Proprotein convertases regulate trafficking and maturation of key proteins within the secretory pathway.

Proprotein Convertases (PCs) are serine endoproteases that regulate the homeostasis of protein substrates in the cell. The PCs family counts 9 members-PC1/3, PC2, PC4, PACE4, PC5/6, PC7, Furin, SKI-1/S1P, and PCSK9. The first seven PCs are known as Basic Proprotein Convertases due to their propensity to cleave after polybasic clusters. SKI-1/S1P requires the additional presence of hydrophobic residues for processing, whereas PCSK9 is catalytically dead after autoactivation and exerts its functions using mechanisms alternative to direct cleavage. All PCs traffic through the canonical secretory pathway, reaching different compartments where the various substrates reside. Despite PCs members do not share the same subcellular localization, most of the cellular organelles count one or more Proprotein Convertases, including ER, Golgi stack, endosomes, secretory granules, and plasma membranes. The widespread expression of these enzymes at the systemic level speaks for their importance in the homeostasis of a large number of biological functions. Among others, PCs cleave precursors of hormones and growth factors and activate receptors and transcription factors. Notably, dysregulation of the enzymatic activity of Proprotein Convertases is associated to major human pathologies, such as cardiovascular diseases, cancer, diabetes, infections, inflammation, autoimmunity diseases, and Parkinson. In the current COVID-19 pandemic, Furin has further attracted the attention as a key player for conferring high pathogenicity to SARS-CoV-2. Here, we review the Proprotein Convertases family and their most important substrates along the secretory pathway. Knowledge about the complex functions of PCs is important to identify potential drug strategies targeting this class of enzymes.

PCSK9 conjugated liposomes for targeted delivery of paclitaxel to the cancer cell: A proof-of-concept study.

Ligand-based targeting of the receptors that are overexpressed explicitly on cancer cells represents an effective drug delivery approach to enhance the chemotherapeutic efficacy. Proprotein convertase subtilisin/kexin type 9 (PCSK9) which is a serine protease enzyme primarily produced by the liver cells, can potentially be used as a targeting ligand. PCSK9 binds to the LDL-r on hepatocytes' surface, leading to endocytosis and endosomal degradation. High LDL-r expression, which is believed to meet the higher demand of the cholesterol and phospholipids to build proliferating cancer cell membrane, ensures selective uptake of the PCSK9 conjugated liposomes. In the present work, the PCSK9 conjugated liposomal system was developed to deliver paclitaxel (PTX) to cancer cells. The protein was conjugated by EDC and NHS in a two-step coupling reaction to the liposomes containing COOH-PEG2000-COOH lipid. Conjugation was confirmed by NMR, and liposomes were further characterized by SEM and zeta sizer. PCSK9-conjugated liposomes showed high encapsulation efficiency of 69.1% with a diameter of 90.0 ± 4.9 nm. Long-term stability (30 days) study (Zeta potential: -9.88) confirmed excellent constancy and significant drug retention (58.2%). Invitro cytotoxicity and targeting efficiency was explored using MTS assay in human embryonic kidney cells (HEK293), liver hepatocellular cells (HEPG2), and a human colon cancer cell line (HCT116) for 24 h. PCSK9 conjugated liposomes exhibited significantly higher growth inhibition than the unconjugated (control) liposomes in HCT116 cell line (p < 0.001). The novel PCSK9 conjugated liposomes presented potent and precise in vitro anticancer activity and, therefore, are suggested for the first time as a promising targeted delivery system for cancer treatment.

The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor.

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.

Mouse Models of Human Proprotein Convertase Insufficiency.

The kexin-like proprotein convertases perform the initial proteolytic cleavages that ultimately generate a variety of different mature peptide and proteins, ranging from brain neuropeptides to endocrine peptide hormones, to structural proteins, among others. In this review, we present a general introduction to proprotein convertase structure and biochemistry, followed by a comprehensive discussion of each member of the kexin-like subfamily of proprotein convertases. We summarize current knowledge of human proprotein convertase insufficiency syndromes, including genome-wide analyses of convertase polymorphisms, and compare these to convertase null and mutant mouse models. These mouse models have illuminated our understanding of the roles specific convertases play in human disease and have led to the identification of convertase-specific substrates; for example, the identification of procorin as a specific PACE4 substrate in the heart. We also discuss the limitations of mouse null models in interpreting human disease, such as differential precursor cleavage due to species-specific sequence differences, and the challenges presented by functional redundancy among convertases in attempting to assign specific cleavages and/or physiological roles. However, in most cases, knockout mouse models have added substantively both to our knowledge of diseases caused by human proprotein convertase insufficiency and to our appreciation of their normal physiological roles, as clearly seen in the case of the furin, proprotein convertase 1/3, and proprotein convertase 5/6 mouse models. The creation of more sophisticated mouse models with tissue- or temporally-restricted expression of specific convertases will improve our understanding of human proprotein convertase insufficiency and potentially provide support for the emerging concept of therapeutic inhibition of convertases.

Effect of K225 residue to the catalytic efficiency of Kex2 protease.

The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Proteases were purified by dialysis and anion exchange chromatography (Q-FF). Their properties were further investigated. For catalysis efficiency, the value of Kcat/Km of Kex2-667-K225L was 3 folds higher than that of Kex2-667. Both were quite stable at 25 °C and 37 °C after 8 h of incubation at pH5.6, while Kex2-667 remained nearly 90% of the total activity while Kex2-667-K225L remained only 80%. The stability of Kex2-667-K225L was lower than that of Kex2-667 from pH4.0 to pH9.0. Due to the mutation site K225 was located at one of the calcium ion binding sites, it resulted in a tighter calcium ion binding region, which may be the reason why the catalytic efficiency of Kex2-667-K225L was improved while the stability was a little decreased.

Detection of anti-Pneumocystis jirovecii antibodies in human serum using a recombinant synthetic multi-epitope kexin-based antigen.

Interest in the detection of specific anti-Pneumocystis jirovecii antibodies has emerged as less-invasive alternative diagnostic approaches. Here is presented the performance of an ELISA based on a recombinant synthetic multi-epitope kexin 1 (Kex1) antigen of P. jirovecii, previously developed. Results showed that IgM anti-Kex1 levels were found significantly increased in patients with Pneumocystis pneumonia (PcP) compared with non-PcP cases (p < 0.001), allowing a diagnostic performance of PcP with a 70.8% sensitivity and a 75.0% specificity. These results suggest that this Kex1-based ELISA is a promising tool toward the serodiagnosis of PcP when the standard methods are difficult to perform.

Combinatorial allosteric modulation of agonist response in a self-interacting G-protein coupled receptor.

The structural plasticity of G-protein coupled receptors (GPCRs) enables the long-range transmission of conformational changes induced by specific orthosteric site ligands and other pleiotropic factors. Here, we demonstrate that the ligand binding cavity in the sphingosine 1-phosphate receptor S1PR1, a class A GPCR, is in allosteric communication with both the ß-arrestin-binding C-terminal tail, and a receptor surface involved in oligomerization. We show that S1PR1 oligomers are required for full response to different agonists and ligand-specific association with arrestins, dictating the downstream signalling kinetics. We reveal that the active form of the immunomodulatory drug fingolimod, FTY720-P, selectively harnesses both these intramolecular networks to efficiently recruit ß-arrestins in a stable interaction with the receptor, promoting deep S1PR1 internalization and simultaneously abrogating ERK1/2 phosphorylation. Our results define a molecular basis for the efficacy of fingolimod for people with multiple sclerosis, and attest that GPCR signalling can be further fine-tuned by the oligomeric state.

Characterisation of early metazoan secretion through associated signal peptidase complex subunits, prohormone convertases and carboxypeptidases of the marine sponge (Amphimedon queenslandica).

Efficient communication between cells requires the ability to process precursor proteins into their mature and biologically active forms, prior to secretion into the extracellular space. Eukaryotic cells achieve this via a suite of enzymes that involve a signal peptidase complex, prohormone convertases and carboxypeptidases. Using genome and transcriptome data of the demosponge Amphimedon queenslandica, a universal ancestor to metazoan multicellularity, we endeavour to bridge the evolution of precursor processing machinery from single-celled eukaryotic ancestors through to the complex multicellular organisms that compromise Metazoa. The precursor processing repertoire as defined in this study of A. queenslandica consists of 3 defined signal peptidase subunits, 6 prohormone convertases and 1 carboxypeptidase, with 2 putative duplicates identified for signal peptidase complex subunits. Analysis of their gene expression levels throughout the sponge development enabled us to predict levels of activity. Some A. queenslandica precursor processing components belong to established functional clades while others were identified as having novel, yet to be discovered roles. These findings have clarified the presence of precursor processing machinery in the poriferans, showing the necessary machinery for the removal of precursor sequences, a critical post-translational modification required by multicellular organisms, and further sets a foundation towards understanding the molecular mechanism for ancient protein processing.

The Proteolytic Regulation of Virus Cell Entry by Furin and Other Proprotein Convertases.

A wide variety of viruses exploit furin and other proprotein convertases (PCs) of the constitutive protein secretion pathway in order to regulate their cell entry mechanism and infectivity. Surface proteins of enveloped, as well as non-enveloped, viruses become processed by these proteases intracellularly during morphogenesis or extracellularly after egress and during entry in order to produce mature virions activated for infection. Although viruses also take advantage of other proteases, it is when some viruses become reactive with PCs that they may develop high pathogenicity. Besides reacting with furin, some viruses may also react with the PCs of the other specificity group constituted by PC4/PC5/PACE4/PC7. The targeting of PCs for inhibition may result in a useful strategy to treat infections with some highly pathogenic viruses. A wide variety of PC inhibitors have been developed and tested for their antiviral activity in cell-based assays.

Enhanced anti-tumor activity of the Multi-Leu peptide PACE4 inhibitor transformed into an albumin-bound tumor-targeting prodrug.

The proprotein convertase PACE4 has been validated as a potential target to develop new therapeutic interventions in prostate cancer (PCa). So far, the most effective compound blocking the activity of this enzyme has been designed based on the structure of a small peptide Ac-LLLLRVKR-NH2 known as the Multi-Leu (ML) peptide. Optimization of this scaffold led to the synthesis of compound C23 (Ac-[DLeu]LLLRVK-amidinobenzylamide) with a potent in vivo inhibitory effect on the tumor growth. However, further developments of PACE4 inhibitors may require additional improvements to counter their rapid renal clearance and to increase their tumor targeting efficiency. Herein, we explored the transformation of the ML-peptide into an albumin-binding prodrug containing a tumor specific release mechanism based on the prostate-specific antigen. Our data confirms that intravenous treatment using the ML-peptide alone has little effect on tumor growth, whereas by using the ML-prodrug in LNCaP xenograft-bearing mice it was significantly reduced. Additionally, excellent in vivo stability and tumor-targeting efficiency was demonstrated using a radiolabelled version of this compound. Taken together, these results provide a solid foundation for further development of targeted PACE4 inhibition in PCa.

Improving the Selectivity of PACE4 Inhibitors through Modifications of the P1 Residue.

Paired basic amino acid cleaving enzyme 4 (PACE4), a serine endoprotease of the proprotein convertases family, has been recognized as a promising target for prostate cancer. We previously reported a selective and potent peptide-based inhibitor for PACE4, named the multi-Leu peptide (Ac-LLLLRVKR-NH2 sequence), which was then modified into a more potent and stable compound named C23 with the following structure: Ac-dLeu-LLLRVK-Amba (Amba: 4-amidinobenzylamide). Despite improvements in both in vitro and in vivo profiles of C23, its selectivity for PACE4 over furin was significantly reduced. We examined other Arg-mimetics instead of Amba to regain the lost selectivity. Our results indicated that the replacement of Amba with 5-(aminomethyl)picolinimidamide increased affinity for PACE4 and restored selectivity. Our results also provide a better insight on how structural differences between S1 pockets of PACE4 and furin could be employed in the rational design of selective inhibitors.

Blackcurrant Extract Ameliorates Hyperglycemia in Type 2 Diabetic Mice in Association with Increased Basal Secretion of Glucagon-Like Peptide-1 and Activation of AMP-Activated Protein Kinase.

Blackcurrants are berries that contain high levels of anthocyanins, particularly delphinidin 3-rutinoside (D3R). Several studies have reported that the consumption of blackcurrant extract (BCE) lowers blood glucose levels and ameliorates glucose tolerance, but the mechanism underlying this effect remains unclear. Glucagon-like peptide-1 (GLP-1) and AMP-activated protein kinase (AMPK) are considered one of the most significant molecular targets for the prevention and treatment of type 2 diabetes. In this study, we showed that dietary BCE significantly reduced blood glucose concentration and improved glucose tolerance in type 2 diabetic mice (KK-Ay). The basal GLP-1 concentration in plasma was significantly increased in the BCE group accompanied by upregulation of prohormone convertase 1/3 (PC1/3), the enzyme that processes intestinal proglucagon. Moreover, the level of phospho-AMPKα protein in skeletal muscle was significantly increased in the BCE group, and this was increase accompanied by significant upregulation of glucose transporter 4 (Glut4) proteins in the plasma membrane of BCE group. In conclusion, dietary BCE significantly reduced blood glucose concentration and improved glucose tolerance in association with increased basal GLP-1 concentration in plasma, upregulation of PC1/3 expression, and translocation of Glut4 to the plasma membrane of skeletal muscle in type 2 diabetic mice; furthermore, these effects were accompanied by activation of AMPK. Our findings demonstrated that D3R-rich BCE may help prevent diabetes and allow the dosages of diabetes drugs to be reduced.

Oxyntomodulin: Actions and role in diabetes.

Oxyntomodulin is a product of the glucagon precursor, proglucagon, produced and released from the endocrine L-cells of the gut after enzymatic processing by the precursor prohormone convertase 1/3. It corresponds to the proglucagon sequence 33-69 and thus contains the entire glucagon sequence plus a C-terminal octapeptide, comprising in total 37 amino acids. As might have been expected, it has glucagon-like bioactivity, but also and more surprisingly also activates the receptor for GLP-1. This has given the molecule an interesting status as a glucagon-GLP-1 co-agonist, which is currently attracting considerable interest for its potential in the treatment of diabetes and obesity. Here, we provide an update on oxyntomodulin with a focus on its potential role in metabolic diseases.

Enzymatic sources and physio-pathological functions of soluble (pro)renin receptor.

PURPOSE OF REVIEW: (Pro)renin receptor (PRR) belongs to type I transmembrane receptor family and binds both prorenin and renin, representing a potential regulator of the activity of the renin-angiotensin system. Soluble form of PRR (sPRR) is generated by intracellular protease-mediated cleavage of full-length PRR. The purpose of this review is to highlight recent advances in understanding the mechanisms of action and production of sPRR. RECENT FINDINGS: It has recently been demonstrated that site-1-protease (S1P) plays a dominant role in the generation of sPRR. New evidence is also emerging to support a biological function of sPRR in the physiological regulation of fluid homeostasis as well as pathogenesis of chronic kidney disease. SUMMARY: sPRR is a 28 kDa product of PRR cleavage via S1P-mediated protease activity. Not only does sPRR regulate renal tubular water transport, but it also mediates pathogenic responses to renal cellular injury. sPRR is likely involved in a wide range of physio-pathological processes.

Functional analysis of corin protein domains required for PCSK6-mediated activation.

Atrial natriuretic peptide (ANP) is a cardiac hormone essential for normal blood pressure and cardiac function. Corin is a transmembrane serine protease that activates ANP. Recently, we identified proprotein convertase subtilisin/kexin-6 (PCSK6), also called PACE4, as the long-sought corin activator. Both corin and PCSK6 are expressed in cardiomyocytes, but corin activation occurs only on the cell surface. It remains unknown if cell membrane association is needed for PCSK6 to activate corin. Here we expressed corin deletion mutants in HEK293 cells to analyze the domain structures required for PCSK6-mediated activation. Our results show that soluble corin lacking the transmembrane domain was activated by PCSK6 in the conditioned medium but not intracellularly. Recombinant PCSK6 also activated the soluble corin under cell-free conditions. Moreover, PCSK6-mediated corin activation was not enhanced by cell membrane fractions. These results indicate that cell membrane association is unnecessary for PCSK6 to activate corin. Experiments with monensin that blocks PCSK6 secretion and immunostaining indicated that the soluble corin and PCSK6 were secreted via different intracellular pathways, which may explain the lack of corin activation inside the cell. We also found that the protein domains in the corin pro-peptide region were dispensable for PCSK6-mediated activation and that addition of heparan sulfate and chondroitin sulfate or treatment with heparinase or chondroitinase did not alter corin activation by PCSK6 in HEK293 cells. Together, our results provide important insights into the molecular and cellular mechanisms underlying PCSK6-mediated corin activation that is critical for cardiovascular homeostasis.

Proprotein convertase inhibition: Paralyzing the cell's master switches.

Proprotein convertases are serine proteases responsible for the cleavage and subsequent activation of protein substrates, many of them relevant for the development of an ample variety of diseases. Seven of the PCs, including furin and PACE4, recognize and hydrolyze the C-terminal end of the general sequence RXRR/KXR, whereas PCSK-9 recognizes a series of non-basic amino acids. In some systems, PC-mediated substrate activation results in the development of pathological processes, such as cancer, endocrinopathies, and cardiovascular and infectious diseases. After establishing PCs as relevant contributors to disease processes, research efforts were directed towards the development of inhibition strategies, including small and large molecules, anti-sense therapies, and antibody-based therapies. Most of these inhibitors mimic the consensus sequence of PCs, blocking the active site in a competitive manner. The most promising inhibitors were designed as bioengineered proteins; however, some non-protein and peptidomimetic agents have also proved to be effective. These efforts led to the design of pre-clinical studies and clinical trials utilizing inhibitors to PCs. Although the initial studies were performed using non-selective PCs inhibitors, such as CMK, the search for more specific, and compartmentalized selective inhibitors resulted in specific activities ascribed to some, but not all of the PCs. For instance, PACE4 inhibitors were effective in decreasing prostate cancer cell proliferation, and neovascularization. Decreased metastatic ovarian cancer utilizing furin inhibitors represents one of the major endeavors, currently in a phase II trial stage. Antibodies targeting PCSK-9 decreased significantly the levels of HDL-cholesterol, in a phase III trial. The study of Proprotein convertases has reached a stage of maturity. New strategies based on the alteration of their activity at the cellular and clinical level represent a promising experimental pharmacology field. The development of allosteric inhibitors, or specific agents directed against individual PCs is one of the challenges to be unraveled in the future.

Specificity characterization of the α-mating factor hormone by Kex2 protease.

Kex2 is a Ca2+-dependent serine protease from S. cerevisiae. Characterization of the substrate specificity of Kex2 is of particular interest because this protease serves as the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave sites consisting of pairs or clusters of basic residues. Our goal was to study the prime region subsite S' of Kex2 because previous studies have only taken into account non-prime sites using AMC substrates but not the specificity of prime sites identified through structural modeling or predicted cleavage sites. Therefore, we used peptides derived from Abz-KR↓EADQ-EDDnp and Abz-YKR↓EADQ-EDDnp based on the pro-α-mating factor sequence. The specificity of Kex2 due to basic residues at P1' is affected by the type of residue in the P3 position. Some residues in P1' with large or bulky side chains yielded poor substrate specificity. The kcat/KM values for peptides with P2' substitutions containing Tyr in P3 were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in kcat and KM, respectively. The pH profile of Kex2 was fit to a double-sigmoidal pH-titration curve. The specificity results suggest that Kex2 might be involved in the processing of the putative cleavage sites in a polypeptide involved in cell elongation, hyphal formation and the processing of a toxin, which result in host cell lysis. In summary, the specificity of Kex2 is dependent on the set of interactions with prime and non-prime subsites, resulting in synergism.

Improving the Secretory Expression of an -Galactosidase from Aspergillus niger in Pichia pastoris.

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1' residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1' site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.

Polydatin ameliorates lipid and glucose metabolism in type 2 diabetes mellitus by downregulating proprotein convertase subtilisin/kexin type 9 (PCSK9).

BACKGROUND: Abnormalities in lipid and glucose metabolism are constantly observed in type 2 diabetes. However, these abnormalities can be ameliorated by polydatin. Considering the important role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in metabolic diseases, we explore the possible mechanism of polydatin on lipid and glucose metabolism through its effects on PCSK9. METHODS: An insulin-resistant HepG2 cell model induced by palmitic acid (PA) and a db/db mice model were used to clarify the role of polydatin on lipid and glucose metabolism. RESULTS: In insulin-resistant HepG2 cells, polydatin upregulated the protein levels of LDLR and GCK but repressed PCSK9 protein expression, besides, polydatin also inhibited the combination between PCSK9 and LDLR. Knockdown and overexpression experiments indicated that polydatin regulated LDLR and GCK expressions through PCSK9. In the db/db mice model, we found that polydatin markedly enhanced GCK and LDLR protein levels, and inhibited PCSK9 expression in the liver. Molecular docking assay was further performed to analyze the possible binding mode between polydatin and the PCSK9 crystal structure (PDB code: 2p4e), which indicated that steady hydrogen bonds formed between polydatin and PCSK9. CONCLUSIONS: Our study indicates that polydatin ameliorates lipid and glucose metabolism in type 2 diabetes mellitus by downregulating PCSK9.

Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P).

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process.