Recombinant Drosophila melanogaster Furin-like protease 1, isoform 1-CRR (Fur1)

What is Dfurin1-CRR and how does it relate to other furin isoforms in Drosophila melanogaster?

Dfurin1-CRR is one of three main furin-like proteins encoded by the Dfur1 gene in Drosophila melanogaster. The protein consists of 1101 amino acid residues, which makes it intermediate in size between the smaller Dfurin1 (892 residues) and the larger Dfurin1-X (1269 residues). All three isoforms are members of the subtilisin-like proprotein processing enzyme family and are generated through alternative splicing and polyadenylation of the Dfur1 gene transcripts .

The Dfur1 gene produces four distinct transcripts of 7.6, 6.5, 4.5, and 4.0 kb, as detected by Northern blot analysis. Comparative genomic and cDNA sequence analysis has identified 10 coding exons that contribute to these transcripts in varying combinations. The three furin-like proteins share common structural domains but also possess unique regions that contribute to their specific functions and tissue distribution patterns .

What is the structural organization of the Dfur1 gene that produces Dfurin1-CRR?

The Dfur1 gene has a complex organization consisting of 10 coding exons that can be alternatively spliced to generate the different isoforms. Analysis of genomic and cDNA clones revealed that exons 5-13 were identified in the genomic sequence, while the 5'-end exon sequences (nucleotides 1-1495) required additional screening of a Drosophila genomic library. Exon 1 consists of nucleotides 1-552 and is flanked by a 5'-splice site consensus sequence .

The complete understanding of the gene structure came from Northern blot analysis using exon-specific probes (pG6, pG7, PCR1, pIP44, and pIP46), which confirmed that the four different transcripts are generated through a combination of alternative splicing and differential polyadenylation mechanisms .

How is Dfurin1-CRR expression regulated during Drosophila development?

Dfurin1-CRR shows differential expression throughout Drosophila development, with specific temporal patterns observed in Northern blot analysis of RNA from various developmental stages. The transcripts show stage-specific regulation, with the 7.6 kb transcript (which was not initially detected in earlier studies) showing very low expression levels in the 2-8 hour embryo stage .

RNA in situ hybridization experiments have revealed that Dfurin1-CRR and Dfurin1-X isoforms are expressed in completely non-overlapping sets of tissues during Drosophila embryogenesis. This spatial separation suggests distinct roles for these isoforms in the development of different embryonic tissues. The precise regulation of these expression patterns is likely controlled by tissue-specific transcription factors and enhancers that recognize specific regulatory elements in the Dfur1 gene .

What tissue-specific expression patterns does Dfurin1-CRR exhibit compared to other furin isoforms?

Detailed RNA in situ hybridization studies have shown that Dfurin1-CRR and Dfurin1-X are expressed in mutually exclusive tissue sets during embryonic development. This non-overlapping expression pattern strongly indicates that these isoforms have evolved to perform specialized functions in specific tissues .

The tissue-specific expression appears to be tightly regulated, as strong hybridization signals were detected only in those tissues that expressed either Dfurin1-X or Dfurin1-CRR. This suggests that the alternative splicing events leading to the production of these isoforms are tissue-specific and developmentally regulated, rather than occurring randomly throughout the organism .

What are the optimal methods for recombinant expression and purification of Dfurin1-CRR?

Based on the research methodologies described in the literature, the optimal approach for recombinant expression of Dfurin1-CRR involves gene transfer experiments using appropriate expression vectors. In the cited studies, researchers characterized the various Dfurin isoforms through gene transfer approaches followed by immunoprecipitation analysis with anti-Dfurin1 antiserum .

For purification, a combination of techniques is typically employed:

• Design of expression constructs containing the complete Dfurin1-CRR open reading frame

• Transfection into suitable expression systems (typically insect cells for Drosophila proteins)

• Immunoprecipitation using specific antibodies raised against common regions of the Dfurin proteins

• SDS-PAGE and Western blot analysis to confirm protein identity and purity

• Additional chromatography steps (ion exchange, size exclusion) for higher purity if needed

The specific characteristics of Dfurin1-CRR, including its 1101 amino acid length and unique structural domains, should be considered when optimizing expression and purification protocols .

What methods are used to assess the enzymatic activity of recombinant Dfurin1-CRR?

The enzymatic activity of Dfurin1-CRR can be assessed using proprotein processing assays. In the research studies cited, two distinct heterologous proprotein processing assays were employed to evaluate the activity of the various Dfur1-encoded proteins .

Specifically, gene transfer experiments demonstrated that when Dfurin1, Dfurin1-CRR, and Dfurin1-X proteins were expressed at high levels together with either:

• The precursor of the beta A-chain of activin-A (a member of the transforming growth factor beta superfamily)

• The precursor of von Willebrand factor

All three proteins were capable of processing these substrates, indicating that they possess active proprotein processing capabilities despite their structural differences .

A typical enzymatic activity assay would involve:

• Co-expression of Dfurin1-CRR with a known substrate protein

• Collection of cell lysates or culture media

• Analysis of substrate processing by SDS-PAGE, Western blotting, or other detection methods

• Quantification of processed vs. unprocessed substrate to determine enzymatic efficiency

What are the unique structural domains of Dfurin1-CRR and how do they compare to other furin isoforms?

Dfurin1-CRR (1101 residues) contains both domains that are common to all Dfurin isoforms as well as unique structural elements. While detailed structural information from the search results is limited, the comparative analysis of the three isoforms revealed that each possesses common domains essential for catalytic activity, but also unique regions that likely contribute to their specific functions and localization patterns .

The name "CRR" in Dfurin1-CRR suggests the presence of a cysteine-rich region, which is likely important for protein stability, substrate recognition, or regulatory functions. This domain appears to be absent in the basic Dfurin1 isoform (892 residues) but may share some similarities with domains in the larger Dfurin1-X isoform (1269 residues) .

The three isoforms likely share the core catalytic domain characteristic of subtilisin-like proprotein processing enzymes, but differ in their regulatory, targeting, or interaction domains, which explains their ability to perform distinct physiological functions despite recognizing similar substrates in experimental settings .

How do post-translational modifications affect Dfurin1-CRR activity and localization?

While the search results do not specifically address post-translational modifications of Dfurin1-CRR, it is well established that furin-like proteases typically undergo several critical modifications that affect their activity and localization. Based on general knowledge of this protein family, the following modifications likely impact Dfurin1-CRR:

• Auto-proteolytic processing: Like other furin family members, Dfurin1-CRR likely requires removal of its pro-domain through auto-catalysis to become active

• Glycosylation: N-linked glycosylation likely occurs at specific sites and may influence protein folding, stability, and trafficking

• Phosphorylation: Potential phosphorylation sites may regulate enzymatic activity or subcellular trafficking

• Disulfide bond formation: The cysteine-rich regions implied by the "CRR" designation likely form disulfide bonds critical for protein structure and function

The unique tissue distribution pattern of Dfurin1-CRR compared to Dfurin1-X suggests that specific post-translational modifications or trafficking signals may direct these isoforms to different cellular compartments or secretory pathways .

What is the substrate specificity of Dfurin1-CRR and how does it differ from other furin isoforms?

In experimental settings, Dfurin1-CRR demonstrated the ability to process the same test substrates as the other Dfurin isoforms. Gene transfer experiments showed that Dfurin1, Dfurin1-CRR, and Dfurin1-X were all capable of processing the precursor of the beta A-chain of activin-A (a TGF-β superfamily member) and the precursor of von Willebrand factor .

• The specific protein expression patterns in their respective tissues

• Potential differences in their recognition sequences or binding affinity for diverse substrates

• Structural differences that may allow interaction with tissue-specific cofactors or regulatory proteins

• Different subcellular localizations that expose them to distinct sets of potential substrates

The common catalytic capability but distinct tissue distribution suggests an evolutionary strategy to utilize similar enzymatic mechanisms for processing different developmental signals in separate tissues .

What are the known physiological functions of Dfurin1-CRR in Drosophila development?

The research indicates that Dfurin1-CRR has distinct physiological functions in Drosophila development. The non-overlapping expression patterns between Dfurin1-CRR and Dfurin1-X during embryogenesis suggest specialized roles in different developmental processes .

As a member of the subtilisin-like proprotein processing enzyme family, Dfurin1-CRR likely functions in the proteolytic activation of precursor proteins involved in development. Its ability to process TGF-β superfamily members (demonstrated with activin-A) in experimental settings suggests it may activate signaling molecules crucial for tissue differentiation and morphogenesis in the specific embryonic tissues where it is expressed .

The researchers concluded that the Dfur1 gene encodes structurally different subtilisin-like proprotein processing enzymes with distinct physiological functions in Drosophila. This indicates that alternative splicing and differential expression of Dfurin isoforms represent an important mechanism for generating diversity in proprotein processing enzymes to serve specialized developmental roles .

How can CRISPR/Cas9 genome editing be used to study Dfurin1-CRR function?

CRISPR/Cas9 genome editing provides powerful approaches to study Dfurin1-CRR function through several strategies:

• Isoform-specific knockout: Design guide RNAs targeting exons unique to the Dfurin1-CRR isoform to selectively disrupt its expression while preserving other Dfur1 isoforms. This allows isolation of phenotypes specifically attributable to Dfurin1-CRR.

• Domain-specific mutations: Create precise mutations in functional domains of Dfurin1-CRR to assess their contributions to enzyme activity, localization, or substrate specificity.

• Reporter gene knock-in: Insert fluorescent protein tags or epitope tags in-frame with Dfurin1-CRR to monitor its expression, localization, and dynamics in living tissues during development.

• Promoter/enhancer analysis: Target regulatory regions controlling tissue-specific expression of Dfurin1-CRR to identify critical control elements.

Using these approaches would build upon the non-overlapping tissue expression patterns revealed by RNA in situ hybridization and help elucidate the specific developmental processes and signaling pathways regulated by Dfurin1-CRR .

What proteomics approaches are most effective for identifying physiological substrates of Dfurin1-CRR?

For identifying physiological substrates of Dfurin1-CRR, several complementary proteomics approaches would be most effective:

• Quantitative proteomic analysis using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling: Compare proteomes between wild-type and Dfurin1-CRR mutant tissues to identify proteins with altered processing.

• Terminal amine isotopic labeling of substrates (TAILS): This N-terminomics approach enriches for protein N-termini and can identify new cleavage sites generated by Dfurin1-CRR activity.

• Co-immunoprecipitation coupled with mass spectrometry: Identify proteins that interact with catalytically inactive Dfurin1-CRR mutants, which may retain binding to substrates without processing them.

• Bioinformatic prediction and validation: Analyze the Drosophila proteome for proteins containing conserved furin recognition motifs (typically R-X-[K/R]-R↓) that are expressed in tissues where Dfurin1-CRR is active.

• Substrate trapping: Express a catalytically inactive Dfurin1-CRR with enhanced substrate binding properties to trap potential substrates in stable complexes for identification.

These approaches would reveal the specific developmental signaling pathways and processes regulated by Dfurin1-CRR-mediated proteolytic processing in its unique tissue expression domains .

How conserved is Dfurin1-CRR across Drosophila species and other insects?

While the provided search results do not specifically address conservation across species, this question can be approached based on general principles of protein evolution and the functional importance of furin-like proteases:

Furin-like proteases are highly conserved across metazoan evolution due to their essential roles in development and physiology. The Dfurin1-CRR isoform, with its specialized tissue expression pattern and functional capabilities, likely shows varying degrees of conservation:

• The catalytic domain and substrate recognition sites are likely highly conserved across Drosophila species and possibly other dipterans, reflecting functional constraints on these essential regions.

• The cysteine-rich region implied by "CRR" in the name may show intermediate conservation, as these domains often contribute to protein stability and regulation.

• Regulatory regions controlling tissue-specific expression may show greater divergence between species, reflecting different developmental constraints and timing.

• The alternative splicing mechanisms generating the Dfurin1-CRR isoform are likely conserved within the Drosophila genus but may vary in more distant insect lineages.

Evolutionary analysis of Dfurin1-CRR conservation could provide insights into the functional importance of specific domains and the evolutionary history of alternative splicing as a mechanism for generating diversity in proprotein processing enzymes .

What are the functional differences between Drosophila Dfurin1-CRR and mammalian furin homologs?

Dfurin1-CRR and mammalian furins share the fundamental catalytic mechanism characteristic of subtilisin-like proprotein processing enzymes, but likely differ in several important aspects:

• Domain organization: Mammalian furins typically have a more standardized domain structure, while Drosophila has evolved multiple isoforms with unique domain arrangements, including the CRR domain specific to Dfurin1-CRR.

• Tissue specificity: Mammalian furins typically show broader expression patterns, while Drosophila has evolved highly specific expression patterns for different isoforms, with Dfurin1-CRR and Dfurin1-X expressed in non-overlapping tissues.

• Regulatory mechanisms: The alternative splicing mechanisms generating Dfurin1-CRR represent a distinct regulatory strategy compared to the transcriptional and post-translational regulation more commonly observed in mammalian furins.

• Substrate range: While both can process similar test substrates (as shown with activin-A and von Willebrand factor), their physiological substrate ranges likely differ substantially due to differences in their expression patterns and the distinct developmental programs of insects versus mammals.

These differences highlight how evolution has employed similar enzymatic mechanisms but different regulatory and structural strategies to achieve tissue-specific proteolytic processing in different animal lineages .

What are common challenges in expressing recombinant Dfurin1-CRR and how can they be overcome?

Based on the properties of Dfurin1-CRR and general challenges with recombinant expression of complex proteases, researchers may encounter several difficulties:

• Protein misfolding and aggregation: As a large, multi-domain protein (1101 residues) with likely disulfide bonds in the cysteine-rich region, Dfurin1-CRR may fold incorrectly when overexpressed.

  • Solution: Use insect cell expression systems (Sf9, S2) that better recapitulate Drosophila post-translational modifications

  • Include folding chaperones or express at lower temperatures

  • Consider expressing functional domains separately

• Autoproteolysis and self-inactivation: Active proteases often cleave themselves or other proteins in the expression system.

  • Solution: Express as an inactive zymogen with intact pro-domain

  • Use protease inhibitors during purification

  • Design expression constructs with point mutations in the catalytic site for structural studies

• Toxicity to host cells: Proteolytic activity may damage the expression host.

  • Solution: Use tightly controlled inducible expression systems

  • Express as a fusion with self-cleaving inteins for controlled activation

• Low yield: Complex proteins often express at lower levels.

  • Solution: Optimize codon usage for the expression host

  • Screen multiple expression constructs with different tags and fusion partners

  • Develop high-efficiency purification strategies to maximize recovery

The gene transfer and immunoprecipitation approaches used in the cited studies provide a starting point, but optimizations would be needed for large-scale production of active enzyme .

How can the activity and specificity of Dfurin1-CRR be accurately measured in vitro?

To accurately measure the activity and specificity of Dfurin1-CRR in vitro, researchers can employ several complementary approaches:

• Fluorogenic peptide substrates: Design short peptides containing the consensus furin cleavage site (R-X-[K/R]-R↓) with flanking sequences based on known substrates, labeled with fluorophore/quencher pairs that produce fluorescence upon cleavage.

  • Measure kinetic parameters (Km, kcat, kcat/Km)

  • Compare multiple substrate sequences to establish specificity profiles

• Recombinant protein substrates: Express and purify full-length or truncated versions of natural substrates (such as the activin-A and von Willebrand factor precursors used in the cited studies).

  • Analyze processing by SDS-PAGE, Western blotting, or mass spectrometry

  • Quantify processing efficiency under different conditions

• Inhibitor profiling: Test sensitivity to different protease inhibitors to characterize the active site.

  • Compare general serine protease inhibitors vs. furin-specific inhibitors

  • Develop specific inhibitors based on substrate recognition sequences

• pH and calcium dependence: Furin-like proteases typically show pH optima and calcium requirements that reflect their normal cellular compartment.

  • Measure activity across pH range (typically 5.0-8.0)

  • Determine calcium concentration requirements

• Comparative analysis with other isoforms: Parallel testing of Dfurin1, Dfurin1-CRR, and Dfurin1-X under identical conditions to identify subtle differences in specificity or activity that might not be apparent in cellular assays.

These approaches would provide a comprehensive biochemical characterization of Dfurin1-CRR and insight into how its structure relates to its specific functions in Drosophila development .

What are the most promising areas for future research on Dfurin1-CRR?

Based on the current understanding of Dfurin1-CRR and the gaps in knowledge, several promising research directions emerge:

• Comprehensive substrate identification: Apply advanced proteomics approaches to identify the complete set of physiological substrates processed specifically by Dfurin1-CRR in its native tissues. This would connect the enzyme to specific developmental signaling pathways.

• Structural biology: Determine the three-dimensional structure of Dfurin1-CRR through X-ray crystallography or cryo-EM to understand how its unique domains contribute to function and how alternative splicing creates functional diversity in furin-like enzymes.

• Developmental regulation: Elucidate the transcriptional and post-transcriptional mechanisms controlling the tissue-specific expression of Dfurin1-CRR during embryogenesis, including the factors that drive alternative splicing decisions.

• Genetic modeling: Create precise genetic models with isoform-specific mutations or conditional expression systems to dissect the exact developmental processes dependent on Dfurin1-CRR activity.

• Evolutionary analysis: Compare Dfurin1-CRR structure, function, and regulation across insect species to understand how alternative splicing evolves as a mechanism for generating diversity in proprotein processing enzymes.

These research directions would build upon the foundational work described in the cited studies and advance understanding of how proteolytic processing contributes to developmental patterning and signaling specificity .

How might understanding Dfurin1-CRR function contribute to broader insights in developmental biology?

Understanding Dfurin1-CRR function has potential to provide significant insights into several fundamental aspects of developmental biology:

• Proteolytic regulation of developmental signaling: Dfurin1-CRR exemplifies how proteolytic processing creates an additional regulatory layer controlling signal transduction during development. Understanding its specific substrates would illuminate how diverse signaling pathways are coordinated in space and time.

• Alternative splicing as a mechanism for evolutionary innovation: The generation of functionally distinct Dfurin isoforms with unique tissue distributions demonstrates how alternative splicing creates protein diversity without requiring gene duplication, providing insights into evolutionary mechanisms for increasing protein complexity.

• Tissue-specific post-translational processing: The non-overlapping expression patterns of Dfurin1-CRR and Dfurin1-X reveal how organisms achieve tissue-specific protein processing through differential protease expression rather than through substrate specificity alone.

• Integration of transcriptional and post-translational regulation: The precise developmental control of Dfurin1-CRR expression illustrates how organisms coordinate gene expression and protein activation to ensure proper developmental timing.

• Conservation and divergence of proteolytic processing: Comparative studies between Drosophila and vertebrate furin systems may reveal fundamental principles about how similar enzymatic mechanisms have been adapted to serve diverse developmental programs across animal phyla.

These broader insights could influence understanding of how regulatory networks achieve developmental precision and how evolution leverages similar enzymatic mechanisms for diverse developmental outcomes .