STX4 Human
Description
Physiological Roles and Functions
STX4 is widely expressed in human tissues, including skeletal muscle, pancreatic β-cells, and neutrophils. Its functions extend beyond exocytosis to include mitochondrial dynamics and immune modulation.
Exocytosis and Glucose Metabolism
STX4 facilitates the fusion of glucose transporter 4 (GLUT4)-containing vesicles with the plasma membrane in skeletal muscle, enhancing insulin-stimulated glucose uptake . Reduced STX4 levels correlate with insulin resistance and mitochondrial dysfunction in diabetic models .
Mitochondrial Regulation
STX4 localizes to the outer mitochondrial membrane (OMM) and interacts with Drp1, a protein regulating mitochondrial fission-fusion balance. STX4 enrichment in skeletal muscle prevents HFD-induced mitochondrial fragmentation by promoting Drp1 phosphorylation at S637, favoring fusion .
β-Cell Protection and Inflammation
In pancreatic β-cells, STX4 inhibits NF-κB signaling by stabilizing IκBβ, reducing cytokine-induced apoptosis and chemokine (e.g., CXCL9, CXCL10) expression . Overexpression in β-cells enhances insulin secretion and resistance to streptozotocin-induced diabetes .
Research Findings and Mechanisms
Recent studies highlight STX4’s multifaceted roles:
Therapeutic Potential
STX4’s role in metabolic and immune regulation positions it as a candidate for treating diabetes and autoimmune diseases.
Diabetes Management
-
Skeletal Muscle: STX4 enrichment improves insulin sensitivity and mitochondrial function in HFD-fed mice, suggesting potential for reversing insulin resistance .
-
Pancreatic Islets: β-cell-specific STX4 overexpression enhances islet graft survival and function, offering a strategy for islet transplantation .
Autoimmune Disease
STX4 reduces islet inflammation by suppressing CXCL10 expression and boosting regulatory T-cell recruitment, delaying type 1 diabetes progression in NOD mice .
Detection and Experimental Applications
STX4 is detected using specific antibodies (e.g., MAB7894) validated for Western blot, immunoprecipitation, and immunocytochemistry . Key applications include:
Product Specs
Q&A
What is STX4 and what is its primary function in human cells?
STX4 (Syntaxin 4) is a SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein that plays a crucial role in exocytosis processes in human cells. Traditionally, STX4 has been characterized as a regulator of glucose transporter GLUT4 vesicle fusion at the plasma membrane in skeletal muscle, facilitating insulin-stimulated glucose uptake . Recent research has expanded our understanding of STX4 function, revealing its presence on or proximal to mitochondrial membranes and its role in regulating mitochondrial dynamics and function, particularly in the context of insulin resistance and diabetes . This dual localization suggests STX4 has functions beyond its classical role in plasma membrane exocytosis.
Where is STX4 primarily expressed in the human body?
STX4 is expressed in various tissues throughout the human body, with particularly significant expression in skeletal muscle. This is physiologically relevant because skeletal muscle is responsible for over 80% of glucose clearance in prediabetic and healthy individuals, making STX4's role in this tissue critical for whole-body glucose homeostasis . STX4 is also found in dendritic spines near glutamatergic synapses in the brain, where it may play a role in synaptic plasticity and retrograde synaptic signaling . The protein's expression levels can vary in different physiological states, with studies showing approximately 15% decrease in STX4 mRNA levels in skeletal muscle of diabetic versus healthy mice, suggesting potential similar patterns in humans .
How does STX4 contribute to glucose homeostasis?
STX4 contributes to glucose homeostasis primarily through its role in facilitating GLUT4 translocation to the plasma membrane in response to insulin stimulation in skeletal muscle. This process is essential for insulin-stimulated glucose uptake, which accounts for over 80% of glucose clearance in healthy individuals . Research has shown that global upregulation of STX4 causes a twofold increase in glucose uptake, highlighting its importance in preserving glucose homeostasis . Furthermore, skeletal muscle-specific STX4 enrichment has been demonstrated to reverse established insulin resistance in mouse models, restoring insulin sensitivity to normal levels even in the continued presence of a high-fat diet . This effect occurs without altering body weight, body composition, or food consumption, suggesting a direct metabolic effect on muscle tissue.
What methodologies are used to study STX4 localization in mitochondria?
Several complementary methodologies have been employed to study STX4 localization in mitochondria:
a) Immunogold labeling with high-resolution transmission electron microscopy (TEM): This technique involves using gold particle-conjugated antibodies specific to STX4, allowing visualization of the protein at the ultrastructural level. In studies, this method revealed STX4 localization on or proximal to the outer mitochondrial membrane in skeletal muscle .
b) Specificity controls: To confirm the specificity of mitochondrial STX4 localization, researchers used blocking peptides with wild-type muscle and muscle from STX4 knockout mice, which eliminated the immunogold signal at mitochondrial membranes .
c) Subcellular fractionation: Biochemical isolation of mitochondrial fractions from skeletal muscle tissue, followed by immunoblotting for STX4, provided additional evidence for STX4's presence in mitochondria .
This multi-method approach provides robust evidence for STX4's mitochondrial localization, challenging the traditional view that STX4 acts solely at the plasma membrane.
How does skeletal muscle-specific STX4 enrichment affect insulin resistance?
Skeletal muscle-specific STX4 enrichment (skmSTX4tg) has been shown to reverse established insulin resistance in mouse models of diet-induced obesity. In studies utilizing a high-fat diet (HFD) paradigm, transgenic mice with inducible STX4 expression in skeletal muscle demonstrated remarkable improvements in insulin sensitivity after just 4 weeks of STX4 induction, despite continued consumption of the HFD .
| Parameter | HFD-fed Control Mice | HFD + STX4 Mice | Chow-fed Mice |
|---|---|---|---|
| Insulin Sensitivity | Reduced | Restored to normal | Normal |
| Respiratory Exchange Ratio | ~0.77 (fat metabolism) | >0.81 (increased carbohydrate use) | ~0.88 (carbohydrate metabolism) |
| Spontaneous Activity | Reduced | Similar to chow-fed | Normal |
| Body Weight | Increased | Unchanged by STX4 induction | Normal |
| Food Consumption | Unchanged | Unchanged by STX4 induction | Normal |
These data demonstrate that skmSTX4tg induction is sufficient to restore insulin sensitivity to levels comparable to those of chow-fed mice, effectively reversing the diabetogenic effects of the HFD without altering body weight or food intake .
What mechanisms link STX4 to mitochondrial dynamics and function?
Several interconnected mechanisms link STX4 to mitochondrial dynamics and function:
a) Regulation of mitochondrial fission/fusion balance: STX4 enrichment promotes mitochondrial fusion and prevents fragmentation by interacting with the mitochondrial fission protein Dynamin-related protein 1 (Drp1) and affecting its phosphorylation state .
b) AMPK-mediated signaling: STX4 enhances AMPK activity, which leads to increased phosphorylation of Drp1 at serine 637 (S637). This phosphorylation inhibits Drp1's fission activity, favoring fusion and resulting in more elongated mitochondria .
c) Mitochondrial morphology: Under high-fat diet conditions, STX4 enrichment prevents mitochondrial fragmentation and vacuolization, preserving mitochondrial structure with organized cristae .
d) Respiratory capacity: STX4 enrichment increases maximal mitochondrial respiration, as measured by oxygen consumption rates in primary myofibers, indicating improved mitochondrial function .
These findings collectively suggest that STX4's mitochondrial effects contribute significantly to its insulin-sensitizing properties and may represent a novel therapeutic target for improving metabolic health.
How does STX4 interact with Drp1 and AMPK signaling pathways?
STX4 interacts with Drp1 (Dynamin-related protein 1) and AMPK (AMP-activated protein kinase) signaling pathways through several mechanisms:
a) STX4-Drp1 direct interaction: Research has demonstrated that STX4 physically interacts with Drp1, a key regulator of mitochondrial fission. This interaction may influence Drp1's localization and activity at the mitochondrial membrane .
b) AMPK activation: STX4 enrichment in skeletal muscle leads to increased AMPK activity, as evidenced by elevated phosphorylation of AMPK at threonine 172 (T172), a marker of AMPK activation .
c) Drp1 phosphorylation: Activated AMPK phosphorylates Drp1 at serine 637 (S637), which inhibits Drp1's fission activity. STX4 enrichment increases this inhibitory phosphorylation, shifting the balance toward mitochondrial fusion rather than fission .
The exact mechanism by which STX4 activates AMPK remains unclear. Possibilities include: 1) STX4 may modulate the AMP to ATP ratio in skeletal muscle to activate AMPK, or 2) STX4 might activate AMPK independent of AMP/ATP ratios via modulating cellular glucose levels . These mechanistic details represent important areas for future research.
What experimental models are used to study STX4 function in vivo?
Several experimental models have been developed to study STX4 function in vivo:
a) Transgenic mouse models:
-
Skeletal muscle-specific STX4 enrichment (skmSTX4tg) mice: These mice contain doxycycline-inducible STX4 expression specifically in skeletal muscle, allowing temporal control of STX4 overexpression .
-
Global STX4 transgenic (gbSTX4tg) mice: These mice overexpress STX4 in all tissues, useful for comparing tissue-specific versus global effects .
-
STX4 knockout (skmSTX4-KO) mice: These mice lack STX4 expression specifically in skeletal muscle, serving as important controls and for loss-of-function studies .
b) Diet-induced obesity models:
-
High-fat diet (HFD) feeding: Typically using a 45% fat diet to induce insulin resistance and metabolic dysfunction, creating a model to test STX4's therapeutic potential .
c) Metabolic phenotyping approaches:
-
Insulin tolerance tests (ITT) to assess whole-body insulin sensitivity .
-
Metabolic caging units to measure respiratory exchange ratio, energy expenditure, and spontaneous physical activity .
-
Seahorse XFe24 flux analyzer to measure mitochondrial respiration in isolated primary myofibers .
These models collectively provide powerful tools to investigate STX4's role in metabolic health and mitochondrial function in physiologically relevant contexts.
How can researchers differentiate between plasma membrane and mitochondrial STX4 functions?
Differentiating between plasma membrane and mitochondrial STX4 functions requires specialized approaches:
a) Subcellular fractionation: Isolating purified plasma membrane and mitochondrial fractions followed by immunoblotting for STX4 can help attribute specific activities to STX4 in each compartment .
b) Immunogold electron microscopy: This technique provides ultrastructural localization of STX4, allowing visualization of the protein at both plasma membrane and mitochondrial sites with high precision .
c) Functional readouts: Using specific assays for plasma membrane functions (GLUT4 translocation, glucose uptake) versus mitochondrial functions (respiratory capacity, fission/fusion dynamics) can help attribute phenotypes to STX4 at different locations .
d) Blocking peptides and knockout controls: These provide essential validation of antibody specificity when determining subcellular localization of STX4 .
These complementary approaches allow researchers to delineate the distinct roles of STX4 pools at different subcellular sites, advancing our understanding of this protein's multifaceted functions.
What techniques are used to measure STX4-mediated changes in mitochondrial respiration?
Several techniques are used to measure STX4-mediated changes in mitochondrial respiration:
a) Seahorse XFe24 flux analyzer: This platform measures oxygen consumption rates (OCR) in real-time in intact cells or isolated mitochondria. In STX4 research, this technique revealed that primary myofibers from STX4-enriched mice had greater maximal mitochondrial respiration compared to controls .
b) Parameters typically measured include:
-
Basal respiration
-
ATP-linked respiration
-
Maximal respiratory capacity
-
Spare respiratory capacity
-
Proton leak
-
Non-mitochondrial respiration
c) Electron transport chain (ETC) subunit quantification: Western blotting for ETC complex components can assess mitochondrial respiratory capacity at the protein level .
d) Respiratory exchange ratio (RER) measurements: Using metabolic caging units to measure the ratio of CO2 produced to O2 consumed, which indicates whether carbohydrates or fats are the predominant metabolic fuel source .
These complementary approaches allow researchers to comprehensively assess how STX4 influences mitochondrial function at multiple levels.
How do STX4 levels change in diabetic versus healthy human skeletal muscle?
Research has shown that STX4 expression is altered in diabetic conditions:
a) mRNA expression: STX4 mRNA levels are decreased by approximately 15% in skeletal muscle of diabetic versus healthy mice, suggesting a similar pattern may exist in humans .
b) Correlation with insulin sensitivity: Lower STX4 levels correlate with decreased insulin sensitivity, supporting the hypothesis that STX4 reduction contributes to skeletal muscle insulin resistance .
c) Therapeutic implications: The observation that STX4 levels are reduced in diabetic muscle provides rationale for therapeutic approaches aimed at restoring or enhancing STX4 expression or function .
These findings suggest that STX4 reduction may be both a marker and a contributor to diabetic pathophysiology, making it a potential therapeutic target for developing new interventions for prediabetes and type 2 diabetes.
How can researchers assess the impact of STX4 on spontaneous physical activity and metabolism?
Researchers can assess the impact of STX4 on spontaneous physical activity and metabolism through several methodologies:
a) Metabolic caging systems: These specialized cages continuously monitor:
-
Respiratory exchange ratio (RER): Measures the ratio of CO2 produced to O2 consumed, indicating whether carbohydrates (higher RER ~0.9) or fats (lower RER ~0.7) are the predominant fuel source
-
Energy expenditure: Calculated from oxygen consumption and carbon dioxide production
-
Spontaneous activity: Infrared beam breaks quantify horizontal and vertical movement
-
Distance traveled: Average distance traveled per hour, particularly during active periods (night for nocturnal animals)
b) Correlation analyses: Statistical methods to assess relationships between:
-
STX4 expression levels and activity parameters
-
Activity levels and metabolic outcomes (insulin sensitivity, body composition)
This data suggests that STX4 enrichment not only improves metabolic parameters but also increases spontaneous physical activity in HFD-fed mice to levels similar to those of healthy controls, providing a comprehensive assessment of STX4's physiological effects .
What are the parallels between STX4 enrichment and exercise effects on skeletal muscle?
Research has identified intriguing parallels between the effects of STX4 enrichment and those of endurance exercise on skeletal muscle metabolism:
a) Mitochondrial adaptations: Both interventions improve maximum respiratory capacity and change mitochondrial dynamics. Exercise induces mitochondrial elongation, similar to the effects observed with STX4 enrichment .
b) Metabolic improvements: Both exercise and STX4 enrichment improve glucose tolerance and insulin sensitivity .
c) Spontaneous activity: Exercise-trained mice show increased spontaneous activity levels compared to untrained controls, similar to the increased spontaneous activity observed in skmSTX4tg mice .
d) Key differences: Unlike exercise, STX4 enrichment does not cause weight loss or reduced percentage of abdominal body fat. Mice remain obese after STX4 induction, whereas exercise typically causes weight loss .
These parallels suggest that STX4 may mediate some of the beneficial effects of exercise on metabolism, though not all. Understanding these similarities and differences could help develop "exercise mimetics" that target STX4 pathways to provide some exercise benefits for individuals unable to exercise conventionally.
Product Science Overview
Syntaxin-4 is widely expressed in various tissues and cells. The human Syntaxin-4 protein consists of 297 amino acids and includes several important domains and motifs :
- Basolateral targeting motif: Located at amino acids 24-29, this motif is crucial for targeting the protein to the basolateral membrane in epithelial cells.
- Coiled-coil domain (Habc): Spanning amino acids 43-163, this domain is involved in protein-protein interactions.
- S-nitrosylation site: Found at amino acid 141, this site regulates the fusion activity of Syntaxin-4.
- t-SNARE domain: Located at amino acids 200-262, this domain is essential for the formation of the SNARE complex.
- C-terminal transmembrane domain: Spanning amino acids 276-296, this domain anchors the protein to the membrane.
In the process of membrane fusion, Syntaxin-4 interacts with other SNARE proteins, including VAMPs (Vesicle-Associated Membrane Proteins) and SNAPs (Soluble NSF Attachment Proteins) . These interactions facilitate the fusion of vesicles with target membranes, allowing the transfer of cargo between cellular compartments. SM (Sec1/Munc18-like) accessory proteins act as catalysts in this process .
Recombinant human Syntaxin-4 is produced using E. coli expression systems. The recombinant protein typically includes a C-terminal 6-His tag for purification purposes . It is available in various formulations, including carrier-free versions that do not contain Bovine Serum Albumin (BSA), which can interfere with certain applications .
Recombinant human Syntaxin-4 is used in various research applications, including:
- Cell adhesion studies: It has been shown to induce adhesion of ATDC5 mouse chondrogenic cells .
- Protein-protein interaction studies: The recombinant protein can be used to study interactions with other SNARE proteins and regulatory factors.
- Membrane fusion assays: Researchers use recombinant Syntaxin-4 to investigate the mechanisms of vesicle fusion and exocytosis.
Recombinant human Syntaxin-4 is typically lyophilized from a filtered solution in PBS and TCEP. It should be reconstituted at a concentration of 250 μg/mL in PBS . The protein is stable for up to 12 months when stored at -20 to -70°C, and for shorter periods at 2 to 8°C under sterile conditions after reconstitution .
