ANXA7 Human

What is the molecular structure of human ANXA7 and how does it compare to other annexins?

ANXA7 belongs to the annexin family of calcium-dependent membrane-binding proteins. The protein exists in two isoforms (47 kDa and 51 kDa) and contains four characteristic annexin repeats (endonexin-fold repeats) with the consensus sequence GX(X)GT . These repeats are responsible for calcium and phospholipid binding. ANXA7 differs from other annexins like ANXA1 and ANXA5 in its translocation dynamics to injury sites, with ANXA7 showing slower translocation velocity (peaking at 80-120 seconds post-injury) compared to ANXA1 and ANXA5 (which peak at approximately 30 seconds) . This distinctive kinetic profile suggests specialized functions for ANXA7 that may not be shared with other annexin family members.

How does ANXA7 regulate calcium homeostasis in human cells?

ANXA7 functions as a critical regulator of intracellular calcium homeostasis and vesicle fusion events . Methodologically, researchers investigate this function through:

• Calcium imaging using fluorescent indicators in ANXA7 knockdown or overexpression models

• Electrophysiological measurements of calcium currents

• Analysis of calcium-dependent protein interactions

ANXA7 appears to mediate calcium-dependent membrane fusion and participates in maintaining proper calcium signaling through interaction with calcium channels and calcium storage compartments. Dysfunction in this regulatory role has been linked to both neurological conditions and cancer progression, with experimental evidence showing that ANXA7's calcium-binding properties are directly linked to its tumor suppressor function .

What protein interactions are most critical for ANXA7's cellular functions?

ANXA7 engages in several key protein-protein interactions that determine its biological functions:

• SRI (Sorcin) interaction: ANXA7 interacts with SRI to regulate epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma, contributing to tumor aggressiveness .

• ALG-2 and ALIX recruitment: ANXA7 is required for the recruitment of ALG-2 and ALG-2-interacting protein X (ALIX) to damaged membranes, initiating ESCRT III machinery buildup for membrane repair .

• IP3 receptor modulation: Research indicates ANXA7 function is associated with differential IP3 receptor expression, affecting calcium signaling pathways .

Methodologically, these interactions can be studied using co-immunoprecipitation, proximity ligation assays, FRET analysis, and live cell imaging techniques with fluorescently tagged proteins.

How does ANXA7 contribute to epithelial-mesenchymal transition in hepatocellular carcinoma?

ANXA7 has been identified as a promoter of epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) through its interaction with Sorcin (SRI). This relationship has significant implications for HCC progression and metastasis.

Research findings demonstrate that:

• ANXA7 and SRI are co-overexpressed in HCC tissues and cells

• The ANXA7-SRI interaction regulates EMT marker expression

• Overexpression of ANXA7 promotes migration, invasion, and proliferation in HCC cells

• In vivo models confirm ANXA7's role in promoting tumorigenicity and EMT

Experimental approaches to study this mechanism include:

• RNA interference to knock down ANXA7 expression

• Overexpression studies using plasmid constructs

• Co-immunoprecipitation to confirm protein interactions

• Western blotting to analyze EMT marker expression

• Transwell migration and invasion assays

• Xenograft models to assess in vivo effects

What evidence supports ANXA7's role as a tumor suppressor versus an oncogene?

ANXA7's role in cancer appears context-dependent, with research showing contradictory functions:

Evidence for tumor suppressor role:

• ANXA7 contains tumor suppressor gene (TSG) properties and is located on chromosome 10q21

• Loss of ANXA7 expression correlates with greater malignancy in glioblastoma multiforme

• The dominant-negative triple mutant of ANXA7 inhibits tumor cell proliferation and sensitizes cells to cell death

Evidence for oncogenic role:

• ANXA7 promotes EMT and contributes to aggressiveness in HCC

• ANXA7 overexpression enhances tumorigenicity in vivo in certain cancer models

This apparent contradiction may be explained by:

• Cancer type-specific effects

• Differential expression of ANXA7 isoforms

• Context-dependent protein interactions

• Variations in calcium signaling environments across cancer types

Researchers should employ multiple cancer cell lines and patient-derived samples when studying ANXA7 function to account for these context-dependent effects.

How do mutations in ANXA7 affect its function in tumor progression?

Mutations in ANXA7 can dramatically alter its functional properties and impact on tumor progression. The dominant-negative triple mutant (DNTM/DN-ANXA7) identified in research has revealed critical insights:

• The DNTM mutation affects the four C-terminal endonexin-fold repeats (GX(X)GT) within ANXA7's annexin repeats

• This mutation suppresses ANXA7's membrane fusion ability while simultaneously inhibiting tumor cell proliferation

• The mutant alters membrane fusion rates and calcium/phospholipid binding capacity

• ANXA7 mutations affect downstream signaling through PI3K/AKT/mTOR pathways

For researchers investigating ANXA7 mutations, methodological approaches should include:

• Site-directed mutagenesis of specific calcium-binding domains

• Membrane fusion assays using artificial membranes

• Phospholipid binding assays

• Cell proliferation and apoptosis measurements

• Signaling pathway activation analysis using phospho-specific antibodies

What signaling pathways are modulated by ANXA7 in cancer cells?

ANXA7 intersects with several critical signaling pathways in cancer cells:

Research indicates that ANXA7's calcium and phospholipid binding properties are essential for its ability to modulate these pathways, as demonstrated by studies with the dominant-negative triple mutant that alters phosphatidylserine exposure, membrane permeabilization, and cellular apoptosis .

What role does ANXA7 play in neuronal apoptosis after subarachnoid hemorrhage?

ANXA7 has been identified as a critical mediator of neuronal apoptosis following subarachnoid hemorrhage (SAH). Research findings demonstrate:

• ANXA7 protein levels significantly increase after experimental SAH in rats, peaking at 48 hours post-injury

• ANXA7 is primarily localized in neurons, not astrocytes, as confirmed by double immunofluorescence staining

• ANXA7 knockdown via lenti-ANXA7-siRNA significantly reduces neuronal apoptosis after SAH

The molecular mechanism involves:

• Modulation of the intrinsic apoptotic pathway

• Regulation of the Bax/Bcl-2 ratio (ANXA7 knockdown increases anti-apoptotic Bcl-2 while decreasing pro-apoptotic Bax)

• Reduction of cleaved caspase-3 expression

Experimental approaches for investigating this phenomenon include:

• Western blot analysis of ANXA7 expression at various time points after SAH

• TUNEL staining to assess neuronal apoptosis

• Double immunofluorescence staining for localization studies

• RNA interference using lenti-ANXA7-siRNA for knockdown experiments

• Analysis of apoptotic markers by Western blot

How does ANXA7 regulate glutamate release in neurological conditions?

ANXA7 plays a significant role in regulating glutamate release following neurological injury:

• Research has shown that glutamate concentration in cerebrospinal fluid (CSF) increases significantly after subarachnoid hemorrhage (SAH)

• ANXA7 knockdown dramatically decreases glutamate release in experimental SAH models

• This regulatory effect on glutamate may represent a key mechanism by which ANXA7 mediates early brain injury after SAH

For researchers investigating this phenomenon, methodological approaches include:

• Measurement of glutamate concentration in CSF samples

• Use of ANXA7 knockdown models via siRNA or CRISPR/Cas9

• Calcium imaging to assess the relationship between calcium signaling and glutamate release

• Electrophysiological recordings to measure excitatory postsynaptic potentials

• Isolation of synaptic vesicles to study ANXA7's role in vesicle fusion and neurotransmitter release

How does ANXA7 knockdown affect blood-brain barrier integrity and brain edema?

ANXA7 knockdown has demonstrated substantial protective effects on blood-brain barrier (BBB) integrity and brain edema following neurological injury:

• Experimental data shows that ANXA7 knockdown significantly improves BBB disruption after SAH, as measured by Evans blue extravasation

• Brain edema, quantified by brain water content, is markedly reduced in ANXA7 knockdown models after SAH

• These improvements in BBB integrity and reduced edema correlate with enhanced neurological outcomes

Methodological approaches to study these effects include:

• Evans blue dye extravasation assays to quantify BBB permeability

• Brain water content measurement via wet/dry weight analysis

• Immunohistochemistry for tight junction proteins

• Transmission electron microscopy to visualize BBB ultrastructure

• Neurological testing using validated scoring systems like the Garcia test

Researchers should note the temporal progression of BBB disruption and consider multiple timepoints (24-72 hours post-injury) when evaluating intervention efficacy.

What are the most effective techniques for visualizing ANXA7 translocation during membrane repair?

Live-cell imaging techniques provide the most effective approach for visualizing ANXA7 dynamics during membrane repair processes:

• Fluorescent protein tagging: Expressing ANXA7-GFP or ANXA7-RFP fusion proteins allows real-time tracking of ANXA7 translocation. Research has shown that ANXA7-GFP begins accumulating at repair sites within 20 seconds of membrane injury, reaching maximum accumulation by 80-120 seconds .

• Multi-color imaging: Co-expressing ANXA7 with other annexins (e.g., ANXA1-GFP, ANXA5-GFP) or repair machinery components (e.g., ALG-2-GFP, ALIX-GFP) enables comparative analysis of translocation kinetics and co-localization .

• Focal laser injury models: Using focal laser to create localized membrane injuries provides a controlled system for studying repair dynamics .

• Spinning disk confocal microscopy: This technique offers the temporal resolution needed to capture the rapid translocation events during membrane repair.

For optimal results, researchers should:

• Use physiologically relevant cell models (e.g., HeLa cells, MCF7-p95ErbB2 cells)

• Maintain appropriate calcium concentrations in imaging media

• Employ quantitative analysis of fluorescence intensity at injury sites

• Compare translocation timing with control proteins

How can researchers effectively establish ANXA7 knockdown or knockout models?

Multiple approaches can be employed to create ANXA7 knockdown or knockout models, each with specific advantages:

RNA interference (siRNA/shRNA):

• Lenti-ANXA7 siRNA with sequence 5′-GACCAGAGGCAACAAATTAAA-3′ has proven effective in rat models

• Administration via intraventricular injection (1 × 10^6 TU in 10 μl) demonstrated successful knockdown

• Timing: Administer 5 days before experimental procedures for optimal effect

CRISPR/Cas9 gene editing:

• Successfully implemented in MCF7-p95ErbB2 cells to generate complete ANXA7 knockout (A7-CRISPR cells)

• Verification of knockout should be performed via Western blot analysis

• Rescue experiments with ANXA7-RFP expression provide important controls

Dominant-negative mutants:

• The dominant-negative triple mutant (DNTM/DN-ANXA7) targeting the C-terminal endonexin-fold repeats offers an alternative approach

• This mutant suppresses wild-type ANXA7 function without completely eliminating the protein

For all approaches, researchers should:

• Verify knockdown/knockout efficiency via Western blot or qPCR

• Include appropriate controls (scramble siRNA, vector-only, etc.)

• Conduct rescue experiments when possible to confirm specificity

• Consider potential compensatory mechanisms by other annexin family members

What assays are most reliable for measuring ANXA7-mediated membrane repair?

Several complementary assays can effectively evaluate ANXA7's role in membrane repair:

• Laser injury and FM dye influx assay: This approach allows quantification of membrane repair kinetics by measuring the influx of membrane-impermeable dyes following focal laser injury .

• Calcium imaging during repair: Since ANXA7 function is calcium-dependent, simultaneous imaging of calcium dynamics and ANXA7 translocation provides insights into the relationship between calcium signaling and repair mechanisms .

• ESCRT III component recruitment: Measuring the recruitment kinetics of ALG-2, ALIX, and other ESCRT III machinery components in ANXA7 knockout/knockdown cells versus controls .

• Shedding assay: Quantifying the release of membrane vesicles following injury can assess ANXA7's role in ESCRT III-mediated membrane shedding .

• Cell survival assays: Comparing survival rates following membrane damage in ANXA7-normal versus ANXA7-deficient cells provides functional outcomes measurement.

For reliable results, researchers should:

• Standardize injury parameters (laser power, exposure time)

• Control for calcium concentration in experimental media

• Conduct multiple technical and biological replicates

• Employ appropriate statistical analyses for time-course data

What are the best methods for studying ANXA7-protein interactions?

Multiple complementary techniques can be employed to study ANXA7's interactions with other proteins:

• Co-immunoprecipitation (Co-IP): Effective for confirming direct protein-protein interactions, as demonstrated in studies of ANXA7-SRI interaction in HCC .

• Proximity ligation assay (PLA): Provides in situ visualization of protein interactions at the subcellular level with high specificity.

• Fluorescence resonance energy transfer (FRET): Enables real-time monitoring of protein interactions in living cells with high sensitivity.

• Live-cell co-localization imaging: As demonstrated in studies of ANXA7 with ALG-2 and ALIX, this approach reveals the spatial and temporal dynamics of interactions during cellular processes like membrane repair .

• Yeast two-hybrid screening: Useful for identifying novel ANXA7 binding partners in an unbiased manner.

• Protein domain mapping: Through truncation and mutation analysis, researchers can identify specific domains responsible for protein interactions, as demonstrated with the dominant-negative triple mutant approach .

When designing interaction studies, researchers should:

• Include appropriate negative controls (non-interacting proteins)

• Verify interactions using multiple complementary methods

• Consider the calcium dependence of many ANXA7 interactions

• Validate in vitro findings with in situ or in vivo approaches

How does the dominant-negative mutant of ANXA7 affect calcium signaling pathways?

The dominant-negative triple mutant (DNTM/DN-ANXA7) provides valuable insights into ANXA7's calcium-dependent functions:

• The DNTM targets the four C-terminal endonexin-fold repeats (GX(X)GT) within ANXA7's annexin repeats, which are critical for calcium binding .

• This mutation dramatically alters calcium and phospholipid binding properties, resulting in:

  • Suppressed ability to fuse with artificial membranes

  • Altered membrane fusion rates

  • Changes in phosphatidylserine exposure on the cell surface

• The mutant affects calcium signaling through:

  • Differential IP3 receptor expression

  • Modified calcium-dependent cellular processes including membrane permeabilization and apoptosis

  • Altered PI3K/AKT/mTOR pathway modulation

These findings highlight the critical importance of ANXA7's calcium binding domains for its tumor suppressor function, suggesting that the calcium signaling and membrane fusion activities of ANXA7 are integral to its role in preventing tumorigenesis .

For researchers investigating the dominant-negative mutant approach, recommended methodologies include:

• In vitro membrane fusion assays

• Calcium imaging with fluorescent indicators

• Phospholipid binding studies

• Western blot analysis of calcium-dependent signaling pathways

• Cell viability and apoptosis assays

What is the molecular mechanism by which ANXA7 recruits ESCRT III machinery to damaged membranes?

ANXA7 plays a crucial role in the recruitment of ESCRT III machinery to damaged plasma membranes through a well-defined molecular mechanism:

• Following membrane injury, ANXA7 rapidly translocates to the damaged site (within 20 seconds) in a calcium-dependent manner .

• ANXA7 is specifically required for the recruitment of ALG-2 (calcium-binding protein) to the injury site. In ANXA7 knockout cells:

  • ALG-2-GFP translocation to injury sites is delayed

  • ALG-2 accumulation becomes spatially diffuse rather than localized

  • This defect can be rescued by re-expressing ANXA7-RFP

• ANXA7-dependent ALG-2 recruitment is essential for the subsequent recruitment of ALIX (ALG-2-interacting protein X) to the damaged membrane:

  • In control cells lacking ANXA7, ALIX-GFP accumulates in a spatially diffuse manner

  • Expression of ANXA7-RFP causes co-localized accumulation of ALIX-GFP at the damage site

• The ANXA7-ALG-2-ALIX interaction initiates the buildup of ESCRT III components at the injury site, enabling the shedding of damaged membrane during the repair process .

This sequential recruitment process (ANXA7 → ALG-2 → ALIX → ESCRT III components) represents a critical pathway for plasma membrane repair following injury.

How do contradictory findings about ANXA7 in different cancers inform our understanding of context-dependent protein function?

The apparently contradictory roles of ANXA7 across different cancer types provide important insights into context-dependent protein function:

• Opposing roles in different cancers:

  • In hepatocellular carcinoma (HCC), ANXA7 promotes EMT and contributes to tumor aggressiveness

  • In glioblastoma multiforme, loss of ANXA7 expression correlates with greater malignancy

  • In prostate cancer, the dominant-negative ANXA7 mutant inhibits tumor cell proliferation

• Potential explanations for these contradictions:

  a) Tissue-specific protein interactions:

  • In HCC, ANXA7 interacts with SRI to promote EMT

  • Different binding partners in other tissues may result in different functional outcomes

  b) Calcium signaling environment:

  • Variations in calcium homeostasis across tissue types may affect ANXA7 function

  • The PI3K/AKT/mTOR pathway modulation by ANXA7 may differ between cancer types

  c) Isoform expression:

  • The balance between the 47 kDa and 51 kDa isoforms may vary between tissues

  • Different isoforms may have different functions or binding partners

• Methodological considerations for researchers:

  • Studies should include multiple cancer cell lines

  • Tissue-specific knockdown/knockout models should be employed

  • Interaction studies should identify tissue-specific binding partners

  • Calcium signaling should be assessed in a tissue-specific context

This apparent contradiction highlights the importance of cellular context in protein function and emphasizes the need for comprehensive studies across multiple model systems.