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PubMed2005

Both PA63 and PA83 are endocytosed within an anthrax protective antigen mixed heptamer: a putative mechanism to overcome a furin deficiency.

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.

Animals↗

PubMed2004

Role of beta-cell prohormone convertase (PC)1/3 in processing of pro-islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) (amylin), the major component of islet amyloid, is produced by cleavage at the COOH- and NH(2)-termini of its precursor, proIAPP, likely by the beta-cell prohormone convertases (PC) 1/3 and PC2. Mice lacking PC2 can process proIAPP at its COOH- but not its NH(2)-terminal cleavage site, suggesting that PC1/3 is capable of initiating proIAPP cleavage at its COOH-terminus. To determine the precise role of PC1/3 in proIAPP processing, Western blot analysis was performed on islets isolated from mice lacking PC1/3 (PC1/3(-/-)). These islets contained not only fully processed IAPP as in PC1/3(+/+) islets, but also elevated levels of a COOH-terminally unprocessed intermediate form, suggesting impaired processing at the COOH-terminus. Next, GH3 cells that do not normally express proIAPP or detectable levels of PC1/3 or PC2 were cotransduced with adenoviruses expressing rat proIAPP and either PC2 or PC1/3. As expected, in GH3 cells transduced to express only proIAPP, no processing was observed. Coexpression of proIAPP and PC2 resulted in production of mature IAPP, whereas in cells that coexpressed proIAPP and PC1/3 only a 6-kDa intermediate was produced. We conclude that PC1/3 is important for processing of proIAPP at the COOH-terminus, but in its absence, PC2 can initiate complete processing of proIAPP to IAPP by cleaving the precursor at either its NH(2)- or COOH-terminal cleavage sites.

Amyloid↗

PubMed1999

Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins.

Site-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum lumen, thereby initiating a process by which the transcriptionally active NH(2)-terminal fragments of SREBPs are released from membranes. In the current experiments, we transfected cDNAs encoding epitope-tagged hamster S1P into HEK-293 cells or mutant hamster cells that lack S1P. Protease protection assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH(2)-terminal signal sequence of S1P generates S1P-A (amino acids 23-1052), which is inactive. The protein is self-activated by an intramolecular cleavage at Site-B, generating S1P-B (amino acids 138-1052) and liberating a 115-amino acid propeptide that is secreted intact into the medium. The sequence at Site-B is RSLK, which differs from the RSVL sequence at the cleavage site in SREBP-2. S1P-B is further cleaved at an internal RRLL sequence to yield S1P-C (amino acids 187-1052). Mutational analysis suggests that S1P-B and S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it appears to be transported to the Golgi, a site at which SREBP-2 cleavage may not normally occur. These data provide the initial description of the processing of a subtilisin-related protease that controls the level of cholesterol in blood and cells. In an accompanying paper (Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999), J. Biol. Chem., 274, 22805-22812), we develop an in vitro assay to characterize the activity of purified recombinant S1P.

Amino Acid Sequence↗

PubMed1993

Rapid increases in peptide processing enzyme expression in hippocampal neurons.

Recent studies have demonstrated that seizure activity causes a dramatic increase in neuropeptide expression in specific regions of the rat hippocampus. In this study we investigated the effect of electroconvulsive treatment (ECT) on the expression of three posttranslational processing enzymes involved in the production of many bioactive peptides from their inactive precursors. Peptidylglycine alpha-amidating monooxygenase (PAM) converts peptidylglycine substrates into alpha-amidated products and prohormone convertases 1 and 2 perform the tissue-specific endoproteolytic cleavage of many prohormones. After a single ECT, in situ hybridization demonstrated a rapid increase in the level of PAM mRNA in the dentate granule cells of the hippocampus, reaching peak levels between 1 and 4 h and then returning to near baseline levels within 24 h. Northern blot analysis confirmed the changes in PAM mRNA expression seen by using in situ hybridization. Similar rapid changes in PAM mRNA expression were seen after repeated ECT, suggesting that chronic ECT did not affect the regulation of PAM expression in the hippocampus. Immunohistochemical staining demonstrated an increase in PAM protein in the molecular layer of the dentate gyrus at 4 and 8 h after a single ECT. Based on in situ hybridization, levels of mRNA for the prohormone convertases 1 and 2 were also increased in dentate granule cells after a single ECT. Prohormone convertase 2 mRNA levels exhibited a slower response to ECT, not reaching maximal levels until 8 h after ECT. The response of the dentate granule cells of the hippocampus to ECT provides a model system for studying the rapid, coordinate regulation of peptide-processing enzymes.

Animals↗

PubMed1998

The proprotein convertases.

The major endoproteolytic processing enzymes of the secretory pathway are the subtilisin-like proprotein convertases (SPCs). Furin (SPC1) has emerged as one of the major processing enzymes of the constitutive secretory pathway and its localization in the trans-Golgi network and mechanism of autoactivation have been studied in considerable detail. Recent gene disruption experiments and the study of naturally-occurring mutations underscore the importance of PC2 (prohormones convertase 2, or SPC2) and PC1/PC3 (prohormone convertase 1/3, or SPC3) in the processing of a wide variety of hormone and neuropeptide precursors. The role of Carboxypeptidase E (CPE) in the removal of carboxy-terminal basic residues exposed by the endoproteases was shown to be necessary for efficient endoproteolytic processing of proinsulin and several other protein precursors. Many biologically active peptides are also amidated after their proteolytic processing by peptidylglycine alpha-amidating monooxygenase (PAM) and recent X-ray studies of the peptidyl alpha-hydroxylating monooxygenase component of PAM have shed new light on the role of copper in the mechanism of this reaction.

Furin↗

PubMed2000

The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments.

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl(2)-responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr(1802)GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.

Amino Acid Motifs↗

PubMed1999

Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p).

The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation. Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg. No cleavage was seen when both Lys and Arg were absent. No significant enhancement was seen with multiple basic residues. However, cleavage always occurred carboxyl to the most COOH-terminal basic residue. V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely. These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)'. The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0. However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast.

Amino Acid Sequence↗

PubMed1997

Eukaryotic protein processing: endoproteolysis of precursor proteins.

Limited endoproteolysis of biologically inactive polypeptide precursors is a general mechanism generating a diversity of biologically active peptides and proteins in all eukaryotic phyla. One of the major recognition motifs involves cleavage at either specific single or pairs of basic residues of the general formula (R/K) - Xn - (R/K) decreases, where n = 0, 2, 4 or 6. Such sites are found in a variety of protein precursors in all eukaryotes, including those of endocrine and neural polypeptide hormones, enzymes, growth factors, receptors, adhesion molecules, viral glycoproteins, coagulation factors and even cell signaling molecules. A family of seven mammalian proteinases responsible for the processing of these proproteins has been recently identified. It comprises the proprotein convertases PC1/PC3, PC2, furin/PACE, PC4, PACE4, PC5/PC6 and PC7/SPC7/LPC/PC8. In a combinatorial fashion, these enzymes determine the cell-type and time at which biologically active products are derived from a given inactive precursor protein, thereby profoundly affecting cellular communication, differentiation and metabolic activity.

Amino Acid Sequence↗

PubMed1994

A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor.

The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.

3T3 Cells↗

PubMed1999

Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1-42 maturase.

The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2's subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.

Amino Acid Sequence↗

PubMed2002

Molecular characterization of a metastatic neuroendocrine cell cancer arising in the prostates of transgenic mice.

The features and functions of prostatic neuroendocrine (NE) cells remain ill-defined. Neuroendocrine differentiation (NED) in adenocarcinoma of the human prostate (CaP) is associated with more aggressive disease, but the underlying mediators are poorly understood. We examined these issues in transgenic mice that utilize regulatory elements from the cryptdin-2 gene (Defcr2) to express simian virus 40 large T antigen (TAg) in prostatic NE cells. CR2-TAg mice develop prostatic intraepithelial neoplasia at 8 weeks of age, 1 week after the onset of TAg expression. An invasive phase follows 2-4 weeks later, with lymph node, liver, lung, brain, and bone metastases appearing within 16 weeks. DNA microarray studies revealed 122 mRNAs that were increased >/=2-fold in duplicate assays of 16-week-old CR2-TAg versus normal prostates. Thirty two transcripts encode proteins associated with neurons and endocrine cells (e.g. basic helix loop helix, SRY-related high mobility group box and sine-oculis homeobox transcription factors, Hu RNA-binding proteins, neuronatin, Racgap1, collapsin response mediator protein-1, synaptotagmin-1, proprotein convertase, and secretogranins). Follow-up studies of candidate mediators and biomarkers of differentiation/growth in the microarray data set involved real time quantitative reverse transcriptase-PCR assays of laser capture microdissected NE cells from CR2-TAg prostates plus liver metastases, and immunohistochemical comparisons of transgenic mouse prostates and 35 human CaP samples. Our findings include (a) expression of the bHLH mouse achaete-scute homolog (mASH1) in normal and CR2-TAg NE cells and foci of NED in human CaP, (b) glutamic acid decarboxylase and its product (gamma-aminobutyric acid) in neoplastic NE cells juxtaposed next to cohorts of normal gamma-aminobutyric acid receptor expressing secretory cells (a potential route for paracrine interactions between these two epithelial lineages), and (c) aromatic l-amino-acid decarboxylase, but not its dopamine/serotonin products, in CR2-TAg NE cells and NED. These results underscore the value of CR2-TAg mice for characterizing normal NE cell biology and tumorigenesis.

Animals↗

PubMed1991

Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2.

Using a 796-basepair cDNA fragment obtained from a mouse pituitary library we have screened two mouse insulinoma libraries and isolated a full-length cDNA clone (2516 basepairs; 753 amino acids), designated mPC1. The cDNA sequence of mPC1 codes for a protein containing 753 amino acids and three potential N-glycosylation sites. This cDNA encodes a putative novel subtilisin-like proteinase, exhibiting within its presumed catalytic domain 64%, 55%, and 47% amino acid sequence identity to the recently characterized candidate prohormone convertases human Furin, mouse PC2, and yeast Kex2 gene products, respectively. An identical sequence to mPC1 was derived from a cDNA library of mouse corticotroph AtT-20 tumor cells. An ArgGlyAsp tripeptide identical to the recognition sequence of integrins was observed in the structures of the mammalian PC1, PC2, and Furin. In situ hybridization results demonstrated a distinct localization of the mPC1 and mPC2 transcripts in pituitary and brain. Thus, whereas both mPC1 and mPC2 are found in the intermediate lobe of the pituitary, only mPC1 is easily detected in the anterior lobe. In extrahypothalamic regions of the brain, including cortex, hippocampus, thalamus, and spinal cord, mPC2 transcripts predominate over mPC1. Both mRNAs are found in only a fraction of hypothalamic neurons, with greater abundance of mPC1 over mPC2 in the supraoptic nucleus. The genes coding for mPC1 and mPC2 map to the murine chromosomes 13 (band 13c) and 2 (2F3-2H2 region), respectively.

Amino Acid Sequence↗

PubMed2001

Direct measurement of acylenzyme hydrolysis demonstrates rate-limiting deacylation in cleavage of physiological sequences by the processing protease Kex2.

Saccharomyces cerevisiae Kex2 protease is the prototype for the family of eukaryotic proprotein convertases that includes furin, PC1/3, and PC2. These enzymes belong to the subtilase superfamily of serine proteases and are distinguished from degradative subtilisins by structural features and by their much more stringent substrate specificity. Pre-steady-state studies have shown that both Kex2 and furin exhibit an initial burst of 7-amino-4-methylcoumarin release in cleavage of peptidyl methylcoumarinamide substrates that are based on physiological cleavage sites. Thus, in cleavage of such substrates, formation of the acylenzyme intermediate is fast relative to some later step (deacylation or N-terminal product release). This behavior is significant, because Kex2 also exhibits burst kinetics in cleavage of peptide bonds. k(cat) for cleavage of a tetrapeptidyl methylcoumarinamide substrate based on the physiological yeast substrate pro-alpha-factor exhibits a weak solvent isotope effect, but neither this isotope effect nor temperature dependence studies with this substrate conclusively identify the rate-limiting step for Kex2 cleavage of this substrate. We therefore developed an assay to measure deacylation directly by pulse-chase incorporation of H(2)(18)O in a rapid-quenched-flow mixer followed by mass spectrometric quantitation. The results given by this assay rule out rate-limiting product release for cleavage of this substrate by Kex2. These experiments demonstrate that cleavage of the acylenzyme ester bond, as opposed to either the initial attack on the amide bond or product release, is rate-limiting for the action of Kex2 at physiological sequences. This work demonstrates a fundamental difference in the catalytic strategy of proprotein processing enzymes and degradative subtilisins.

Acylation↗

PubMed2023

An N terminomics toolbox combining 2-pyridinecarboxaldehyde probes and click chemistry for profiling protease specificity.

Proteomic profiling of protease-generated N termini provides key insights into protease function and specificity. However, current technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an N terminomics toolbox that combines selective N-terminal biotinylation using 2-pyridinecarboxaldehyde (2PCA) reagents with chemically cleavable linkers to enable efficient enrichment of protein N termini. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (chemical enrichment of protease substrates with purchasable, elutable reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.

Peptide Hydrolases↗

PubMed2000

Diminished prohormone convertase 3 expression (PC1/PC3) inhibits progastrin post-translational processing.

Gastrin is initially synthesized as a large precursor that requires endoproteolytic cleavage by a prohormone convertase (PC) for bioactivation. Gastric antral G-cells process progastrin at Arg(94)Arg(95) and Lys(74)Lys(75) residues generating gastrin heptadecapeptide (G17-NH(2)). Conversely, duodenal G-cells process progastrin to gastrin tetratriacontapeptide (G34-NH(2)) with little processing at Lys(74)Lys(75). Both tissues express PC1/PC3 and PC2. Previously, we demonstrated that heterologous expression of progastrin in an endocrine cell line that expresses PC1/PC3 and little PC2 (AtT-20) resulted in the formation of G34-NH(2). To confirm that PC1/PC3 was responsible for progastrin processing in AtT-20 cells and capable of processing progastrin in vivo we coexpressed either human wild-type (Lys(74)Lys(75)) or mutant (Arg(74)Arg(75), Lys(74)Arg(75), and Arg(74)Lys(75)) progastrins in AtT-20 cells with two different antisense PC1/PC3 constructs. Coexpression of either antisense construct resulted in a consistent decrease in G34-NH(2) formation. Gastrin mRNA expression and progastrin synthesis were equivalent in each cell line. Although mutation of the Lys(74)Lys(75) site within G34-NH(2) to Lys(74)Arg(75) resulted in the production of primarily G17-NH(2) rather than G34-NH(2), inhibition of PC1/PC3 did not significantly inhibit processing at the Lys(74)Arg(75) site. We conclude that PC1/PC3 is a progastrin processing enzyme, suggesting a role for PC1/PC3 progastrin processing in G-cells.

Aspartic Acid Endopeptidases↗

PubMed2004

Structural basis for differences in substrate selectivity in Kex2 and furin protein convertases.

Kex2 is the yeast prototype of a large family of serine proteases that are highly specific for cleavage of their peptide substrates C-terminal to paired basic sites. This paper reports the 2.2 A resolution crystal structure of ssKex2 in complex with an Ac-Arg-Glu-Lys-Arg peptidyl boronic acid inhibitor (R = 19.7, R(free) = 23.4). By comparison of this structure with the structure of the mammalian homologue furin [Henrich, S., et al. (2003) Nat. Struct. Biol. 10, 520-526], we suggest a structural basis for the differences in substrate recognition at the P(2) and P(4) positions between Kex2 and furin and provide a structural rationale for the lack of P(6) recognition in Kex2. In addition, several monovalent cation binding sites are identified, and a mechanism of activation of Kex2 by potassium ion is proposed.

Binding Sites↗

PubMed2000

Effect of amino acid substitution at the P(3) and P(4) subsites of fusion proteins on kex2 protease activity.

The effect of amino acid substitution at the P(3) and P(4) subsites on the cleavage activity of a recombinant secretory-type Kex2 protease (Kex2-660) was investigated using recombinant fusion proteins. They were constructed from a truncated Escherichia coli beta-galactosidase (beta G-117S) followed by human parathyroid hormone amino acids 1-34 [hPTH(1-34)] with a junction designed to allow cleavage with Kex2-660. Kex2-660 preferentially cleaved the fusion protein with Arg-His at the P(3)-P(4) subsite, whereas it poorly cleaved the fusion proteins with Val-Asp, Gly-His, Cys-His and Leu-His compared with the original sequence, Val-Lys. As for Asp and Glu at P(4), they precluded the cleavage almost completely. Some of the substitutions were investigated kinetically using the fusion proteins and peptide-substrate mimics corresponding to the 15 amino acids contained in the junction. The substitution Val-Lys to Arg-His resulted in a 2-fold increase in k(cat) and the introduction of Asp at P(4) of the peptide substrate resulted in a large increase in K(m) and a change in the optimum pH. The ordered cleavage of the fusion protein was attained by introducing the two dibasic sites, Arg-His and Asp-His, at P(4)-P(3). The fusion protein was cleaved with Kex2 only at the former site in 3.0 M urea at pH 8.0 and the liberated peptides containing the latter site could be cleaved further with Kex2 at pH 6.5 once purified in urea-free solution. Thus Kex2 exhibited extended substrate specificity beyond P(2)-P(1) in the cleavage of the fusion proteins, although it depended on the reaction conditions.

Amino Acid Sequence↗

PubMed2006

The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors.

Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations < or = 100 microM these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

Amino Acid Sequence↗