PRSS3 Human

What is the genomic organization of the PRSS3 gene in humans?

The PRSS3 gene encoding mesotrypsinogen is located on chromosome 9p13, unlike the genes for cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) which are found at proximal loci on chromosome 7q35 . The gene contains 5 exons and intron/exon junctions that have been subject to comprehensive genetic analysis in various patient cohorts . The cDNA sequence is recorded in GenBank as NM_002771.2, and mutations are described according to the nomenclature recommended by the Human Genome Variation Society .

How does mesotrypsin differ structurally and functionally from other human trypsins?

Mesotrypsin is distinguished by its exceptional resistance to traditional trypsin inhibitors and unique substrate specificity . Unlike other human trypsins, mesotrypsin possesses the extraordinary catalytic capability to hydrolyze the reactive sites of canonical trypsin inhibitors such as SPINK1 and soybean trypsin inhibitor . Originally isolated as "zymogen X" by Rinderknecht et al. in 1979, it was later renamed mesotrypsinogen based on its relative isoelectric point . Mesotrypsin accounts for a small fraction (<0.5%) of proteins in human pancreatic juice, compared to cationic trypsinogen (~13%) and anionic trypsinogen (~6%) .

What is the normal tissue distribution of PRSS3/mesotrypsin expression?

Normal expression of the PRSS3 gene is highest in pancreas and brain with very limited expression elsewhere . This restricted expression pattern is significant when studying its pathological roles, as PRSS3 upregulation has been observed in various epithelial cancers including lung, colon, breast, pancreas, and prostate during cancer progression . Within the skin epidermis, mesotrypsin primarily expresses in the upper layers of the stratified epidermis .

What role does mesotrypsin play in epidermal differentiation and morphology?

Within the skin epidermis, mesotrypsin plays a crucial role in processing pro-filaggrin (Pro-FLG) . Research using HaCaT keratinocytes has demonstrated that inducing Venus-mesotrypsin expression results in a flattened phenotype and reduced proliferative capacity . Additionally, these cells displayed altered F-actin assembly, enhanced E-cadherin adhesive activity, and facilitated tight junction formation without overtly influencing epidermal differentiation . These findings underscore mesotrypsin's potentially pivotal role in shaping the characteristic cellular morphology of upper epidermal layers, particularly in the transition from granular to cornified layers.

How can researchers effectively study mesotrypsin function in keratinocyte models despite its rapid degradation?

Studying mesotrypsin in keratinocyte models presents challenges due to the ready degradation of active mesotrypsin. Researchers have overcome this limitation by developing a fusion protein approach. Specifically, fusion with Venus fluorescent protein, flanked by a peptide linker, enables evasion from the protein elimination machinery, thus facilitating activation of the Pro-FLG processing system . This methodological approach allows for sustained expression and functional analysis of mesotrypsin in keratinocyte models, particularly in HaCaT cells which are characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states .

What evidence links PRSS3/mesotrypsin to cancer progression and metastasis?

Multiple studies have established strong links between PRSS3/mesotrypsin and cancer progression:

Experimental validation has shown that over-expression of PRSS3 promotes pancreatic cancer cell proliferation and invasion in vitro, as well as tumor progression and metastasis in vivo . Conversely, knockdown of PRSS3 by RNA interference correlates with suppression of malignant growth in 3D cultures .

Through what molecular mechanisms does PRSS3/mesotrypsin promote cancer metastasis?

Several molecular mechanisms have been identified:

• PAR1-mediated ERK pathway: In pancreatic cancer, PRSS3 upregulates VEGF expression via the PAR1-mediated ERK pathway . ERK inhibitor significantly delayed metastasis progression and prolonged survival in animal models (p<0.05) .

• PAR2 activation: Shear stress triggers PRSS3 to cleave the N-terminal inhibitory domain of PAR2 within 2 hours . As a G protein-coupled receptor, PAR2 activates the G𝛼i protein to turn on the Src-ERK/p38/JNK-FRA1/cJUN axis, promoting gene expression including PRSS3 itself, creating a feed-forward loop facilitating metastasis .

• CD109 shedding: In breast cancer, the cell surface glycoprotein CD109 was identified as a potential mesotrypsin substrate involved in driving malignancy .

How does shear stress influence PRSS3/mesotrypsin function in circulating tumor cells?

Circulating tumor cells (CTCs) experience significant mechanical forces while traveling in bloodstream. Research using microfluidic circulatory systems that generate arteriosus shear stress has revealed that shear stress upregulates mesotrypsin (PRSS3), protease-activated receptor 2 (PAR2), and Fos-related antigen 1 (FOSL1) . Approximately half of cancer cells survive shear stress damage and show higher invasion ability .

Mechanistically, shear stress triggers PRSS3 to cleave the N-terminal inhibitory domain of PAR2 within 2 hours, activating downstream signaling pathways that promote invasion and metastasis . PAR2 functions as a shear stress-specific mechanosensor cleavable by PRSS3 in circulation, providing new insights for targeting metastasis-initiating CTCs .

What is the relationship between PRSS3/mesotrypsin and chronic pancreatitis?

Mesotrypsin accounts for 3–10% of trypsin activity in human pancreatic juice and exhibits remarkable resistance to naturally occurring trypsin inhibitors . PRSS3 can hydrolyze the reactive-site peptide bond of trypsin inhibitors including SPINK1, suggesting a potential role in degradation of protective inhibitors . This ability raised the possibility that premature PRSS3 activation might contribute to chronic pancreatitis (CP) development .

How can researchers functionally analyze PRSS3/mesotrypsin variants?

Researchers can employ several methodological approaches:

• Site-directed mutagenesis: Introduction of specific mutations (e.g., p.T167A) into expression plasmids (e.g., pcDNA3.1(–)_PRSS3) using PCR mutagenesis .

• Cell-based expression systems: Transfection of human embryonic kidney (HEK) 293T cells with wild-type and mutant PRSS3 plasmids .

• Activity assays: Measurement of enzymatic activity after enterokinase activation at specific time points (e.g., 24 and 48 hours post-transfection) .

• Protein visualization: Confirmation of mesotrypsinogen expression by SDS-PAGE and Coomassie Blue staining .

• Inhibitor resistance studies: Analysis of the variant's resistance to natural trypsin inhibitors and its ability to degrade these inhibitors .

For genetic studies, PCR amplification of all 5 exons and intron/exon junctions followed by direct sequencing represents the standard approach, with subsequent confirmatory functional studies for identified variants .

What experimental systems are optimal for studying mesotrypsin's role in epidermal differentiation?

Studying mesotrypsin in epidermal differentiation presents challenges due to the differentiation-activated cell death program in primary cultured keratinocytes . HaCaT keratinocytes represent an optimal experimental system because they are characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states .

To overcome rapid degradation of active mesotrypsin, researchers have developed a Venus-mesotrypsin fusion protein approach where a peptide linker enables evasion from cellular protein elimination machinery . This methodology facilitates activation of the Pro-FLG processing system and allows for sustained experimental observation .

The experimental workflow typically involves:

• Generation of Venus-mesotrypsin fusion constructs

• Transfection into HaCaT keratinocytes

• Assessment of morphological changes (flattened phenotype)

• Analysis of proliferative capacity

• Examination of F-actin assembly, E-cadherin activity, and tight junction formation

How can researchers effectively study the role of PRSS3/mesotrypsin in circulating tumor cells?

To study PRSS3/mesotrypsin in circulating tumor cells (CTCs), researchers have developed microfluidic circulatory systems that generate arteriosus shear stress while controlling for detachment effects . This approach allows comparison of transcriptome profiles of circulating cancer cells with or without shear stress exposure.

The experimental workflow involves:

• Development of a microfluidic circulatory system mimicking arterial shear stress

• Exposure of cancer cells to controlled shear stress conditions

• Transcriptome profiling to identify differentially expressed genes

• Validation of PRSS3, PAR2, and FOSL1 upregulation

• Analysis of N-terminal PAR2 cleavage by PRSS3 (occurring within 2 hours)

• Investigation of downstream signaling pathways (Src-ERK/p38/JNK-FRA1/cJUN axis)

• Assessment of invasion and metastatic potential

This methodology has revealed that shear stress triggers PRSS3-mediated PAR2 activation, initiating signaling cascades that promote metastasis .

What are the emerging targets and applications of PRSS3/mesotrypsin research in cancer therapy?

Several promising therapeutic targets have emerged:

The correlation between PRSS3 expression and poor clinical outcomes in multiple cancer types suggests that targeting PRSS3/mesotrypsin signaling pathways could represent an effective approach for cancer treatment, particularly for preventing metastasis .

What contradictions or paradoxes exist in the current understanding of PRSS3/mesotrypsin biology?

Several notable contradictions exist:

• Tissue-specific roles: PRSS3/mesotrypsin plays important physiological roles in epidermal differentiation and morphology , yet has pathological roles in cancer progression and metastasis . This duality suggests context-dependent functions requiring further investigation.

• Genetic contribution to pancreatitis: Despite biochemical evidence that PRSS3 can degrade pancreatic trypsin inhibitors , comprehensive genetic analysis found no disease-associated PRSS3 variants in chronic pancreatitis patients . This contradiction suggests either limited pathophysiological relevance of this biochemical activity or compensatory mechanisms.

• Expression paradox: Normal PRSS3 expression is highest in pancreas and brain , yet its upregulation in various epithelial cancers suggests either that cancer cells co-opt its normal functions or that it has undiscovered roles that become apparent only in disease states.

• Functional evaluation challenges: While in vitro studies clearly demonstrate mesotrypsin's ability to degrade inhibitors, translating these findings to in vivo physiological and pathological significance remains challenging, particularly given the contradictory genetic evidence in pancreatitis studies .

Resolving these paradoxes represents an important frontier in PRSS3/mesotrypsin research and may lead to new insights into both its physiological functions and pathological roles.

What are the recommended approaches for detecting and quantifying PRSS3/mesotrypsin expression in clinical samples?

For clinical research involving PRSS3/mesotrypsin, several detection methods have been validated:

• Immunohistochemistry: Used to detect PRSS3 protein in human pancreatic cancer tissues, with 40.54% of samples (n=74) showing positive expression . This method allows correlation with clinical outcomes and metastatic status.

• Quantitative PCR (qPCR): PRSS3 expression in human pancreatic cancer cell lines can be detected by qPCR to quantify mRNA levels .

• Immunoblotting: Western blotting provides protein-level confirmation of PRSS3 expression and can be used alongside qPCR for comprehensive analysis .

• ELISA: For analyzing secreted proteins related to PRSS3 signaling, such as VEGF expression induced by PRSS3 activity .

When interpreting PRSS3 expression in clinical samples, researchers should consider both the sensitivity of detection methods and the specific splice variants being detected, as these may have functional differences.

What experimental design considerations are critical when manipulating PRSS3 expression in model systems?

When designing experiments to manipulate PRSS3 expression, researchers should consider:

• Cell line selection: Different cell lines may have varying baseline expression of PRSS3 and related pathway components. HaCaT keratinocytes have minimal endogenous mesotrypsin expression, making them suitable for overexpression studies .

• Expression stability: Due to rapid degradation of active mesotrypsin in some systems, fusion approaches (e.g., Venus-mesotrypsin) may be necessary to achieve stable expression .

• Functional validation: Beyond confirming altered expression, enzymatic activity should be verified using appropriate substrates to ensure functional relevance of experimental manipulations.

• Pathway analysis: When studying PRSS3's role in cancer, comprehensive analysis of downstream pathways (PAR1/ERK, PAR2/G𝛼i/Src) is essential for mechanistic understanding .

• In vivo validation: Findings from in vitro studies should be validated in appropriate in vivo models to confirm relevance to complex physiological conditions .