FURIN Human

What is human FURIN and what is its fundamental role in cellular biology?

Human FURIN (FUR) is a mammalian proprotein convertase that catalyzes the proteolytic maturation of a diverse array of prohormones and proproteins in the secretory pathway. Identified in 1990 as the first mammalian proprotein convertase, FURIN cleaves large precursor proteins at consensus recognition sites (typically -Arg-X-Lys/Arg-Arg↓-) to generate biologically active proteins . Its substrates include growth factors, receptors, hormones, cytokines, and extracellular matrix proteins that play crucial roles in embryonic development, tissue homeostasis, and various pathological conditions . The proteolytic activation catalyzed by FURIN is essential for numerous cellular processes, including signal transduction, protein trafficking, and maintenance of tissue architecture.

What is the gene structure and transcriptional regulation of human FURIN?

The human FURIN gene is located on chromosome 15q26.1 and contains 16 exons that encode eight different transcript variants driven by three distinct promoters: P1, P1A, and P1B . These transcript variants differ only in their first untranslated exon and therefore generate identical FURIN precursor proteins. While the P1A and P1B promoters resemble constitutively expressed housekeeping genes, the P1 promoter can bind multiple transcription factors, including hypoxia-inducible factor-1 (HIF-1), C/EBPβ, and cAMP-responsive element binding protein (CREB) . This complex promoter structure enables tissue-specific and condition-dependent regulation of FURIN expression, allowing for precise control of proteolytic activity across different physiological contexts and cellular environments.

How does FURIN activation and maturation occur?

FURIN is initially synthesized as an inactive zymogen (profurin) that undergoes sequential autocatalytic processing to become active. This self-activation process involves multiple steps:

• The signal peptide is removed in the endoplasmic reticulum

• The pro-domain initially acts as an intramolecular chaperone aiding in proper folding

• Primary autocatalytic cleavage occurs in the endoplasmic reticulum

• The pro-domain remains non-covalently associated with the enzyme, maintaining it in an inactive state

• A second autocatalytic cleavage in the acidic environment of the trans-Golgi network releases the pro-domain

• The fully mature, active FURIN then proceeds through its trafficking itinerary

This "measure once, cut twice" mechanism appears to be a common theme in the processing of other proteins as well, including bone morphogenetic protein-4 (BMP-4) during embryogenesis, where ordered processing at consensus and minimal furin sites ensures proper morphogen activity .

What trafficking pathways does FURIN follow in different cell types?

FURIN follows distinct trafficking pathways depending on the cell type and secretory pathways present:

In cells with constitutive secretion, FURIN primarily cycles between the trans-Golgi network (TGN), the cell surface, and endosomes. This cycling is directed by signals in FURIN's cytoplasmic tail, including a tyrosine-based motif for internalization and casein kinase 2 (CK2)-phosphorylated acidic cluster that mediates TGN retrieval via interaction with PACS-1 (phosphofurin acidic cluster sorting protein-1) .

In endocrine and neuroendocrine cells with regulated secretory pathways, FURIN exhibits additional trafficking steps :

• FURIN initially enters immature secretory granules (ISGs) along with hormones

• During granule maturation, FURIN is selectively removed through a brefeldin A-sensitive, ADP ribosylation factor-1 (ARF1)-dependent mechanism

• This retrieval requires CK2-phosphorylated acidic cluster and PACS-1

• After removal, FURIN is returned to the TGN

This complex trafficking pattern ensures FURIN can process substrates in different cellular compartments while maintaining proper spatial regulation of its activity.

What methodological approaches are most effective for studying FURIN localization in living cells?

Multiple complementary approaches are effective for studying FURIN localization in living cells:

• Fluorescent protein fusions: Tagging FURIN with GFP or other fluorescent proteins enables real-time visualization of its dynamic movements between compartments .

• Compartment-specific markers: Co-expression with markers for TGN, endosomes, secretory granules, and other compartments helps identify FURIN's precise localization.

• pH-sensitive fluorophores: Fusion proteins with pHluorin can monitor FURIN movement between compartments with different pH values.

• Photoactivatable or photoconvertible fluorescent proteins: These enable pulse-chase experiments tracking specific pools of FURIN molecules through the cell.

• FRAP (Fluorescence Recovery After Photobleaching): This technique measures FURIN mobility and exchange rates between different cellular compartments.

• Super-resolution microscopy: Techniques like STORM, PALM, or STED overcome diffraction limits to visualize FURIN with nanometer precision.

• Correlative light and electron microscopy: This combines fluorescence imaging with electron microscopy to examine FURIN localization at ultrastructural resolution.

These methods have revealed that FURIN's compartmentalization is dynamically regulated and critical for controlling access to its various substrates.

How do researchers identify and validate authentic FURIN substrates?

Identifying and validating authentic FURIN substrates requires multiple complementary approaches:

• Consensus sequence analysis: FURIN preferentially cleaves after -Arg-X-Lys/Arg-Arg↓-, though other proprotein convertases recognize similar motifs. Bioinformatic analysis can identify candidate sites .

• In vitro cleavage assays: Purified FURIN is incubated with potential substrates to demonstrate direct cleavage. Comparing cleavage efficiency with other purified proprotein convertases helps determine specificity.

• Cell-based processing assays: Cells expressing FURIN are transfected with tagged substrate proteins, and processing is monitored by Western blot. FURIN-deficient cell lines provide valuable negative controls.

• Site-directed mutagenesis: Mutating potential FURIN cleavage sites in substrate proteins confirms the exact location of proteolytic processing and its functional significance.

• Inhibitor studies: Specific FURIN inhibitors (such as decanoyl-RVKR-chloromethylketone) are used to block processing and confirm FURIN dependency.

• Conditional knockout models: Tissue-specific FURIN knockout animals help identify physiological substrates in vivo.

• Mass spectrometry: Proteomics approaches identify FURIN-dependent changes in the cellular proteome or secretome.

These approaches collectively help distinguish direct FURIN substrates from proteins processed by other proteases or affected indirectly by FURIN deficiency.

What factors determine FURIN's substrate specificity and processing efficiency?

Multiple factors determine FURIN's substrate specificity and processing efficiency:

• Primary sequence: The canonical FURIN recognition motif is -Arg-X-Lys/Arg-Arg↓-, but processing efficiency varies based on surrounding residues.

• Structural context: The accessibility of the cleavage site within the tertiary structure affects processing efficiency. Some substrates require partial unfolding for FURIN access.

• Post-translational modifications: Glycosylation near the cleavage site can enhance or inhibit FURIN processing depending on the substrate.

• Compartmentalization: Co-localization of FURIN and substrates in the same cellular compartment is necessary for processing. Trafficking defects in either can prevent interaction.

• pH dependency: FURIN activity is optimal at the slightly acidic pH found in the TGN and endosomes, affecting substrate processing in different compartments.

• Cofactors: Some FURIN-substrate interactions require additional proteins or cofactors.

• Competitive inhibition: High concentrations of preferred substrates can competitively inhibit processing of other substrates.

Understanding these factors is crucial for predicting which proteins will be processed by FURIN in vivo and for developing strategies to selectively modulate particular substrate processing events.

What evidence links FURIN to Alzheimer's disease, and what are the potential mechanisms involved?

Several lines of evidence link FURIN to Alzheimer's disease (AD):

• Expression changes: FURIN mRNA expression is reduced in the brains of AD patients, suggesting altered proteolytic processing capacity in affected regions .

• Animal models: Decreased protein levels of FURIN are found in the cortex of AD mouse models .

• APP processing: FURIN influences the α-, β-, and γ-secretase-mediated processing of β-amyloid precursor protein (APP), which determines whether APP-derived peptides enhance nerve growth factor (NGF) signaling to innervating neurons or cause neurodegeneration associated with AD .

• Neuroprotective pathways: When APP is cleaved by α-secretase, it produces soluble APPs that enhance anti-apoptotic and neuroprotective activities of NGF. FURIN may indirectly influence these pathways .

• Notch signaling: FURIN cleaves the transmembrane receptor Notch, which is required for the release of the Notch intracellular domain by γ-secretase proteolysis. This process influences cell-cell communication during development and neuronal function .

These findings suggest FURIN dysregulation may alter the balance of APP processing pathways and other neuroprotective mechanisms, potentially contributing to AD pathogenesis, though the exact mechanisms remain to be fully characterized.

How might altered FURIN activity contribute to schizophrenia pathophysiology?

Evidence suggests several mechanisms through which altered FURIN activity may contribute to schizophrenia pathophysiology:

• Prefrontal cortex dysfunction: FURIN mRNA expression is decreased in the prefrontal cortex of schizophrenia patients, a brain region critical for cognitive functions disrupted in the disorder .

• Neurodevelopmental factors: FURIN processes crucial neurotrophins and their receptors, which regulate neuronal differentiation, survival, and synaptic plasticity. Altered processing of these factors could affect brain development and synaptic connectivity.

• Notch signaling: FURIN processes the Notch receptor, which controls cell-cell communication during neurodevelopment. In the mature brain, Notch signaling regulates synaptic plasticity and memory formation .

• Other substrates: FURIN processes numerous other substrates involved in neural development, including growth factors and cell adhesion molecules that could affect brain circuit formation.

Understanding these connections may provide new insights into schizophrenia pathophysiology and potentially identify novel therapeutic targets. Research methodologies including post-mortem tissue analysis, genetic association studies, animal models, and cell culture systems have all contributed to establishing these links, though the precise mechanisms remain under investigation.

What experimental approaches have proven most valuable for studying FURIN's role in neuronal function?

Several experimental approaches have proven particularly valuable for studying FURIN's role in neuronal function:

• Conditional genetic models: Neuron-specific or brain region-specific FURIN deletion or overexpression models have revealed phenotypes related to neuronal development, function, and degeneration. Notably, increasing FURIN expression in the mouse brain enhances BDNF maturation, promotes dendritic spine density, and improves memory performance .

• Electrophysiological studies: Manipulating FURIN expression affects neuronal excitability. Inhibiting FURIN reduces spontaneous rhythmic electrical activity of cerebral neurons and suppresses epileptic seizure activity in epileptic mice .

• Primary neuronal cultures: These allow precise manipulation of FURIN levels and observation of effects on neurite outgrowth, synapse formation, and responses to neurotrophic factors or neurotoxic insults.

• Brain slice models: These preserve local circuits while allowing pharmacological and genetic manipulation of FURIN activity.

• Proteomics approaches: Mass spectrometry-based methods identify FURIN substrates in neuronal tissues under normal and pathological conditions.

• Human induced pluripotent stem cells (iPSCs): Patient-derived neurons can be used to study how FURIN dysregulation affects neuronal development and function in human cellular models of disease.

These complementary approaches have collectively demonstrated FURIN's importance in neuronal development, synaptic plasticity, and responses to neurological insults.

What is the relationship between FURIN levels and hypertension development?

The relationship between FURIN and hypertension appears complex, with evidence suggesting a negative correlation in early-stage disease:

• Clinical observations: Lower serum levels of FURIN are associated with high blood pressure in humans .

• Natriuretic peptide processing: FURIN processes B-type natriuretic peptide (BNP), which has vasodilatory and diuretic effects that lower blood pressure. Reduced FURIN activity could decrease mature BNP levels, contributing to hypertension .

• Renin-angiotensin system regulation: FURIN mediates the shedding of membrane (pro)renin receptor (PRR), which when membrane-bound facilitates activation of the renin-angiotensin-aldosterone system (RAAS). Decreased FURIN activity might reduce PRR shedding, leading to enhanced RAAS activation and subsequently increased blood pressure .

• Vascular tone regulators: FURIN processes other substrates involved in vascular tone regulation, including endothelin-1 (ET-1) and transforming growth factor-β1 (TGF-β1) .

How does FURIN contribute to atherosclerosis progression and plaque stability?

Unlike its relationship with early hypertension, FURIN appears to increase with atherosclerosis progression, contributing through multiple mechanisms:

• Lipid metabolism: Upregulation of FURIN promotes maturation of membrane type 1-matrix metalloproteinase (MT1-MMP), which cleaves low-density lipoprotein receptor (LDLR), potentially contributing to dyslipidemia—a major risk factor for atherosclerosis .

• Inflammation: FURIN processes pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), which enhance vascular inflammation and promote atherosclerotic plaque formation .

• Cell proliferation: FURIN activates growth factors involved in vascular smooth muscle cell proliferation, a key component of atherosclerotic lesion development .

• Extracellular matrix remodeling: FURIN activates matrix metalloproteinases and other enzymes that degrade extracellular matrix components, potentially contributing to plaque instability and rupture .

• Endothelial dysfunction: FURIN processing of various substrates affects endothelial cell function and permeability, potentially promoting lipid accumulation in the vessel wall .

These mechanisms suggest targeting FURIN might be beneficial in advanced atherosclerosis, though the stage-dependent effects of FURIN would need to be carefully considered in any therapeutic approach.

What methodological approaches are most effective for studying FURIN's role in myocardial infarction and heart failure?

Several methodological approaches have proven effective for studying FURIN's role in myocardial infarction (MI) and heart failure:

• Clinical biomarker studies: Measuring FURIN levels in blood samples from patients with MI or heart failure and correlating with disease severity, progression, and outcomes.

• Animal models: Several approaches are particularly informative:

  • Myocardial infarction models with cardiac-specific FURIN manipulation

  • Pressure overload models (e.g., transverse aortic constriction) in FURIN-modified animals

  • Genetic heart failure models combined with FURIN modulation

• Ex vivo approaches:

  • Isolated perfused hearts (Langendorff preparations) with FURIN inhibitors

  • Myocardial tissue slices from normal and diseased hearts treated with FURIN modulators

• Cellular models:

  • Primary cardiomyocytes with FURIN knockdown or overexpression

  • Cardiac fibroblasts examining how FURIN affects fibrotic responses

  • Co-culture systems studying cell-cell interactions in the presence of FURIN manipulation

• Molecular approaches:

  • Proteomics to identify FURIN substrates altered during cardiac injury and remodeling

  • Targeted analysis of key substrates including TNF-α, ET-1, and TGF-β1, which mediate inflammation, hypertrophy, and fibrosis in MI and heart failure

These approaches collectively demonstrate that FURIN and its substrates contribute to post-MI inflammation, adverse remodeling, and progression to heart failure, highlighting potential therapeutic opportunities.

What are the challenges and approaches in developing selective FURIN inhibitors?

Developing selective FURIN inhibitors faces several significant challenges:

• Selectivity issues: FURIN belongs to a family of structurally similar proprotein convertases, making it difficult to develop inhibitors that don't cross-react with other family members.

• Essential physiological roles: FURIN processes numerous physiologically important substrates, raising concerns about potential side effects of systemic inhibition.

• Multiple cellular compartments: FURIN operates in different cellular compartments, which may be differentially accessible to inhibitors depending on their chemical properties.

• Context-dependent effects: FURIN's role varies by disease stage and tissue. For example, it shows negative correlation with early hypertension but positive correlation with atherosclerosis progression .

Promising approaches to address these challenges include:

• Structure-based design: Using crystal structures of FURIN to design highly selective inhibitors that exploit unique features of its active site or allosteric regions.

• Tissue-specific delivery: Developing strategies to target FURIN inhibitors to specific tissues where its activity contributes to pathology while sparing essential functions elsewhere.

• Substrate-selective inhibition: Creating inhibitors that block FURIN processing of pathological substrates while permitting cleavage of physiologically important ones.

• Temporal control: Designing inhibition strategies that can be activated only during specific disease stages when FURIN inhibition would be beneficial.

These approaches could potentially lead to novel therapeutic strategies for various diseases where FURIN dysregulation plays a role.

How can computational approaches advance our understanding of FURIN substrate prediction and inhibitor design?

Computational approaches offer powerful tools for advancing FURIN research:

• Substrate prediction algorithms:

  • Machine learning models trained on known FURIN substrates can predict novel candidates

  • Sequence-based approaches identifying canonical and non-canonical cleavage sites

  • Structural models predicting accessibility of potential cleavage sites

  • Systems biology approaches integrating proteomic data with FURIN expression patterns

• Molecular dynamics simulations:

  • Modeling FURIN-substrate interactions to understand binding determinants

  • Simulating conformational changes during substrate recognition and catalysis

  • Investigating how different cellular environments affect FURIN activity

• Drug design approaches:

  • Virtual screening of compound libraries against FURIN structures

  • Structure-based design of selective inhibitors targeting unique FURIN features

  • Optimization of lead compounds for improved potency, selectivity, and pharmacokinetics

  • Prediction of potential off-target effects based on structural similarities

• Network analysis:

  • Modeling how FURIN fits into broader proteolytic networks

  • Predicting systemic consequences of FURIN inhibition

  • Identifying optimal intervention points in disease-specific networks

These computational approaches complement experimental methods by generating testable hypotheses, prioritizing candidate substrates or inhibitors, and providing mechanistic insights that might be difficult to obtain experimentally.

What are the emerging therapeutic opportunities for targeting FURIN across different disease contexts?

Emerging therapeutic opportunities for targeting FURIN vary across disease contexts:

• Neurodegenerative disorders:

  • Increasing FURIN activity in Alzheimer's disease might enhance neuroprotective APP processing

  • FURIN upregulation could promote BDNF maturation, potentially improving memory and neural plasticity

  • Temporal and regional specificity would be crucial for therapeutic success

• Neuropsychiatric disorders:

  • Normalizing FURIN levels in schizophrenia might restore proper processing of neurodevelopmental factors

  • Modulating FURIN activity could potentially affect neurotransmitter systems through indirect mechanisms

• Cardiovascular diseases:

  • Early hypertension might benefit from FURIN upregulation to enhance BNP processing and reduce PRR-mediated RAAS activation

  • Advanced atherosclerosis might benefit from FURIN inhibition to reduce inflammation and matrix degradation

  • Heart failure treatment might target specific FURIN substrates like TGF-β1 rather than FURIN itself

• Infectious diseases:

  • FURIN inhibition could reduce pathogen activation, as FURIN processes viral proteins including SARS-CoV-2 spike protein

  • Targeted delivery to affected tissues could minimize systemic side effects

These diverse therapeutic opportunities highlight the need for context-specific approaches rather than general FURIN modulation. Future therapeutic strategies will likely focus on targeted, tissue-specific, and temporally controlled modulation of FURIN activity or the selective inhibition of disease-specific substrate processing events.