distinct protein functions, providing insight into the mechanistic basis of disease severity and potential therapeutic approaches.

What research models best represent human MMP14 function in normal and pathological conditions?

For studying MMP14 function in normal physiology and disease:

Cell-Based Models:

• MRC-5V1 fibroblasts for studying MMP14 processing and ECM degradation

• Primary human osteoblasts and chondrocytes for bone and cartilage-related studies

• Cancer cell lines (e.g., nasopharyngeal carcinoma) for studying MMP14's role in invasion and EMT

• 3D organoid cultures that better recapitulate tissue architecture and cell-matrix interactions

Animal Models:

• MMP14 knockout mice: display severe skeletal abnormalities, dwarfism, osteopenia, and premature death

• MMP14 point mutation models (Sabe, Cartoon): harbor specific mutations (p.R92C, p.S466P) that mimic human disease

• Conditional and tissue-specific knockout/knockin models to study tissue-specific functions

• Xenograft models using cells with modified MMP14 expression for cancer studies

Human Samples:

• Patient-derived cells from individuals with MMP14 mutations provide the most relevant model but are extremely rare

• Tissue microarrays from various pathological conditions (e.g., cancer) to study MMP14 expression patterns

• Induced pluripotent stem cells (iPSCs) from patients, differentiated into relevant cell types

Advantages and Limitations:

• Cell models allow detailed mechanistic studies but lack physiological context

• Animal models provide systemic effects but have species-specific differences

• Human samples are most relevant but limited in availability and experimental flexibility

The choice of model should be guided by the specific research question, with emerging technologies like gene editing and organoid culture offering new possibilities for studying MMP14 biology.

How can researchers distinguish between catalytic and non-catalytic functions of MMP14?

MMP14 exhibits both proteolytic and non-proteolytic functions that can be challenging to differentiate:

Experimental Approaches:

• Domain-specific mutants: Compare His-tagged MMP14 constructs with:

  • Catalytic domain mutations (E240A) that abolish enzymatic activity

  • Hemopexin domain mutations that affect protein interactions but preserve catalysis

  • Cytoplasmic domain mutations/truncations that affect intracellular signaling

• Selective inhibitors:

  • Use highly selective MMP14 catalytic inhibitors (e.g., GM6001 derivatives)

  • Compare inhibitor effects with those of dominant-negative MMP14 expression

  • Time-course studies to distinguish immediate (likely non-catalytic) from delayed (likely catalytic) effects

• Substrate vs. binding partner analysis:

  • Identify proteins cleaved by MMP14 using proteomics approaches

  • Identify binding partners using crosslinking mass spectrometry or proximity labeling

  • Compare these datasets to distinguish substrates from signaling partners

• Rescue experiments:

  • In MMP14-depleted cells, compare rescue with wild-type vs. catalytically inactive MMP14

  • Functions rescued only by wild-type are likely catalytic

  • Functions rescued by both versions are likely scaffold/signaling related

Research Insights:
MMP14 regulates EMT not only through ECM degradation (catalytic) but also by influencing the expression of EMT-associated genes like E-cadherin and vimentin through signaling mechanisms that may be independent of its catalytic activity . Understanding these dual functions is crucial for developing targeted therapeutic approaches.

What are the current challenges in achieving crystal structures of full-length human MMP14?

Obtaining crystal structures of full-length human MMP14 presents several technical challenges:

Key Obstacles:

• Membrane association: The transmembrane domain makes the full protein highly hydrophobic and prone to aggregation during purification

• Flexible regions: The linker between catalytic and hemopexin domains is highly flexible, complicating crystallization

• Post-translational modifications: Glycosylation is important for function but introduces heterogeneity

• Multiple conformational states: MMP14 exists in both pro and active forms, and undergoes conformational changes upon substrate binding

• Autolytic degradation: Active MMP14 can self-digest, creating heterogeneous samples

Current Technical Approaches:

Future Directions:
Integrative structural biology approaches combining multiple techniques are most likely to succeed in determining the full-length structure of MMP14, providing crucial insights into its activation mechanism and substrate recognition.

What emerging technologies show promise for advancing MMP14 research?

Several cutting-edge technologies are poised to transform our understanding of MMP14 biology:

Single-Cell Analysis:

• Single-cell RNA-seq and proteomics to understand cell-specific MMP14 expression patterns

• Spatial transcriptomics to map MMP14 expression in tissue contexts

• These approaches will reveal heterogeneity in MMP14 expression and function within tissues

Advanced Imaging:

• Super-resolution microscopy to visualize MMP14 clustering and dynamics at the cell surface

• FRET-based biosensors to monitor MMP14 activity in real-time in living cells

• Intravital imaging to observe MMP14 function in vivo

Structural Biology Advances:

• Cryo-electron microscopy for visualization of full-length MMP14 in different conformational states

• Molecular dynamics simulations to understand MMP14 dynamics and substrate interactions

• Integrative structural approaches combining multiple experimental techniques

Gene Editing Technologies:

• CRISPR-based approaches for precise engineering of MMP14 mutations

• Base editing for introducing specific amino acid changes to study structure-function relationships

• In vivo somatic gene editing to study tissue-specific MMP14 functions

Proteomics and Interactomics:

• Proximity labeling methods (BioID, APEX) to identify MMP14 interaction partners in living cells

• Degradomics to comprehensively identify MMP14 substrates in different contexts

• Crosslinking mass spectrometry to map protein interaction surfaces

These technologies promise to provide unprecedented insights into MMP14 biology, potentially leading to new therapeutic strategies for MMP14-related disorders.

How might therapeutic targeting of MMP14 evolve based on current research insights?

The therapeutic landscape for MMP14 is evolving based on deeper understanding of its structure and function:

Current Limitations in MMP Targeting:

• Previous broad-spectrum MMP inhibitors failed in clinical trials due to lack of specificity

• Catalytic site similarity across MMPs makes selective inhibition challenging

• Dual roles of MMP14 in disease (both promoting and protective) complicate therapeutic approaches

Emerging Strategies:

• Highly selective catalytic inhibitors:

  • Structure-based design targeting unique features of the MMP14 catalytic pocket

  • Allosteric inhibitors that bind outside the catalytic site for greater selectivity

  • Exosite inhibitors targeting substrate-binding regions specific to MMP14

• Function-selective approaches:

  • Inhibitors that block specific MMP14 activities (e.g., pro-MMP2 activation) while preserving others

  • Targeting MMP14 in specific contexts (e.g., cancer-specific glycoforms)

  • Hemopexin domain-targeting agents that modulate protein-protein interactions

• Cell-specific delivery:

  • Antibody-drug conjugates targeting MMP14-expressing cells

  • Nanoparticle delivery of MMP14 inhibitors to specific tissues

  • Exploiting MMP14 itself as a trigger for targeted drug release

• Gene therapy approaches:

  • Correction of MMP14 mutations for rare genetic disorders like Winchester syndrome

  • Modulation of MMP14 expression in specific tissues using non-coding RNAs

  • CRISPR-based approaches for precise gene editing

Future Perspectives:
The development of context-specific MMP14 modulators rather than global inhibitors represents the most promising avenue for therapeutic development. Understanding how different MMP14 mutations impact specific functions (as seen with p.R111H vs. p.T17R) provides crucial insights for designing precision medicines for MMP14-related disorders.