KLK1 Human, HEK

What is Human KLK1 and what are its primary physiological functions?

Human Tissue Kallikrein 1 (KLK1) is a serine protease belonging to the kallikrein-kinin system (KKS). Its primary function involves cleaving low molecular weight kininogen (LMWK) at Met-Lys and Arg-Ser bonds to produce kinins, particularly bradykinin (BK) and Lys-bradykinin . These kinins then bind to B1 and B2 bradykinin receptors, activating downstream signaling cascades including nitric oxide (NO), cyclic guanosine monophosphate (cGMP), prostacyclin, and cyclic adenosine monophosphate (cAMP) . Through these pathways, KLK1 exerts anti-inflammatory, antifibrotic, and antioxidative effects in various tissues . Additionally, KLK1 plays roles in various physiological processes including vascular function, inflammation regulation, and potentially glucose metabolism . The enzyme's actions have been studied extensively in contexts of aging-related tissue changes, showing protective effects against fibrosis and oxidative stress .

What is the relationship between KLK1 expression and aging in humans?

KLK1 expression demonstrates a significant age-dependent decline in human tissues, particularly in the prostate. Research has shown a negative correlation between age and KLK1 expression in human prostate tissue with a correlation coefficient of r = -0.347 and statistical significance of P = 0.018 . This age-related reduction in KLK1 expression may contribute to various pathophysiological changes observed in aging tissues. In experimental models, aged transgenic rats harboring the human KLK1 gene showed milder fibrosis, reduced oxidative stress, and generally healthier tissue morphology compared to aged wild-type rats . These findings suggest that the natural decline in KLK1 with aging may lay the groundwork for age-associated conditions such as benign prostatic hyperplasia (BPH), and that maintaining KLK1 levels could potentially protect against these age-related changes .

Why are HEK 293 cells commonly used for recombinant human KLK1 expression?

HEK 293 cells represent an optimal expression system for recombinant human KLK1 production for several scientific reasons. As a human-derived cell line, HEK 293 cells provide the correct cellular machinery for proper folding and post-translational modifications of human proteins. For KLK1 specifically, these cells accurately produce the O-linked glycosylation patterns on Ser-93, Ser-104, and Ser-167, which are typically mucin-type glycans linked to N-acetylgalactosamine (GalNAc) . These modifications are critical for maintaining the protein's stability, enzymatic activity, and physiological function. Recombinant human KLK1 expressed in HEK 293 cells consistently achieves high purity (>95%) with minimal endotoxin contamination (<0.1 EU/μg), making it suitable for both in vitro mechanistic studies and potential therapeutic applications . The HEK 293 expression system also facilitates the production of properly folded KLK1 with the correct conformation of its catalytic triad, ensuring optimal enzymatic function for research applications.

What are the known physiological substrates for human KLK1?

Human KLK1 interacts with several physiological substrates that mediate its diverse biological functions. The primary and most well-characterized substrate is low molecular weight kininogen (LMWK), which KLK1 cleaves at Met-Lys and Arg-Ser bonds to release Lys-bradykinin . This reaction represents the canonical function of KLK1 within the kallikrein-kinin system. Beyond kininogen processing, KLK1 has been demonstrated to cleave Neisseria meningitidis NHBA in saliva, suggesting a role in mucosal immunity against this bacterial pathogen . KLK1 also interacts with components of the TGF-β1 signaling pathway, where it suppresses TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation . This occurs through a mechanism where KLK1 cleaves LMWK to produce bradykinin, which then upregulates endothelial nitric oxide synthase (eNOS) expression and nitric oxide production in endothelial cells . Additionally, the enzyme influences the TGF-β1/RhoA/ROCK1 signaling pathway, providing another mechanism through which it exerts antifibrotic effects in tissues .

How is the kallikrein-kinin system organized and what is KLK1's specific role?

The kallikrein-kinin system (KKS) comprises a complex network of proteins including kininogens (substrates), kallikreins (enzymes), kinins (bioactive peptides), kinin-degrading enzymes, and kinin receptors . Within this system, KLK1 functions as a key enzymatic component that operates independently of the contact system (which activates plasma kallikrein) . KLK1 specifically cleaves low molecular weight kininogen to produce kinins, particularly bradykinin and Lys-bradykinin. These kinins then bind to bradykinin receptors (B1 and B2), triggering downstream signaling cascades with diverse physiological effects . Unlike plasma kallikrein, which generates bradykinin through the contact activation pathway, KLK1 provides an alternative route for kinin production that maintains essential signaling even when contact activation is compromised . Through bradykinin generation, KLK1 activates several downstream pathways including the NO/cGMP and COX-2/PTGIS/cAMP pathways, while inhibiting the TGF-β1/RhoA/ROCK1 pathway . This balanced modulation of multiple signaling cascades underlies KLK1's protective effects in various tissues.

What mechanisms underlie KLK1's protective effects against age-related tissue fibrosis?

KLK1's protection against age-related tissue fibrosis involves a sophisticated network of molecular pathways. Central to this protection is KLK1's ability to cleave low molecular weight kininogen (LMWK) to produce bradykinin (BK), which then activates specific signaling cascades that counteract fibrotic processes . In experimental models, KLK1 suppresses TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation, a key cellular event in tissue fibrosis development . This occurs through a mechanism where bradykinin upregulates endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production in endothelial cells . The increased NO then activates the NO/cGMP pathway, which has established antifibrotic effects. Simultaneously, KLK1 activation leads to upregulation of the COX-2/PTGIS/cAMP signaling pathway while inhibiting the pro-fibrotic TGF-β1/RhoA/ROCK1 signaling cascade . Studies in aged transgenic rats harboring the human KLK1 gene have demonstrated that these animals exhibit significantly milder fibrosis and less oxidative stress compared to age-matched wild-type rats, particularly in prostate tissue . Additionally, bradykinin slightly stimulates the proliferation ability of prostatic stromal cells while upregulating inducible nitric oxide synthase (iNOS) and inhibiting TGF-β1 expression in these cells .

How can researchers distinguish between activities of KLK1 and other kallikrein family members?

Distinguishing between KLK1 and other kallikrein family members in experimental systems requires a multi-faceted methodological approach:

• Selective inhibitors: Specific inhibitors targeting different kallikreins can help differentiate their activities. For example, SPINK6 shows family-specific inhibition of kallikreins, allowing researchers to distinguish KLK1 activity from other proteases .

• Genetic manipulation: RNA interference techniques using short hairpin RNAs (shRNAs) targeting specific KLK genes can selectively suppress individual kallikreins and attribute observed effects to specific family members . This approach has been successfully used to demonstrate the specific role of KLK13 in viral entry, providing a methodological template for KLK1 studies .

• Substrate specificity profiling: Each kallikrein has distinct substrate preferences. KLK1 specifically cleaves Met-Lys and Arg-Ser bonds in kininogen to produce Lys-bradykinin , while other KLKs have different cleavage site preferences. Using selective substrates can help identify which kallikrein is responsible for observed proteolytic activities.

• Bradykinin receptor activation: As KLK1 specifically generates bradykinin, monitoring activation of B1 and B2 receptors can help distinguish KLK1-mediated effects from those of other kallikreins that may not produce kinins . Notably, KLK1 is the only stimulus leading to inconsistent B1R stimulation, providing another distinguishing characteristic .

• Recombinant protein standards: Using purified recombinant kallikreins with defined activities as standards can help calibrate detection methods and establish specificity.

What experimental models are most appropriate for studying KLK1's role in aging?

Several experimental models have proven valuable for investigating KLK1's role in age-related conditions:

• Transgenic rodent models: Aged transgenic rats harboring the human KLK1 gene provide a powerful system for studying KLK1's protective effects against age-related alterations . These models allow direct comparison between aged wild-type rats (aWTR) and aged transgenic rats (aTGR) expressing human KLK1, revealing significant differences in tissue fibrosis, oxidative stress, and pathway regulation .

• Age-comparison cohorts: Comparing young wild-type rats (yWTR) with aged wild-type rats (aWTR) provides insights into natural age-related changes in KLK1 expression and associated pathophysiological changes . This approach helps establish baseline alterations that occur with aging in the absence of genetic manipulation.

• Primary cell co-culture systems: In vitro co-culture of endothelial cells with tissue-specific cells (such as prostatic fibroblasts) allows investigation of KLK1's effects on cell-cell interactions and signaling pathways . This system has been successfully used to demonstrate how KLK1 suppresses TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation via bradykinin production .

• Human tissue biobanks: Analysis of KLK1 expression in human tissue samples across different age groups has revealed significant negative correlations between age and KLK1 levels (r = -0.347, P = 0.018 in prostate tissue) . This approach provides direct translational relevance and validation of findings from animal models.

• Recombinant KLK1 intervention: Administering recombinant KLK1 to aged animals allows assessment of its potential therapeutic effects on age-related pathologies .

What are the potential mechanisms by which KLK1 influences bradykinin receptor signaling?

KLK1 influences bradykinin receptor signaling through several mechanisms with distinct characteristics compared to other kinin-generating proteases. Upon cleaving low molecular weight kininogen (LMWK), KLK1 produces bradykinin that can activate both B1 and B2 bradykinin receptors, though with distinct activation patterns . Studies indicate that KLK1 is the only stimulus leading to inconsistent B1R stimulation, suggesting unique receptor activation properties compared to other proteases in the kallikrein-kinin system . The B2 receptor, being constitutively expressed, responds more consistently to KLK1-generated bradykinin, activating the NO/cGMP and COX-2/PTGIS/cAMP signaling pathways . KLK1 works independently of the contact system to generate bradykinin, providing an alternative pathway for kinin production that maintains signaling even when contact activation is compromised . In blood, KLK1 rapidly (within 5 minutes) induces significant bradykinin generation (>100 ng/mL), creating a rapid onset of receptor activation . Additionally, in specific tissue contexts such as prostatic stroma, bradykinin produced by KLK1 not only slightly stimulates cell proliferation but also upregulates inducible nitric oxide synthase (iNOS) and inhibits TGF-β1 expression, modulating downstream signaling cascades beyond direct receptor activation .

What purification strategies yield the highest purity of recombinant KLK1 from HEK 293 cells?

Achieving high-purity recombinant human KLK1 from HEK 293 expression systems requires a strategic purification protocol optimized for this serine protease. Commercial recombinant human KLK1 produced in HEK 293 cells consistently achieves >95% purity with endotoxin levels below 0.1 EU/μg, making it suitable for research applications including SDS-PAGE and HPLC analysis . A typical purification workflow begins with the collection of conditioned media from transfected HEK 293 cells expressing the KLK1 protein (amino acids 19-262) with an appropriate affinity tag, often a C-terminal polyhistidine tag . Initial capture typically employs affinity chromatography using immobilized metal affinity chromatography (IMAC) resins. This is followed by intermediate purification via ion exchange chromatography to separate KLK1 from HEK 293 cell proteins with different charge properties. A final polishing step using size exclusion chromatography removes aggregates and achieves final purification. Throughout the purification process, it's critical to maintain conditions that preserve enzymatic activity, often requiring the inclusion of stabilizing agents and careful pH and temperature control. For applications requiring extremely low endotoxin levels, specific endotoxin removal steps may be incorporated. The purified protein should be validated not only for purity via SDS-PAGE and HPLC but also for functional activity using appropriate enzymatic assays that confirm its ability to cleave known substrates such as low molecular weight kininogen or synthetic peptides containing Met-Lys and Arg-Ser bonds .

How can researchers validate the enzymatic activity of recombinant KLK1?

Validating the enzymatic activity of recombinant KLK1 requires multiple complementary approaches to ensure both specificity and physiological relevance. A comprehensive validation strategy includes:

• Substrate cleavage assays: Testing KLK1's ability to cleave its canonical substrate, low molecular weight kininogen (LMWK), at Met-Lys and Arg-Ser bonds to release Lys-bradykinin . This can be monitored using mass spectrometry to identify specific cleavage products or immunoassays to detect bradykinin release.

• Synthetic peptide substrates: Using fluorogenic or chromogenic peptides containing KLK1's preferred cleavage sites to quantitatively measure enzymatic activity through fluorescence or absorbance changes upon substrate hydrolysis.

• Bradykinin receptor activation: Monitoring B1 and B2 receptor activation in cell-based assays following treatment with KLK1-generated kinins . This functional readout confirms that the enzyme produces biologically active peptides.

• Inhibitor sensitivity profiling: Verifying that KLK1 activity is blocked by known kallikrein inhibitors. This approach can also help distinguish KLK1 activity from other proteases based on differential inhibitor sensitivity patterns.

• Comparison with commercial standards: Benchmarking activity against established recombinant KLK1 standards with known specific activity, such as those expressed in HEK 293 cells with >95% purity .

• Physiological activity assays: Testing KLK1's ability to stimulate NO production in endothelial cells or affect TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation in co-culture systems .

• Proteome-wide substrate profiling: Using proteomic approaches to identify the range of proteins cleaved by KLK1 in complex biological samples, confirming both canonical and potentially novel substrates.

What techniques are optimal for measuring KLK1 expression in human tissue samples?

Several complementary techniques can be employed to measure KLK1 expression in human tissue samples, each with specific advantages:

• Quantitative RT-PCR (qRT-PCR): This technique allows precise quantification of KLK1 mRNA expression. When studying age-related changes in KLK1 expression, qRT-PCR can reveal significant correlations, such as the documented negative correlation between age and prostate KLK1 expression (r = -0.347, P = 0.018) . Primers must be carefully designed to distinguish KLK1 from other highly homologous kallikrein family members.

• RNA-seq: For a more comprehensive transcriptomic analysis, RNA-seq provides insights into KLK1 expression relative to other genes, including potential regulatory factors and downstream targets. This approach is particularly valuable for exploring pathway interactions.

• Immunohistochemistry (IHC): IHC allows visualization of KLK1 protein distribution within tissue sections, providing crucial information about cell-type specificity and localization patterns. This technique has been valuable in comparing KLK1 expression between young and aged tissues .

• Western blotting: For quantitative protein analysis, western blotting with validated anti-KLK1 antibodies enables measurement of total KLK1 protein levels and potential post-translational modifications.

• ELISA: Enzyme-linked immunosorbent assays provide sensitive quantification of KLK1 protein in tissue homogenates or biological fluids, allowing comparison across multiple samples.

• Enzymatic activity assays: Beyond expression, measuring KLK1 enzymatic activity in tissue extracts using specific substrates provides functional information that may not always correlate with expression levels.

• In situ hybridization: This technique localizes KLK1 mRNA within tissue sections, complementing protein detection methods and confirming cell-specific expression patterns.

What approaches are effective for studying KLK1-mediated signaling pathways?

Investigating KLK1-mediated signaling pathways requires a multi-faceted methodological approach that captures both immediate signaling events and downstream effects:

• Bradykinin receptor signaling analysis:

  • Selective receptor antagonists can distinguish B1 versus B2 receptor-mediated effects following KLK1 treatment

  • Calcium flux assays detect immediate receptor activation responses

  • Phosphorylation studies of receptor-associated kinases reveal early signaling events

• NO/cGMP pathway investigation:

  • Direct measurement of nitric oxide production using Griess reagent or fluorescent indicators

  • Analysis of eNOS and iNOS expression and phosphorylation status by western blotting

  • Quantification of cGMP levels using specific immunoassays or reporter systems

  • Pharmacological inhibition of NOS to confirm pathway involvement in KLK1 effects

• COX-2/PTGIS/cAMP pathway assessment:

  • Monitoring COX-2 expression changes following KLK1 treatment

  • Measuring prostaglandin levels to confirm pathway activation

  • Quantifying cAMP production using specific assays

  • Using pathway inhibitors to establish causal relationships

• TGF-β1/RhoA/ROCK1 pathway analysis:

  • Assessing TGF-β1 expression levels and RhoA activation status

  • Measuring ROCK1 phosphorylation and activity

  • Co-culture systems demonstrating KLK1's inhibitory effects on TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation

• Integrative approaches:

  • Co-immunoprecipitation to identify KLK1-associated signaling proteins

  • Phosphoproteomic analysis to map signaling cascades comprehensively

  • Transcriptomic profiling to identify downstream gene expression changes

What are the critical considerations when designing inhibitors specific for human KLK1?

Designing specific inhibitors for human KLK1 requires consideration of several key factors to achieve selectivity, potency, and appropriate pharmacological properties:

• Structural specificity: While KLK1 shares the catalytic triad common to serine proteases, its substrate binding pocket contains unique features that can be exploited for selective inhibitor design. Crystal structures of KLK1 reveal distinctive subsites that determine its preference for cleaving Met-Lys and Arg-Ser bonds .

• Selectivity challenges: The high sequence homology between KLK1 and other kallikrein family members (particularly in the catalytic domain) presents a significant challenge for inhibitor specificity. Successful inhibitor design must target regions that differ between KLK1 and related proteases.

• Activity validation: Inhibitor candidates should be tested against multiple kallikreins and other serine proteases to confirm selectivity. The approach used for KLK13-specific inhibitors, where inhibitory effects are tested against related proteases, provides a useful methodological template .

• Binding mode characterization: Understanding whether inhibitors act competitively (binding the active site) or allosterically (inducing conformational changes that affect activity) is crucial for optimization.

• Physiological context: Since KLK1 exerts beneficial effects in many contexts, highly specific inhibitors are needed to avoid disrupting these functions while targeting pathological KLK1 activity. Additionally, inhibitors should be tested in physiologically relevant systems such as co-culture models .

• Delivery considerations: For potential therapeutic applications, inhibitor design must account for stability in biological fluids, tissue penetration, and pharmacokinetic properties.

• Chemical scaffolds: Natural kallikrein inhibitors like SPINK6 can provide starting points for designing synthetic inhibitors with improved specificity profiles.

What evidence supports KLK1 as a therapeutic target for age-related conditions?

Multiple lines of evidence support KLK1 as a promising therapeutic target for age-related conditions:

• Age-dependent expression decline: Human studies have demonstrated a significant negative correlation between age and KLK1 expression in prostate tissue (r = -0.347, P = 0.018) . This natural decline suggests that restoring or supplementing KLK1 might counteract age-related pathological changes.

• Protective effects in animal models: Aged transgenic rats harboring the human KLK1 gene (aTGR) show significantly milder fibrosis, reduced oxidative stress, and healthier tissue morphology compared to aged wild-type rats (aWTR) . These observations provide direct evidence that KLK1 can protect against age-related tissue alterations.

• Molecular pathway modulation: KLK1 upregulates protective NO/cGMP and COX-2/PTGIS/cAMP signaling pathways while inhibiting the pro-fibrotic TGF-β1/RhoA/ROCK1 signaling pathway . This multi-pathway modulation addresses several mechanisms implicated in age-related tissue dysfunction.

• Antifibrotic mechanisms: KLK1 suppresses TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation, a key process in age-related fibrosis development . This effect was demonstrated in co-culture experiments with endothelial cells and prostatic stromal cells, providing mechanistic insight into KLK1's protective actions.

• Favorable safety profile: Experimental data indicate that the side effects of KLK1 on prostate cells are not obvious, suggesting potentially minimal adverse effects in therapeutic applications .

• Potential metabolic benefits: Some studies report that recombinant KLK1 treatment lowers blood glucose in experimental animals, suggesting additional metabolic benefits that could address age-related metabolic dysfunction .

How does KLK1 activity differ across various human tissues?

KLK1 demonstrates distinct tissue-specific activity patterns that influence its physiological functions and potential therapeutic applications:

What challenges exist in developing KLK1-based therapeutics?

Developing KLK1-based therapeutics faces several significant challenges that must be addressed for clinical translation:

• Protein stability and half-life: As a serine protease, recombinant KLK1 is susceptible to degradation and inactivation in vivo. Strategies to extend its half-life without compromising activity are needed for therapeutic applications.

• Production complexity: Recombinant human KLK1 requires expression in systems like HEK 293 cells to ensure proper post-translational modifications, particularly O-linked glycosylation on Ser-93, Ser-104, and Ser-167 . These requirements increase production complexity and costs compared to simpler therapeutic proteins.

• Delivery challenges: Ensuring KLK1 reaches target tissues in sufficient concentrations presents a significant hurdle. The protein's size and charge characteristics limit passive diffusion across biological barriers, potentially necessitating advanced delivery systems.

• Dosing precision: KLK1 activates multiple signaling pathways with complex feedback mechanisms. Determining optimal therapeutic dosing regimens that provide benefits without unintended consequences requires extensive preclinical investigation.

• Context-dependent effects: KLK1's effects vary across tissues and pathophysiological states, as evidenced by contradictory findings regarding its impact on glucose metabolism . This context-dependency complicates therapeutic development and necessitates targeted approaches.

• Monitoring challenges: Establishing reliable biomarkers to monitor therapeutic efficacy presents difficulties, as KLK1 affects multiple pathways simultaneously. This complexity must be addressed for clinical trial design and patient monitoring.

• Regulatory considerations: As a multi-functional protease with diverse physiological roles, KLK1 therapeutics may face intensive regulatory scrutiny regarding safety and off-target effects, requiring robust preclinical safety data.

• Competing therapeutic approaches: For conditions like age-related fibrosis, multiple therapeutic strategies are under development. KLK1-based approaches must demonstrate advantages over competing therapeutics targeting the same conditions.

How do interactions between KLK1 and other proteases influence inflammatory responses?

KLK1's interactions with other proteases create a complex network that modulates inflammatory responses through several mechanisms:

• Independent bradykinin generation: Unlike plasma kallikrein which requires contact system activation, KLK1 operates independently to generate bradykinin . This provides an alternative pathway for kinin production during inflammation that persists even when the contact system is inhibited.

• Rapid kinetics: KLK1 rapidly (within 5 minutes) induces significant bradykinin generation (>100 ng/mL) in blood, comparable to contact system activation but through a distinct mechanism . This rapid response capability allows KLK1 to influence the early phases of inflammatory processes.

• Selective receptor activation: KLK1 is the only stimulus that leads to inconsistent B1R stimulation while more reliably activating B2 receptors . This selective receptor activation profile differs from other kinin-generating proteases and creates unique downstream signaling patterns during inflammation.

• TGF-β1 pathway interaction: KLK1 inhibits TGF-β1 expression in prostatic stromal cells , potentially modulating the anti-inflammatory and pro-fibrotic effects of this cytokine during tissue repair and chronic inflammation.

• Neutrophil and platelet independence: Unlike some inflammatory contexts where neutrophil or platelet activation generates kinins, studies indicate that stimulating neutrophils or platelets does not generate immunoreactive bradykinin . This highlights KLK1's distinctive role in kinin production during inflammation.

• Tissue-specific protease networks: In different tissues, KLK1 may interact with tissue-specific proteases, creating unique inflammatory microenvironments with tissue-specific responses.

What methodological advances have improved the study of recombinant human KLK1?

Recent methodological advances have significantly enhanced the study of recombinant human KLK1:

• Optimized HEK 293 expression systems: Modern expression protocols consistently produce recombinant human KLK1 with high purity (>95%) and low endotoxin contamination (<0.1 EU/μg) . These improvements ensure reliable experimental results and reduce variability between studies.

• Full-length protein production: Current methods enable expression of full-length KLK1 (amino acids 19-262) with proper post-translational modifications, particularly O-linked glycosylation on Ser-93, Ser-104, and Ser-167 . This represents a significant improvement over truncated or improperly modified versions used in earlier studies.

• Transgenic animal models: The development of transgenic rats harboring the human KLK1 gene has provided valuable tools for studying KLK1's protective effects against age-related conditions in vivo . These models allow direct comparison between wild-type and KLK1-expressing animals under identical conditions.

• Sophisticated co-culture systems: Advanced co-culture models combining endothelial cells with tissue-specific cells (such as prostatic fibroblasts) allow investigation of KLK1's effects on cell-cell interactions and pathway modulation in controlled environments . These systems have revealed mechanisms such as KLK1's suppression of TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation.

• Improved activity assays: Modern enzymatic assays with greater sensitivity and specificity enable precise quantification of KLK1 activity across different experimental conditions. This has facilitated more detailed characterization of KLK1's enzymatic properties and substrate preferences.

• Human tissue correlation studies: Methodological advances in quantifying KLK1 expression in human tissue samples have revealed significant correlations with age and pathological conditions . These approaches bridge the gap between basic research and clinical relevance.

• Multi-omics integration: The combination of proteomics, transcriptomics, and metabolomics provides comprehensive insights into KLK1's effects across multiple cellular processes and signaling networks.