ASPRV1 Human

How is ASPRV1 structurally characterized and activated?

ASPRV1 is synthesized as a 28 kDa zymogen (SASP28) containing a putative transmembrane domain and a conserved catalytic domain with a key aspartic acid residue essential for enzymatic activity . This zymogen undergoes autocleavage under slightly acidic conditions, releasing the 14 kDa catalytic domain (SASP14) that homodimerizes to form the active protease . Structural analysis through homology modeling reveals similarities to retroviral proteases, though ASPRV1 demonstrates lower dimer stability compared to HIV-1 protease, as indicated by urea dissociation experiments . The self-processing of the SASP28 precursor is a critical regulatory step in enzyme activation. The mature SASP14 enzyme functions optimally at slightly acidic pH (pH 6.27) and shows higher activity at increased ionic strength, biochemical properties that align with its physiological environments in skin and potential inflammatory microenvironments .

What phenotypes are associated with ASPRV1 deficiency in model systems?

Genetic studies have provided important insights into ASPRV1 function through the characterization of deficiency models. In skin, ASPRV1-deficient mice exhibit relatively mild phenotypes, including fine wrinkles and reduced skin hydration in adulthood, indicating a role in maintaining normal water conditions and skin barrier function . These phenotypes align with ASPRV1's known role in profilaggrin processing, which affects skin barrier integrity. More dramatically, in experimental autoimmune encephalomyelitis (EAE) models, mice lacking ASPRV1 develop a significantly less severe chronic phase of disease when immunized with a B cell-dependent myelin antigen . In many ASPRV1-deficient animals, the chronic inflammatory phase completely resolves, suggesting a critical role for this protease in sustaining chronic inflammation, specifically in B cell-dependent autoimmune conditions . These collective findings position ASPRV1 as a potential therapeutic target in certain inflammatory and autoimmune conditions.

What are the optimal experimental conditions for studying ASPRV1 enzymatic activity in vitro?

Establishing optimal conditions for ASPRV1 activity measurements is critical for accurate biochemical characterization. Research indicates that GST-SASP14 exhibits highest activity at near-neutral pH (pH optimum = 6.27 ± 0.02), which aligns with the physiological environment of the stratum granulosum in skin . This pH optimum differs from that of HIV-1 protease (pH 4-6) but is similar to human foamy virus protease (pH 6.6-6.8) . Additionally, ASPRV1 activity is significantly enhanced by high ionic strength buffer conditions, a property shared with HIV-1 and human foamy virus proteases .

For substrate selection, synthetic oligopeptides derived from HIV-1 MA/CA cleavage sites with specific modifications have proven effective. Notably, variants containing hydrophobic residues (particularly leucine) at the P2 position are efficiently cleaved by SASP14, while the wild-type sequence and P3 variants show much lower cleavage efficiency . Researchers should note that extended incubation times (overnight rather than 1 hour) may be necessary to detect cleavage of less optimal substrates . These parameters provide a methodological framework for in vitro studies of ASPRV1 activity, substrate specificity, and inhibitor screening.

How does ASPRV1 contribute to neuroinflammation in multiple sclerosis models?

ASPRV1 plays a previously unrecognized role in neuroinflammation, particularly in experimental autoimmune encephalomyelitis (EAE) and potentially in multiple sclerosis. In EAE models, ICAM1+ neutrophils with macrophage-like properties infiltrate the CNS parenchyma and express high levels of ASPRV1 . These neutrophils form synapses with T and B cells in situ, suggesting direct immune cell interactions . Most significantly, ASPRV1 appears essential for the progression from acute to chronic inflammation in B cell-dependent forms of EAE .

Mice lacking ASPRV1 develop a less severe chronic phase of EAE when immunized with B cell-dependent myelin antigens, with many animals showing complete resolution of chronic symptoms . This effect is specific to B cell-dependent EAE models and is not observed with traditional myelin oligodendrocyte glycoprotein peptide immunization . These findings parallel observations in human multiple sclerosis, where ASPRV1 levels are elevated in brain lesions of patients with severe disease compared to those with mild or moderate MS . Collectively, these observations suggest ASPRV1 may be a key molecular mediator in the chronicity of neuroinflammation, potentially through proteolytic processing of yet-unidentified substrates that influence immune cell function and inflammatory persistence.

What methods are most effective for isolating and studying distinct neutrophil populations expressing ASPRV1?

Studying ASPRV1 in neutrophils requires distinguishing between different neutrophil subpopulations. Flow cytometry using cell-surface markers (CD45, CD11b, Ly6G, ICAM1) has proven effective for separating neutrophil populations in the spinal cord and blood . ICAM1 expression serves as a critical marker distinguishing parenchyma-infiltrating neutrophils (ICAM1+) from intravascular neutrophils (ICAM1-) .

For in vivo visualization, genetic approaches using reporter systems have been valuable. Studies have successfully employed Ly6G-Cre transgenic mice (with Cre recombinase expressed under the neutrophil-specific Ly6G promoter) crossed with fluorescent reporter mice (Ai6, expressing ZsGreen fluorescent protein upon Cre activity) . This system enables confocal imaging of neutrophils in tissue sections, with additional immunostaining for ICAM1 and Ly6G confirming neutrophil identity and subtype .

Research shows that ICAM1+ neutrophils are concentrated in inflammatory foci near the central canal and in meningeal/submeningeal areas of the spinal cord during EAE . These cells exhibit distinct transcriptional profiles compared to intravascular neutrophils, with increased activation markers and macrophage-like properties . These methodological approaches provide a framework for isolating and characterizing ASPRV1-expressing neutrophils in various disease models and potentially in human samples.

What is known about the substrate specificity of ASPRV1 and how does it compare to other retroviral-like proteases?

The substrate specificity of ASPRV1 shows both similarities and differences compared to retroviral proteases, reflecting its evolutionary relationship while highlighting its specialized functions. While profilaggrin remains the only confirmed natural substrate in the skin, in vitro studies have provided insights into ASPRV1's cleavage preferences.

ASPRV1 demonstrates distinct substrate preferences:

This data indicates ASPRV1 strongly prefers hydrophobic residues at the P2 position , a preference shared with several retroviral proteases including human T-cell leukemia virus type 1 (HTLV-1), human foamy virus (HFV), and bovine leukemia virus (BLV) proteases . Unlike HIV-1 protease, which accommodates polar residues at P2, ASPRV1 requires hydrophobic residues at this position for efficient cleavage . This specificity profile provides a foundation for predicting potential natural substrates in neutrophils and other tissues, which could help elucidate ASPRV1's role in inflammatory processes.

How can ASPRV1 inhibition be achieved and measured in experimental settings?

ASPRV1 inhibition can be approached through multiple experimental strategies with specific considerations for each method:

Pharmacological inhibition:
ASPRV1, as an aspartic protease, is susceptible to inhibition by classic aspartic protease inhibitors including pepstatin A and acetyl-pepstatin . Additionally, some HIV-1 protease inhibitors used in antiretroviral therapies may inhibit ASPRV1, potentially explaining certain cutaneous side effects observed in patients . Activity assays using synthetic oligopeptide substrates with hydrophobic P2 residues provide a reliable method for measuring inhibition efficacy, with cleavage products quantified by HPLC or mass spectrometry .

Genetic approaches:
ASPRV1 function can be disrupted through knockout models, which have been successfully generated and characterized . Complete knockout mice show skin phenotypes (fine wrinkles, reduced hydration) and significant alterations in inflammatory responses in EAE models . For cell-specific or inducible approaches, CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown systems can be employed, with phenotypic outcomes compared to controls.

Measurement of inhibition effects:
Inhibition can be assessed through multiple readouts including: (1) direct measurement of enzymatic activity using synthetic substrates, (2) quantification of natural substrate processing (e.g., profilaggrin processing in skin models), (3) neutrophil functional assays in inflammatory models, and (4) disease phenotyping in models like EAE, where ASPRV1 deficiency significantly alters disease progression . The choice of readout depends on the specific research question and experimental system.

What expression systems are most effective for producing recombinant ASPRV1 for structural and functional studies?

For recombinant expression of functional ASPRV1, GST-fusion protein systems have proven most effective. Both SASP28 (28 kDa zymogen) and SASP14 (14 kDa active form) can be successfully expressed as GST-fusion proteins . The GST tag provides multiple advantages including enhanced solubility, simplified purification through glutathione affinity chromatography, and retention of enzymatic activity in the fusion state .

When expressing SASP28, researchers should account for the self-processing that leads to autoactivation of the protease under slightly acidic conditions . For direct expression of the active enzyme, GST-SASP14 constructs can be employed, which yield the catalytic domain capable of homodimerization and enzymatic activity . Site-directed mutagenesis can be used to create inactive variants (e.g., by mutating the catalytic aspartic acid residue) for control experiments or to prevent autoproteolysis when studying the zymogen form .

The functional activity of recombinant ASPRV1 should be verified using synthetic oligopeptide substrates containing hydrophobic residues at the P2 position . Expression in bacterial systems has been successfully reported, though eukaryotic expression systems may be considered for studies requiring post-translational modifications not present in bacterial systems .

How can researchers differentiate between intravascular and tissue-infiltrating neutrophils when studying ASPRV1 in neuroinflammatory conditions?

Distinguishing between intravascular neutrophils and those that have infiltrated the tissue parenchyma is critical when studying ASPRV1 in neuroinflammation, as these populations show distinct ASPRV1 expression patterns. Several complementary approaches can be employed:

Flow cytometry analysis using differential marker expression provides a powerful method for population separation. ICAM1 (CD54) serves as a reliable marker distinguishing parenchyma-infiltrating neutrophils (ICAM1+) from intravascular neutrophils (ICAM1-) . Additional markers including CD45 (expressed at higher levels in infiltrating cells), CD11b, and the neutrophil-specific marker Ly6G enable comprehensive population identification .

Confocal microscopy offers spatial information critically important for distinguishing cell locations. Intravascular neutrophils exhibit characteristic rod-shaped morphology typical of crawling leukocytes and lack ICAM1 expression, while extravascular neutrophils in the meninges and parenchyma display multilobed nuclei and strong ICAM1 positivity (>90% of cells) . The use of transgenic reporter systems (e.g., Ly6G-Cre crossed with fluorescent reporter mice) enables neutrophil visualization with morphological assessment .

Experimental timing is also important, as neutrophils crawl more frequently on the CNS endothelial surface upon exposure to adjuvants but infiltrate the parenchyma only during active disease . This temporal pattern allows experimental designs that capture either intravascular or tissue-infiltrating populations based on disease stage.

What are the recommended controls when evaluating ASPRV1 inhibitors in experimental systems?

When evaluating ASPRV1 inhibitors, comprehensive controls are essential for result validation and interpretation:

Enzymatic assay controls:

• Positive control: Active ASPRV1 enzyme (typically GST-SASP14) with known substrate in optimized conditions (pH ~6.27, high ionic strength)

• Negative control: Heat-inactivated enzyme or catalytic site mutant (D→N mutation in the key aspartic acid residue)

• Specificity control: Testing inhibitor against related aspartic proteases (e.g., cathepsin D) to assess selectivity

• Vehicle control: Ensuring solvent used for inhibitor delivery does not affect enzyme activity

Cellular and tissue controls:

• ASPRV1 knockout/knockdown systems as positive controls for complete inhibition

• Dose-response analysis to establish inhibition curves and IC50 values

• Time-course studies to determine inhibition kinetics and potential compensatory mechanisms

• Assessment of known ASPRV1 substrates (e.g., profilaggrin processing) as functional readouts

In disease models (e.g., EAE):

• Comparison with ASPRV1-deficient animals as reference for complete inhibition effects

• Timing controls: treatment initiation at different disease stages to assess effects on disease onset versus progression

• Analysis of neutrophil ICAM1 expression and function as cellular readouts of inhibition efficacy

• B cell-dependent versus independent EAE models to confirm specificity of effects based on known ASPRV1 involvement in B cell-dependent forms

How can researchers resolve contradictory data regarding ASPRV1 function in different experimental systems?

Resolving contradictory findings regarding ASPRV1 function requires systematic investigation of several variables that may influence experimental outcomes:

Tissue-specific effects:
ASPRV1 functions differ substantially between skin and immune contexts . Experiments should clearly distinguish these systems, as findings in one tissue may not translate to another. The dual role of ASPRV1 in epithelial differentiation and neutrophil function means that phenotypes in different systems may appear contradictory if not properly contextualized.

Methodological differences:
Variations in experimental conditions significantly impact ASPRV1 activity. Researchers should standardize:

• pH conditions (ASPRV1 optimum ~6.27)

• Ionic strength (higher activity at elevated ionic strength)

• Substrate selection (preference for hydrophobic P2 residues)

• Enzyme form used (SASP28 zymogen vs. SASP14 active form)

Animal model considerations:
The EAE model shows ASPRV1 involvement specifically in B cell-dependent forms but not with traditional MOG35-55 peptide immunization . This specificity means results may appear contradictory if researchers use different immunization protocols without recognizing this distinction.

Genetic background effects:
Complete ASPRV1 knockout causes relatively mild skin phenotypes (fine wrinkles and reduced hydration) but significant inflammatory alterations . Contradictory findings might emerge from studies using different knockout strategies or genetic backgrounds, which should be thoroughly documented.

When faced with contradictory data, researchers should conduct side-by-side comparisons using standardized conditions, employ multiple complementary methods to assess ASPRV1 function, and consider developmental compensation that might occur in complete knockout systems versus acute inhibition models.

How might ASPRV1 serve as a biomarker or therapeutic target in multiple sclerosis?

ASPRV1 shows significant potential as both a biomarker and therapeutic target in multiple sclerosis based on recent research findings:

As a biomarker:
ASPRV1 levels are elevated in brain lesions of patients with severe multiple sclerosis compared to those with mild or moderate disease or controls . This correlation with disease severity suggests ASPRV1 could serve as a marker of disease progression or severity. Additionally, ICAM1+ neutrophils expressing ASPRV1 could be detected in peripheral blood or cerebrospinal fluid, potentially providing a less invasive biomarker . Quantitative analysis of ASPRV1 expression or activity might help identify patients likely to develop more severe, chronic forms of MS, enabling earlier intervention strategies.

As a therapeutic target:
Several lines of evidence support ASPRV1 as a promising therapeutic target:

• ASPRV1-deficient mice develop significantly milder chronic phase EAE in B cell-dependent models, with many animals showing complete resolution of chronic symptoms

• ASPRV1 appears specifically involved in the progression from acute to chronic inflammation rather than disease initiation

• ASPRV1 is inhibited by established aspartic protease inhibitors including pepstatin A and acetyl-pepstatin

• The enzyme shows structural similarity to retroviral proteases, for which numerous inhibitors have been developed

Targeting strategies might focus on developing specific ASPRV1 inhibitors, potentially repurposing or modifying existing aspartic protease inhibitors. Therapeutic approaches could specifically target neutrophil ASPRV1 rather than epithelial expression to minimize potential skin-related side effects. The specific involvement of ASPRV1 in B cell-dependent models suggests it might be particularly relevant for MS subtypes with prominent B cell involvement.

What methodological challenges exist in translating ASPRV1 research from mouse models to human patients?

Translating ASPRV1 research from mouse models to human applications faces several methodological challenges:

Species differences in expression and function:
While ASPRV1 shows conserved expression in neutrophils and epithelial tissues across species, functional differences may exist. Human ASPRV1 variants (K199E, R311P, P314T) affect protease function and are associated with ichthyosis , highlighting potential species-specific regulatory mechanisms. Comparative studies of mouse and human ASPRV1 biochemistry, substrate specificity, and regulation are essential before extrapolating findings across species.

Disease model limitations:
EAE models, while valuable, do not fully recapitulate the complexity of human MS. The finding that ASPRV1 is specifically involved in B cell-dependent EAE models suggests its role may be restricted to particular MS subtypes or disease stages. Human MS is heterogeneous, with varying degrees of B cell involvement across patients, requiring careful patient stratification in translational studies.

Diagnostic and sampling challenges:
Detecting ASPRV1 in MS patients presents practical challenges. Brain tissue sampling is highly invasive, limiting biomarker development. Research is needed to determine if ASPRV1 levels in blood neutrophils correlate with CNS expression and disease severity. Flow cytometry protocols for ICAM1+ neutrophil detection and ASPRV1 quantification in peripheral blood need standardization for clinical application.

Therapeutic development considerations:
Developing ASPRV1 inhibitors requires addressing:

• Blood-brain barrier penetration to reach CNS-infiltrating neutrophils

• Specificity to avoid inhibiting related aspartic proteases

• Delivery systems to target neutrophils while minimizing effects on epithelial ASPRV1

• Potential consequences of long-term ASPRV1 inhibition on skin health and neutrophil function in infection defense

These challenges highlight the need for coordinated basic and translational research to advance ASPRV1-based approaches from preclinical models to human applications.

What are the most significant knowledge gaps in ASPRV1 research?

Despite significant advances, several critical knowledge gaps remain in ASPRV1 research:

• Substrate identification beyond profilaggrin, particularly in neutrophils, represents the most pressing knowledge gap. While ASPRV1's role in neuroinflammation is established, the molecular mechanisms and substrates mediating this function remain unknown .

• Regulatory mechanisms controlling ASPRV1 expression in different tissues are poorly understood. The factors governing ASPRV1 upregulation in ICAM1+ neutrophils during inflammation require further characterization .

• The crystal structure of ASPRV1 has not been determined, limiting structure-based drug design. Current structural insights rely on homology modeling rather than experimental structures .

• The precise contribution of ASPRV1 to human multiple sclerosis pathogenesis requires further clarification, including its role in different MS subtypes and disease stages .

• The potential roles of ASPRV1 in other inflammatory or autoimmune conditions beyond MS remain unexplored, despite the enzyme's expression in neutrophils implicated in various inflammatory diseases.

Addressing these knowledge gaps will require interdisciplinary approaches combining structural biology, proteomics, advanced imaging, and clinical studies to fully elucidate ASPRV1's physiological and pathological roles and to harness its therapeutic potential.

What emerging technologies might accelerate ASPRV1 research in the coming years?

Several emerging technologies hold particular promise for advancing ASPRV1 research:

• Single-cell proteomics and transcriptomics could reveal ASPRV1 expression patterns in neutrophil subpopulations and identify co-expressed factors that regulate its function in different contexts, potentially explaining tissue-specific roles .

• CRISPR-based technologies beyond simple knockouts, including base editing and transcriptional modulation, could enable precise manipulation of ASPRV1 expression and function in specific tissues or cell types without complete elimination.

• Advanced imaging techniques like imaging mass cytometry could track ASPRV1+ neutrophils in tissue contexts while simultaneously measuring multiple markers to characterize their interactions with other immune cells .

• Activity-based protein profiling with tailored probes could identify ASPRV1 substrates in neutrophils and inflammatory microenvironments by capturing transient enzyme-substrate interactions.

• Cryo-electron microscopy could determine the high-resolution structure of ASPRV1 in different conformational states, facilitating structure-based drug design for specific inhibitors.

• Patient-derived organoids or humanized mouse models could bridge the gap between basic research and clinical applications, providing more relevant systems for testing ASPRV1 modulators before clinical trials.