APOA1 Human, His

What is the basic structure of human Apolipoprotein A-I?

Human Apolipoprotein A-I (apoA-I) is a 243-amino acid protein with a molecular weight of approximately 28kDa. Following translation and removal of an N-terminal signal peptide, mature apoA-I is secreted as a lipid-poor protein. Its structure contains ten consecutive helical regions that are critical for its biophysical properties, allowing it to spontaneously solubilize lipids in aqueous environments. These amphipathic helical motifs enable apoA-I to function effectively in lipid transport and metabolism. The protein constitutes approximately 70% of the protein component of high-density lipoprotein (HDL) particles in physiological conditions .

What are the primary functions of APOA1 in human physiology?

The APOA1 gene provides instructions for making apolipoprotein A-I, which serves several critical physiological functions. Primarily, apoA-I is a major component of HDL, facilitating the transport of cholesterol and phospholipids through the bloodstream from body tissues to the liver. At the cellular level, apoA-I attaches to cell membranes and promotes the movement of cholesterol and phospholipids from inside cells to their outer surface. Once outside the cell, these substances combine with apoA-I to form HDL particles. Additionally, apoA-I triggers cholesterol esterification, converting cholesterol to a form that can be integrated into HDL and transported through the bloodstream. This process of removing excess cholesterol from cells is fundamental for maintaining cholesterol homeostasis and cardiovascular health . Beyond lipid metabolism, apoA-I also plays roles in modulating inflammatory responses and immune function .

How does APOA1 interact with key transport proteins in cholesterol metabolism?

ApoA-I participates in multiple protein-protein interactions essential for cholesterol metabolism. It stabilizes the ATP-binding cassette transporter 1 (ABCA1) at the cell membrane of hepatocytes and enterocytes, enabling ABCA1 to mediate the efflux of cellular phospholipids and free cholesterol to nascent discoid HDL particles. Each nascent HDL typically contains two to four molecules of apoA-I. ApoA-I also activates lecithin cholesterol acyl transferase (LCAT), an enzyme crucial for HDL maturation. In discoid or mature HDL particles, lipidated apoA-I interacts with ATP-binding cassette subfamily G member 1 (ABCG1), further contributing to reverse cholesterol transport. Additionally, HDL particles undergo remodeling through interaction with cholesteryl ester transfer protein (CETP). Finally, binding of HDL particles to the scavenger receptor class B type 1 (SR-BI) facilitates cholesterol transfer down a concentration gradient .

How is APOA1 gene expression regulated at the transcriptional level?

The regulation of human APOA1 gene expression involves complex control mechanisms at multiple levels. Transcription largely depends on two hormone response elements (HREs) proximal to the transcription start site, which bind members of the hormone nuclear receptor superfamily. Peroxisome proliferator-activated receptor-γ (PPARγ) plays a prominent role in APOA1 transactivation by interacting with these HREs as a heterodimer with RXRα. Other transcription factors involved include hepatocyte nuclear factor 4 (HNF4), which activates the APOA1 promoter, and ApoA-I Regulatory Protein 1 (ARP1/NR2F2), which represses it. HNF4 operates together with Sp1 in facilitating communication between the APOA1 promoter and enhancer sequences that recruit the basal transcriptional machinery .

What role do non-coding RNAs play in APOA1 expression?

ApoA-I expression is controlled in part by a long non-coding RNA called APOA1-AS, which is transcribed in the apolipoprotein gene cluster on chromosome 11q23.3. This lncRNA modulates suppressive epigenetic marks that lead to APOA1 transcriptional repression. Interestingly, tissues where apoA-I is predominantly expressed (liver, small intestine, and colon) show approximately 100-fold higher expression levels of APOA1 mRNA compared to APOA1-AS. In contrast, most other tissues have APOA1/APOA1-AS ratios less than one, suggesting tissue-specific regulation patterns. This differential expression pattern helps explain the tissue-specific production of apoA-I and provides insight into potential regulatory mechanisms that could be targeted in research or therapeutic applications .

What post-transcriptional mechanisms affect APOA1 expression?

Post-transcriptional mechanisms contribute significantly to the regulation of apoA-I expression under certain conditions. For example, research in mice fed high-fat diets has revealed an enrichment of polysomal fractions with APOA1 mRNAs, explaining the observed increase in apoA-I synthesis despite no changes in transcription rates. This finding suggests that translational efficiency, rather than transcriptional activation, may drive increased apoA-I production in response to dietary fat. Other potential post-transcriptional regulatory mechanisms may include mRNA stability factors, RNA-binding proteins, and microRNAs that affect APOA1 mRNA processing, export, stability, or translation efficiency. These mechanisms provide additional layers of control for fine-tuning apoA-I levels in response to physiological or pathological conditions .

Clinical Associations and Biomarkers

Research indicates that serum and plasma levels of apoA-I are significantly reduced in Alzheimer's disease (AD) patients compared to healthy controls. A comprehensive meta-analysis of 18 studies (1992-2017) examined apoA-I levels in 1,077 AD patients and 1,271 healthy controls. The analysis found that both serum (747 AD patients, 680 controls) and plasma (246 AD patients, 456 controls) apoA-I levels were significantly lower in AD patients. The standardized mean difference (SMD) was -1.16 with a 95% confidence interval of (-1.72, -0.59). The mechanistic link appears to involve apoA-I binding to amyloid β (Aβ), potentially affecting AD pathogenesis. This association suggests that apoA-I could serve as a biomarker for AD risk assessment or progression monitoring, though the relationship remains somewhat controversial and requires further investigation .

How do mutations in the APOA1 gene affect HDL levels and cardiovascular disease risk?

Mutations in the APOA1 gene cause familial HDL deficiency, an inherited condition characterized by abnormally low levels of HDL in the blood and an elevated risk for early-onset cardiovascular disease (often before age 50). These mutations produce altered apoA-I proteins that function abnormally in two main ways: some variants are less able to promote the removal of cholesterol and phospholipids from cells, decreasing the substrates available for HDL formation; other variants are less effective at stimulating cholesterol esterification, preventing proper integration of cholesterol into HDL particles. Both mutation types result in reduced HDL levels, which is believed to increase cardiovascular disease risk. Understanding these mutations provides insight into the structure-function relationship of apoA-I and potential therapeutic targets for addressing HDL deficiencies .

What are the best methods for producing recombinant human APOA1 with histidine tags?

For producing recombinant human APOA1 with histidine tags, researchers typically employ bacterial expression systems, particularly Escherichia coli. The protocol involves first cloning the human APOA1 cDNA into an expression vector containing a histidine tag sequence (commonly 6×His) either at the N-terminus or C-terminus of the protein. The N-terminal placement is generally preferred as it avoids interfering with the C-terminal domain involved in lipid interactions.

The expression vector is then transformed into an appropriate E. coli strain such as BL21(DE3) for protein production. After culture growth to optimal density (OD600 ~0.6-0.8), IPTG (isopropyl β-D-1-thiogalactopyranoside) at concentrations of 0.5-1 mM is added to induce protein expression, typically for 4-6 hours at 30°C to balance yield with proper folding.

The cells are then harvested by centrifugation, lysed (using sonication or pressure homogenization), and the His-tagged apoA-I is purified using immobilized metal affinity chromatography (IMAC) with nickel or cobalt resins. Further purification steps may include ion-exchange chromatography and size-exclusion chromatography to achieve high purity. The tag can be removed using specific proteases if necessary for downstream applications. This approach typically yields 5-20 mg of purified protein per liter of bacterial culture .

How can researchers effectively analyze the lipid-binding properties of APOA1?

To analyze the lipid-binding properties of apoA-I, researchers employ several complementary methodologies. One standard approach is the use of circular dichroism (CD) spectroscopy to measure changes in protein secondary structure upon lipid binding, as apoA-I undergoes a conformational shift from approximately 45% α-helical content in the lipid-free state to up to 75% when lipid-bound.

Isothermal titration calorimetry (ITC) provides quantitative measurements of binding affinities and thermodynamic parameters. Surface plasmon resonance (SPR) can measure real-time binding kinetics when lipids are immobilized on biosensor chips.

For more detailed structural analysis, researchers use electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling or hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map specific lipid-binding regions.

Functional lipid binding can be assessed through reconstitution assays where apoA-I is combined with phospholipids to form reconstituted HDL particles, followed by native gel electrophoresis, size-exclusion chromatography, or dynamic light scattering to characterize the resulting particles. Fluorescence-based assays using environment-sensitive probes like 1,6-diphenyl-1,3,5-hexatriene (DPH) can monitor the insertion of apoA-I into lipid membranes. For cellular studies, cholesterol efflux assays using radiolabeled or fluorescently labeled cholesterol measure the ability of apoA-I to promote cholesterol removal from cells .

What are the effective protocols for studying APOA1 interactions with ABC transporters?

To study apoA-I interactions with ABC transporters (particularly ABCA1 and ABCG1), researchers employ multiple complementary approaches. Cell-based assays typically use HEK293 cells or macrophages (like J774, THP-1, or primary cells) transfected with or naturally expressing these transporters. The most common functional assay is the cholesterol efflux assay, where cells are loaded with [³H]-cholesterol or fluorescently labeled cholesterol, then incubated with lipid-free or lipidated apoA-I, followed by measurement of radioactivity or fluorescence in the medium versus cell fractions.

For direct protein-protein interactions, co-immunoprecipitation (co-IP) can identify complexes formed between apoA-I and transporters. This is performed by isolating membrane fractions, solubilizing with mild detergents, then using antibodies against either apoA-I or the transporter, followed by SDS-PAGE and immunoblotting. Cross-linking techniques with reagents like DSP (dithiobis(succinimidyl propionate)) can stabilize transient interactions before co-IP.

Surface plasmon resonance (SPR) with purified components provides quantitative binding kinetics, while fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) systems are valuable for studying interactions in living cells. FRET pairs can be created by tagging apoA-I with a donor fluorophore and the transporter with an acceptor.

For structural studies, cryo-electron microscopy or hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map interaction interfaces. Mutagenesis studies targeting specific amino acids in both apoA-I and transporters help identify critical residues for these interactions .

How does APOA1 contribute to immune modulation and anti-inflammatory effects?

ApoA-I exerts immune-modulatory and anti-inflammatory effects through multiple mechanisms. It suppresses inflammatory responses by inhibiting the activation of monocytes, macrophages, and dendritic cells, partly by reducing the expression and secretion of pro-inflammatory cytokines and chemokines. At the molecular level, apoA-I can inhibit NF-κB activation, a key transcription factor in inflammation, and can modulate toll-like receptor signaling pathways.

The protein also influences adaptive immunity by affecting T cell proliferation and activation, potentially through interaction with T cell receptors or by modulating the function of antigen-presenting cells. In some contexts, apoA-I enhances regulatory T cell development, which helps maintain immune tolerance.

A significant mechanism of apoA-I's anti-inflammatory activity involves its interaction with cellular receptors including ABCA1, ABCG1, and SR-BI. For example, binding of apoA-I to ABCA1 not only facilitates cholesterol efflux but also activates signaling pathways that suppress inflammatory responses. Additionally, apoA-I binds to the beta-chain of ATP F1 synthase at the cell membrane (ecto-F1F0-ATPase), stimulating the hydrolysis of extracellular ATP to ADP and affecting signaling through purinergic receptors, many of which modify inflammatory and immune responses .

What are the molecular mechanisms behind APOA1's tumor-suppressive activities?

The tumor-suppressive activities of apoA-I involve multiple molecular mechanisms. Research suggests that apoA-I's anti-inflammatory properties contribute significantly to its antitumor effects, as chronic inflammation is a known driver of cancer development and progression. By suppressing inflammatory cytokine production and modulating immune cell function, apoA-I may create a less favorable environment for tumor growth.

ApoA-I also interacts with key transport proteins involved in reverse cholesterol transport, including ABCA1, ABCG1, and SR-BI. These interactions appear paradoxical in some research contexts—for instance, mice deficient in ABCG1 and ABCA1 display reduced growth of melanoma or bladder carcinoma tumors when fed a Western-type diet, yet excess apoA-I (which would typically enhance these transporters' function) also contributes to tumor suppression. This suggests that some anti-tumor activities of apoA-I may be mediated through alternative receptors.

One such alternative pathway involves the beta-chain of ATP F1 synthase (ecto-F1F0-ATPase) at the cell membrane, which serves as a high-affinity receptor for lipid-poor apoA-I. Binding of apoA-I to this receptor stimulates the hydrolysis of extracellular ATP to ADP and phosphate, potentially affecting signaling through P2 purinergic receptors that modify tumor growth. One of these receptors, P2Y13, may mediate signaling from ecto-F1F0-ATPase to SR-BI .

How do lipid-binding properties of APOA1 influence its function in different pathological contexts?

The lipid-binding properties of apoA-I significantly influence its function across various pathological contexts through several mechanisms. In cardiovascular disease, apoA-I's amphipathic helices enable efficient removal of excess cellular cholesterol through interactions with ABCA1, forming nascent HDL particles. The protein's ability to activate LCAT allows for cholesterol esterification and HDL maturation, crucial for reverse cholesterol transport. Mutations affecting these lipid-binding domains can lead to familial HDL deficiency and increased cardiovascular risk .

In neurodegenerative disorders like Alzheimer's disease, lipid-binding properties of apoA-I appear to influence its interaction with amyloid-β (Aβ). Research indicates that lipidation status of apoA-I affects its ability to bind Aβ and potentially prevent aggregation or promote clearance. The significantly reduced apoA-I levels in AD patients (SMD = -1.16; 95% CI (-1.72, -0.59)) suggest altered lipid metabolism may contribute to disease pathology .

In cancer contexts, the lipid-binding capacity of apoA-I influences tumor microenvironments by modulating cellular cholesterol homeostasis. This may affect lipid raft formation, which impacts signaling pathways critical for cancer cell survival and proliferation. Additionally, the different lipidation states of apoA-I (lipid-poor versus HDL-associated) appear to have distinct effects on inflammation and immune cell function relevant to tumor progression .

Research techniques that manipulate the lipid-binding properties of apoA-I, such as through site-directed mutagenesis or the development of apoA-I mimetic peptides, have provided valuable insights into structure-function relationships and potential therapeutic applications across these pathological contexts.

What are the current challenges in developing APOA1-based therapeutics?

Developing apoA-I-based therapeutics faces several significant challenges. Production obstacles include the high cost and complexity of manufacturing full-length recombinant apoA-I at clinical scales, with issues in maintaining proper folding and preventing aggregation. Purification processes must eliminate endotoxin contamination, particularly important for inflammatory disease applications.

Pharmacokinetic challenges include the relatively short half-life of apoA-I in circulation (approximately 3-5 days), necessitating frequent administration for chronic conditions. The protein is also susceptible to proteolytic degradation and oxidative modifications that can impair its functionality. Additionally, apoA-I must be delivered in a specific lipidation state depending on the therapeutic application, requiring complex formulation strategies.

Efficacy considerations present further complications. The multifunctional nature of apoA-I means that enhancing one function (like cholesterol efflux) may have unintended consequences on other activities (such as immune modulation). Additionally, genetic variations in patients' endogenous apoA-I or its interactive partners (ABCA1, LCAT, etc.) may lead to variable therapeutic responses.

Regulatory hurdles are also substantial, as apoA-I-based therapies represent a complex biological product requiring extensive characterization and quality control. Clinical trials must be designed to capture the diverse potential benefits of apoA-I across multiple biological systems, from lipid metabolism to inflammation and beyond. Despite these challenges, apoA-I mimetic peptides, which retain key functional domains while being easier to produce, represent a promising alternative approach currently under investigation .

What are the best techniques for studying APOA1 structure-function relationships?

Studying apoA-I structure-function relationships requires a multi-technique approach. X-ray crystallography has provided insights into certain structural domains, though crystallizing the full-length protein remains challenging due to its flexibility. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is particularly valuable for mapping conformational changes upon lipid binding or interaction with protein partners, revealing which regions undergo protective folding or increased exposure.

Site-directed mutagenesis coupled with functional assays remains a cornerstone methodology—systematic replacement of specific residues followed by assessment of lipid binding, cholesterol efflux promotion, or LCAT activation helps identify critical amino acids. Fluorescence spectroscopy with intrinsic tryptophan fluorescence or extrinsic environmentally sensitive probes can monitor conformational changes in real-time.

Molecular dynamics simulations have become increasingly important for predicting protein behavior in different environments, especially for modeling apoA-I-lipid interactions that are difficult to capture experimentally. Cryo-electron microscopy provides structural insights into apoA-I within HDL particles at near-atomic resolution.

Cross-linking mass spectrometry (XL-MS) identifies proximity relationships between amino acids, helping to validate structural models. For functional correlation, cholesterol efflux assays in cell systems with ABCA1 expression measure the primary physiological function, while reconstitution studies with synthetic phospholipids assess the ability to form HDL-like particles. These techniques, used in combination, provide complementary information about how specific structural features of apoA-I relate to its diverse functions in lipid transport and beyond .

How can researchers effectively analyze post-translational modifications of APOA1?

To effectively analyze post-translational modifications (PTMs) of apoA-I, researchers employ a multi-faceted mass spectrometry (MS) approach. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with collision-induced dissociation (CID) or electron transfer dissociation (ETD) fragmentation provides comprehensive PTM mapping. Sample preparation is critical—purification from plasma requires careful immunoprecipitation to maintain PTM integrity, while recombinant sources may have different modification patterns.

For oxidative modifications, which are particularly relevant in inflammatory contexts, targeted MS approaches can quantify specific oxidized residues. Methods like multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) offer sensitive quantification of oxidized methionine, tyrosine, or tryptophan residues. Two-dimensional gel electrophoresis followed by MS can separate apoA-I isoforms based on charge differences from PTMs.

Site-specific antibodies against common apoA-I modifications (such as MPO-mediated chlorination at Tyr192) enable Western blotting and immunohistochemistry applications. For functional analysis, researchers compare modified versus unmodified apoA-I in cholesterol efflux assays, LCAT activation tests, or anti-inflammatory assays to determine how specific PTMs affect different functions.

Top-down proteomics approaches, analyzing intact protein rather than peptide fragments, help maintain the relationship between co-occurring modifications. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can reveal how PTMs alter protein conformation and dynamics. These techniques, often used in combination, provide researchers with comprehensive insights into how apoA-I is modified in different physiological and pathological contexts .

What are the optimal cell models for studying APOA1 function in different research contexts?

The optimal cell models for studying apoA-I function vary based on the specific research context and biological process under investigation. For cholesterol efflux studies, THP-1 or J774 macrophages treated with cAMP to upregulate ABCA1 expression provide robust systems to measure apoA-I-mediated lipid removal. Primary macrophages, while more physiologically relevant, offer consistency challenges across preparations.

Hepatocyte models like HepG2, Huh7, or primary human hepatocytes are essential for studying apoA-I production, secretion, and HDL biogenesis. These models allow researchers to investigate transcriptional and post-transcriptional regulation of APOA1 gene expression. For intestinal apoA-I production, Caco-2 cells differentiated into enterocyte-like cells serve as valuable models.

To study apoA-I's anti-inflammatory functions, endothelial cell lines (HUVECs, HAECs) or activated immune cells (THP-1, RAW264.7 macrophages) can demonstrate how apoA-I modulates inflammatory responses. For cancer-related research, various cancer cell lines (such as MCF-7 breast cancer cells or A549 lung cancer cells) allow investigation of apoA-I's effects on proliferation, migration, and immune evasion.

For neurodegenerative disease contexts, neuronal cell lines (SH-SY5Y) or primary neurons co-cultured with astrocytes and microglia provide insights into apoA-I's neuroprotective effects. Additionally, specialized models like polarized MDCK cells are useful for studying apoA-I transcytosis across barriers.

While cell models offer controlled environments for mechanistic studies, researchers must consider their limitations, including potential differences from in vivo contexts. Complementary approaches using animal models (particularly apoA-I knockout or transgenic mice) and human samples help validate findings from cell-based systems .

What are the most promising directions for APOA1 research in neurodegenerative diseases?

Future research on apoA-I in neurodegenerative diseases should focus on several promising directions. A critical area is elucidating the mechanisms of apoA-I transport across the blood-brain barrier (BBB) and its regulation under both normal and pathological conditions. This includes investigating the role of specific transporters and receptors that facilitate apoA-I movement into the brain parenchyma.

Another important direction involves characterizing brain-specific functions of apoA-I distinct from its peripheral roles. This includes studying how apoA-I interacts with neurons, astrocytes, microglia, and oligodendrocytes, and identifying cell-specific receptors and signaling pathways. Given the significantly reduced apoA-I levels in Alzheimer's disease patients (SMD = -1.16; 95% CI (-1.72, -0.59)), understanding how apoA-I binds to amyloid-β and affects its aggregation, clearance, and neurotoxicity is crucial .

Developing improved biomarker applications represents another promising avenue. This includes examining whether specific post-translational modifications of apoA-I in cerebrospinal fluid or plasma correlate with disease progression or treatment response. Longitudinal studies could establish temporal relationships between apoA-I levels and cognitive decline.

Therapeutically, exploring apoA-I mimetic peptides that can cross the BBB more efficiently than the full-length protein may lead to novel treatments. These peptides could be designed to specifically target pathological processes in neurodegenerative diseases while minimizing off-target effects. Additionally, investigating combinations of apoA-I-based therapies with other approaches targeting different aspects of neurodegenerative pathology may yield synergistic benefits .

How might APOA1 research contribute to emerging cancer immunotherapies?

ApoA-I research has significant potential to contribute to emerging cancer immunotherapies through several mechanisms. Given its role in modulating both innate and adaptive immune responses, apoA-I could enhance immunotherapy efficacy by creating a more favorable tumor microenvironment. Research should focus on how apoA-I affects immune checkpoint inhibitor function, potentially by altering the metabolic state of tumor-infiltrating lymphocytes or modulating PD-1/PD-L1 pathway activity.

Combination approaches represent a promising direction, where apoA-I or mimetic peptides could be administered alongside established immunotherapies to improve their efficacy. Preliminary studies suggest apoA-I may enhance CD8+ T cell infiltration and function in tumors while suppressing immunosuppressive cell populations like MDSCs (myeloid-derived suppressor cells) and Tregs (regulatory T cells) in the tumor microenvironment.

Another avenue involves developing nanoparticle delivery systems incorporating apoA-I or HDL-like structures to target immunomodulatory agents to specific immune cell populations or tumor sites. These biomimetic nanoparticles could improve pharmacokinetics and reduce off-target effects of immunotherapeutic agents.

Mechanistic studies should investigate how apoA-I's cholesterol-modulating effects influence immune cell plasma membrane organization, particularly lipid rafts that serve as signaling platforms for T cell receptor and co-stimulatory molecule function. Additionally, research should explore how apoA-I affects tumor antigen presentation and dendritic cell function, which are critical for initiating effective anti-tumor immune responses.

Finally, biomarker development could identify patients most likely to benefit from combined apoA-I and immunotherapy approaches by correlating serum apoA-I levels or specific isoforms with immunotherapy response rates across different cancer types .

What technological advances could accelerate APOA1 research and therapeutic development?

Several technological advances could significantly accelerate apoA-I research and therapeutic development. CRISPR-Cas9 gene editing enables precise modification of APOA1 and interacting genes in cellular and animal models, allowing detailed investigation of structure-function relationships and potential therapeutic targets. New animal models with humanized lipoprotein metabolism would better recapitulate human physiology for translational studies.

Advanced protein engineering techniques, including directed evolution and computational design, could develop stable apoA-I variants with enhanced therapeutic properties or specificities for particular functions (like improved cholesterol efflux or anti-inflammatory activity). Synthetic biology approaches may enable cost-effective production of recombinant apoA-I at scale, addressing current manufacturing challenges for therapeutic applications.

Nanomedicine advances could create sophisticated apoA-I-based drug delivery systems that mimic HDL particles. These systems could incorporate targeting moieties for tissue-specific delivery of therapeutic agents, particularly valuable for cancer or neurological applications. Novel formulation technologies may improve the stability and half-life of apoA-I-based therapeutics, addressing current pharmacokinetic limitations.

Single-cell technologies, including single-cell RNA-seq and mass cytometry, would enable better understanding of how individual cells respond to apoA-I in complex tissues, revealing cell type-specific effects critical for therapeutic targeting. Advanced imaging technologies like super-resolution microscopy and intravital imaging could visualize apoA-I interactions with cellular receptors and track its trafficking in real time.