S100A13 Human

What is S100A13 and what distinguishes it from other S100 family proteins?

S100A13 is a member of the S100 protein family characterized by two distinct EF-hand calcium-binding motifs with different Ca²⁺ affinities. Unlike some S100 proteins, S100A13 contains a highly charged carboxyl-terminal domain that facilitates specific protein interactions. It lacks a classical signal peptide sequence, which enables its participation in non-classical secretion pathways. S100A13 is widely expressed across various tissues with particularly high expression in the thyroid gland. In smooth muscle cells, it co-expresses with other family members in the nucleus and stress fibers, suggesting diverse functionality . What distinguishes S100A13 is its unique ability to bind copper and form multiprotein complexes that facilitate the stress-dependent release of specific proteins like FGF1 and IL-1α .

How is the S100A13 gene typically expressed in human tissues?

The S100A13 gene is part of a cluster of at least 13 S100 genes located on chromosome 1q21. Expression analysis shows that S100A13 is widely distributed across multiple human tissues, with notably high expression in the thyroid gland . In pathological contexts, S100A13 shows significant upregulation in high-grade vascularized gliomas, where its expression correlates with microvessel density and tumor grading . Research indicates differential expression patterns in normal versus diseased tissues, with particular overexpression noted in various cancer types. The gene responds to cellular stress conditions, which can trigger increased expression levels as part of inflammatory and angiogenic responses .

What are the primary physiological functions of S100A13 in normal human cells?

S100A13 serves several critical physiological functions:

• Calcium homeostasis regulation - S100A13 participates in maintaining cellular calcium balance, with wild-type S100A13 shown to restore calcium homeostasis in cells with defective calcium signaling .

• Non-classical protein secretion - S100A13 acts as a template molecule that facilitates the stress-dependent release of proteins lacking classical signal sequences, including IL-1α, FGF1, and prothymosin-α .

• Inflammatory response modulation - It regulates the secretion of pro-inflammatory cytokines, particularly IL-1α, thus influencing inflammatory cascades .

• Mitochondrial function - S100A13 affects mitochondrial biogenesis, density, and polarization, contributing to cellular energy metabolism and reactive oxygen species (ROS) management .

Under normal conditions, these functions maintain cellular homeostasis, but dysregulation can contribute to pathological states including fibrosis and cancer .

What structural changes occur when S100A13 binds to calcium?

When S100A13 binds calcium, it undergoes significant conformational changes that expose hydrophobic surfaces, facilitating its interaction with target proteins. The protein contains two distinct EF-hand domains with different calcium affinities - the C-terminal canonical EF-hand has higher affinity for Ca²⁺ compared to the N-terminal pseudo EF-hand . Calcium binding alters the orientation of helices within these domains, particularly affecting the hinge region between helices II and III. These structural rearrangements are critical for enabling S100A13 to recognize and bind partner proteins involved in non-classical secretion pathways. The calcium-bound form also facilitates multiprotein complex formation, which is essential for the stress-dependent release of proteins like FGF1 and IL-1α. Additionally, calcium binding enhances S100A13's ability to associate with copper, which further modulates its function in export processes .

What is known about the IL-1α-S100A13 complex structure?

The IL-1α-S100A13 complex forms a heterotetrameric structure that is critical for the non-classical secretion of IL-1α. NMR studies have revealed specific interfacial regions between these proteins. The complex formation involves ambiguous interaction restraints defined by chemical shift perturbations observed upon binding. Active residues at the interface are those experiencing significant chemical shift perturbations and having relative residue accessible surface areas larger than 50% for either side chain or backbone atoms . Passive residues are defined as non-accessible surface residues with relative accessible surface areas smaller than 50%. The solution structure was determined using rigid-body docking with the HADDOCK protocol, which employed 5000 docking trials to establish the most stable configuration . This complex represents the first step in IL-1α export, acting as an assembly platform for additional proteins in the multiprotein aggregate that ultimately facilitates translocation across the plasma membrane. The specific structural features of this complex explain why anti-inflammatory drugs like amlexanox can inhibit the secretion pathway by interfering with these protein-protein interactions .

How is S100A13 involved in the pathogenesis of pulmonary fibrosis?

S100A13 mutations have been identified in patients with atypical and progressive interstitial pulmonary fibrosis. Research shows that digenic mutations in both S100A3 and S100A13 disrupt calcium homeostasis, which plays a pivotal role in the disease pathogenesis . Patient-derived fibroblasts with these mutations exhibit impaired intracellular calcium signaling, with decreased responses to bradykinin stimulation and ionomycin compared to control cells. The mechanistic link between S100A13 dysfunction and fibrosis involves several pathways:

• Dysregulated inflammatory cytokine secretion - Patient cells show over 2-fold higher constitutive IL-1α secretion (42.2 ± 1.7 pg/mL vs. 19 ± 1.1 pg/mL in controls, p < 0.0001), as well as substantially elevated levels of IL-6 (6775 ± 29 pg/mL vs. 445 ± 14 pg/mL), IL-8 (11,716 ± 184 pg/mL vs. 147 ± 7 pg/mL), and MCP-1 (10,331 ± 830 pg/mL vs. 1201 ± 33 pg/mL) .

• Mitochondrial dysfunction - Patient cells demonstrate increased mitochondrial mass and hyperpolarization, which correlate with elevated ROS production .

• Impaired calcium signaling - Mutations disrupt both receptor-mediated calcium signaling and calcium store responses, creating a pro-fibrotic cellular environment .

Most notably, treatment with recombinant wild-type S100A13 can normalize inflammatory cytokine secretion and restore calcium signaling in patient-derived cells, highlighting its potential therapeutic role .

What is the significance of S100A13 in tumor angiogenesis?

S100A13 serves as a key marker and mediator of angiogenesis in human cancers, particularly in astrocytic gliomas. Research has demonstrated that S100A13 expression significantly correlates with tumor grading and microvessel density in these aggressive brain tumors . The protein functions as a key regulator of the stress-dependent release of FGF1, which is a prototypical member of the FGF protein family involved in stimulating blood vessel formation. S100A13's role in angiogenesis involves:

• Formation of a multiprotein aggregate responsible for FGF1 release, enhancing the export of this potent angiogenic factor in response to cellular stress .

• Copper binding capability that facilitates FGF1 export during stress conditions, providing mechanistic support for its pro-angiogenic function .

• Co-expression with vascular endothelial growth factor A (VEGF-A), another powerful angiogenic factor that is significantly upregulated alongside S100A13 in high-grade vascularized gliomas .

Studies examining 26 astrocytic gliomas found that while FGF1 was equally expressed across most tumors, both S100A13 and VEGF-A showed significant upregulation specifically in high-grade, highly vascularized tumors . This pattern suggests that S100A13 plays a critical role in the angiogenic switch during tumor progression, making it both a potential biomarker and therapeutic target in cancer treatment strategies.

How do mutations in S100A13 affect cellular calcium homeostasis?

Mutations in S100A13 significantly disrupt calcium homeostasis through multiple mechanisms. Studies of patient-derived fibroblasts carrying S100A13 mutations revealed impaired responses to both receptor-mediated calcium signaling (via bradykinin) and direct calcium store activation (via ionomycin) . These disruptions manifest as:

• Reduced calcium transients - Patient cells show significantly diminished calcium release in response to stimulation compared to control cells.

• Altered mitochondrial calcium handling - Mutant cells exhibit hyperpolarized mitochondria with greater mitochondrial mass, suggesting dysregulated mitochondrial calcium management .

• Disrupted calcium-dependent signaling pathways - The calcium homeostasis disruption triggers aberrant activation of inflammatory and fibrotic pathways.

Remarkably, transfection of wild-type S100A13 into patient-derived cells restored calcium responses to levels comparable to control cells. Similarly, treatment with recombinant S100A13 protein (500 ng/mL) significantly restored bradykinin response compared to untreated cells (2.94 ± 0.33 fold compared to 1.30 ± 0.08, p < 0.0001), achieving response levels similar to normal control cells (2.77 ± 0.11) . These findings demonstrate that S100A13 is essential for maintaining proper calcium signaling, and that providing functional S100A13 can correct the calcium homeostasis defects caused by mutations.

What are the recommended methods for producing recombinant S100A13 protein?

Recombinant S100A13 protein production requires specific methodological approaches to ensure proper structure and function. Based on established protocols, the recommended method involves:

• Expression system optimization - BL21(DE3) E. coli strains have proven effective for S100A13 expression. The protein can be expressed using standard LB medium for unlabeled protein, or isotopically labeled media for NMR studies .

• Fusion protein strategy - GST-tagged S100A13 fusion constructs facilitate efficient expression and purification. The coding sequence should be preceded by the consensus Kozak sequence for optimal expression .

• Purification protocol:

  • Initial capture using GST-Sepharose column chromatography

  • Washing with PBS to remove non-specifically bound proteins

  • Elution with 10 mM glutathione and 50 mM Tris-HCl (pH 8.0)

  • Buffer exchange into PBS

  • Thrombin cleavage (50 μg for 10-12 hours) to remove the GST tag

  • Second GST column purification to separate the cleaved protein

  • Final polishing via gel filtration on Superdex-75 using 25 mM sodium phosphate (pH 6.5) containing 100 mM NaCl and 2 mM calcium chloride

For mammalian expression, codon-optimized sequences with appropriate tags (such as HA or MYC) have been successfully used. The S100A13 coding sequence can be cloned downstream of a CMV promoter in mammalian expression vectors such as pVQAd5CMVK-NpA or gWIZ Blank Mammalian Expression Vector . Verification of all constructs by bi-directional sequencing is essential to confirm sequence integrity.

What experimental approaches can detect S100A13-mediated calcium signaling changes?

Several experimental approaches are effective for detecting S100A13-mediated calcium signaling changes:

• Live-cell calcium imaging - Utilizing calcium-sensitive fluorescent dyes (such as Fura-2 AM) to monitor real-time changes in intracellular calcium concentrations in response to stimuli like bradykinin or ionomycin. This approach can directly measure the impact of S100A13 on receptor-mediated calcium signaling and calcium store responses .

• Calcium flux assays - Measuring calcium mobilization in response to specific agonists, with readouts normalized to baseline levels. The fold increase in calcium signal provides quantitative assessment of calcium signaling efficiency .

• Complementation experiments - Transfection of wild-type or mutant S100A13 into cells, followed by calcium response measurements to determine functional recovery. This approach has successfully demonstrated that wild-type S100A13 can restore calcium signaling in cells with mutant S100A13 .

• Recombinant protein treatment - Application of purified recombinant S100A13 (typically at 500 ng/mL) to cells followed by stimulation with calcium mobilizing agents. This method has shown that extracellular S100A13 can normalize calcium responses in cells with defective S100A13 function .

• Mitochondrial functional assays - Since S100A13 affects mitochondrial calcium handling, measuring mitochondrial membrane potential, mitochondrial mass, and calcium uptake provides insights into the broader impact of S100A13 on cellular calcium homeostasis .

These approaches should be used in combination to comprehensively characterize S100A13's role in calcium signaling under both normal and pathological conditions.

What techniques effectively characterize S100A13-protein interactions?

Several advanced techniques have proven effective for characterizing S100A13-protein interactions:

• Nuclear Magnetic Resonance (NMR) Spectroscopy - This has been the gold standard for determining the solution structure of S100A13 complexes. Specific experiments include:

  • 13C and 15N (F1)-filtered, 13C (F2)-edited, and 12C (F3)-filtered NOESY experiments

  • Chemical shift perturbation analysis to identify binding interfaces

  • Ambiguous interaction restraints defined by residues showing significant chemical shift changes

• Isothermal Titration Calorimetry (ITC) - Provides thermodynamic parameters of binding interactions between S100A13 and its partners, offering quantitative data on binding affinity, stoichiometry, and energetics .

• Co-immunoprecipitation assays - Using tagged versions of S100A13 (HA-tagged or MYC-tagged) to pull down interacting proteins from cell lysates, followed by immunoblotting to identify specific interaction partners .

• Molecular docking - Computational approaches such as HADDOCK protocol with rigid-body docking trials have been successfully employed to model S100A13-protein complexes based on experimental constraints .

• Fluorescence-based interaction assays - Including fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) to visualize protein interactions in living cells.

• Cross-linking coupled with mass spectrometry - To identify interaction interfaces and specific residues involved in S100A13-protein complexes.

For optimal results, a combination of these techniques should be employed to provide complementary data on the structural, thermodynamic, and functional aspects of S100A13 interactions with partner proteins.

Could recombinant S100A13 serve as a potential therapeutic for S100A13-deficient conditions?

Research indicates substantial promise for recombinant S100A13 as a therapeutic agent for conditions associated with S100A13 deficiency, particularly pulmonary fibrosis. Studies with patient-derived cells carrying S100A13 mutations have demonstrated that treatment with recombinant S100A13 protein can normalize multiple cellular dysfunction markers:

• Calcium signaling restoration - Recombinant S100A13 (500 ng/mL) significantly restored bradykinin-mediated calcium responses in patient cells to levels comparable to healthy controls (2.94 ± 0.33 fold increase vs. 2.77 ± 0.11 in controls) .

• Inflammatory cytokine normalization - Treatment with recombinant wild-type S100A13 reduced the pathologically elevated IL-1α secretion in patient cells to levels comparable to control fibroblasts .

• Mitochondrial function improvement - Combined S100A13 and S100A3 treatment reduced both abnormal mitochondrial density and hyperpolarization toward levels seen in control cells .

The research directly states that "treatment of patients' cells with recombinant S100A3 and S100A13 proteins is sufficient to normalize most of cellular responses, and may therefore suggest the use of these recombinant proteins in the treatment of this devastating disease" . This provides strong preliminary evidence supporting therapeutic potential. Future research should focus on delivery methods, optimal dosing regimens, and potential side effects of recombinant S100A13 therapy. Additionally, animal models of S100A13 deficiency would be valuable for preclinical evaluation before human clinical trials can be considered.

How might S100A13 inhibition affect angiogenesis in cancer treatments?

S100A13 inhibition represents a promising strategy for targeting angiogenesis in cancer treatments, particularly in aggressive tumors like high-grade gliomas. The mechanistic rationale for this approach is multifaceted:

• Disruption of FGF1 secretion - S100A13 is a key regulator of stress-dependent FGF1 release, a potent angiogenic factor. Inhibiting S100A13 would impair the non-classical export of FGF1, potentially reducing tumor vascularization .

• Correlation with tumor vascularity - Research has demonstrated that S100A13 expression significantly correlates with microvessel density and tumor grading in astrocytic gliomas, suggesting it plays a causative role in tumor angiogenesis .

• Co-expression with VEGF-A - S100A13 is upregulated alongside VEGF-A specifically in highly vascularized tumors, indicating it may participate in a coordinated angiogenic program that could be disrupted through inhibition .

What are the current knowledge gaps in S100A13 research that require further investigation?

Despite significant advances in S100A13 research, several critical knowledge gaps remain that warrant further investigation:

• Complete signaling network - While S100A13's role in non-classical secretion and calcium homeostasis is established, the full extent of its signaling network and downstream effectors remains incompletely characterized. Research is needed to identify all binding partners and how these interactions change under different physiological and pathological conditions .

• Regulatory mechanisms - The factors controlling S100A13 expression, post-translational modifications, and activity regulation are not fully understood. Investigation into transcriptional, translational, and post-translational regulation would provide valuable insights into how S100A13 function is modulated in different contexts .

• Tissue-specific functions - Despite known widespread expression, the tissue-specific roles of S100A13 remain largely unexplored beyond limited contexts like fibrosis and cancer. Research into its function in different organs and cell types would provide a more comprehensive understanding of its biological significance .

• Precise mechanism of membrane translocation - While S100A13 is known to facilitate non-classical protein secretion, the exact mechanism by which the S100A13-containing complexes cross the plasma membrane remains unclear and requires detailed characterization .

• Role in additional pathologies - Beyond pulmonary fibrosis and astrocytic gliomas, S100A13's potential involvement in other diseases warrants investigation, particularly in conditions involving dysregulated calcium signaling, inflammation, or protein secretion .

• Therapeutic targeting strategies - Development of specific modulators (both inhibitors and activators) of S100A13 function would provide valuable tools for research and potential therapeutics. Current knowledge of drug interactions with S100A13 is limited to a few compounds like amlexanox .

Addressing these knowledge gaps will require multidisciplinary approaches combining structural biology, cell signaling, animal models, and translational research.