Biosynthesis and Cellular Trafficking of the Convertase SKI-1/S1P

Elagöz, Aram; Benjannet, Suzanne; Mammarbassi, Aida; Wickham, L. Alexandra; Seidah, Nabil G.

doi:10.1074/jbc.m109011200

Cited by 108 publications

(121 citation statements)

References 46 publications

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“…4 for gene model, and supporting information for details). The growth factor cytokine receptor (GFCR) motif of S1P is essential for autoprocessing of the proprotein and formation of active protease in vitro (33). The GFCR motif, which is well conserved in zebrafish (see supporting information), is absent or disrupted in both goz mutant alleles goz tr6722 and goz tr6721 , resulting in an S1P protein that is predicted to be inactive (Fig.…”

Section: Resultsmentioning

confidence: 99%

Site-1 protease is required for cartilage development in zebrafish

Schlombs¹,

Wagner²,

Scheel³

2003

Proc. Natl. Acad. Sci. U.S.A.

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gonzo (goz) is a zebrafish mutant with defects in cartilage formation. The goz phenotype comprises cartilage matrix defects and irregular chondrocyte morphology. Expression of endoderm, mesoderm, and cartilage marker genes is, however, normal, indicating a defect in chondrocyte morphogenesis. The mutated gene responsible for the goz phenotype, identified by positional cloning and confirmed by phosphomorpholino knockdown, encodes zebrafish site-1 protease (s1p). S1P has been shown to process and activate sterol regulatory element-binding proteins (SREBPs), which regulate expression of key enzymes of lipid biosynthesis or transport. This finding is consistent with the abnormal distribution of lipids in goz embryos. Knockdown of site-2 protease, which is also involved in activation of SREBPs, results in similar lipid and cartilage phenotypes as S1P knockdown. However, knockdown of SREBP cleavage-activating protein, which forms a complex with SREBP and is essential for S1P cleavage, results only in lipid phenotypes, whereas cartilage appears normal. This indicates that the cartilage phenoptypes of goz are caused independently of the lipid defects.T he vertebrate skeleton is comprised of cartilage and bone and is derived from multiple embryologic origins, including neural crest, dorsal paraxial mesoderm, cephalic mesoderm, and lateral mesoderm (for review, see ref. 1). Skeletal development in the vertebrate head is regulated by sonic hedgehog (Shh) (2-5), which is expressed in the rostral axial mesendoderm and the prechordal plate (6). The coexpression of shh and bone morphogenetic proteins (bmps) observed in many developing vertebrate organs suggests a closely regulated relationship between Shh and BMPs (7). It has been shown that BMP-4 induces sox9 and collagen II (Col II) expression and leads to chondrogenesis in mandibular organ culture systems of chick and mouse (8).Mature cartilage consists of chondrocytes and the surrounding extracellular matrix. Matrix collagens and proteoglycans are synthesized by chondrocytes. They are hydroxylated, glycosylated, and proteolytically cleaved in the secretory pathway and further processed and assembled into cartilage matrix at the cell surface (9). Large complexes of proteoglycans are restricted in their mobility and swelling by a network of Col II and XI fibrils of high tensile strength. Interactions between the extracellular matrix and chondrocytes have been shown to be essential for cartilage development (10), and mutations in collagen genes can cause human chondrodysplasias (11,12).In addition to protein factors, lipids play an essential role in cartilage development. Deficiencies in cholesterol metabolism during embryogenesis can lead to severe skeletal abnormalities like Smith-Lemli-Opitz syndrome (for review, see ref. 13 and references therein). Similar chondrodysplasias have been observed in other vertebrates, including zebrafish (14, 15), in which the molecular mechanisms of skeletal development are conserved, including the roles of shh (16), patched (ptc) (17), bmp-...

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Section: Resultsmentioning

confidence: 99%

Site-1 protease is required for cartilage development in zebrafish

Schlombs¹,

Wagner²,

Scheel³

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…2-4). It may well be that the RRLL sequence that is best recognized by SKI-1 in a number of substrates (7,44,48) may actually be cleaved when present in the RSL of ␣ 1 -AT. Thus, whereas only an RSL with a P1 Leu in combination with either P2 Val, Ile, and Tyr resulted in an inhibitory serpin, we cannot eliminate the possibility that a P1 Val or Ile with a P2 Val, Ile, or Tyr may not also be inhibitory.…”

Section: Discussionmentioning

confidence: 99%

“…The absence of a P4 Arg should block the B-site (7,37) but could still allow the BЈ to interact with the SKI-1 catalytic pocket. It should be noted that the R134E mutant of the full-length SKI-1 is ϳ50% less processed into the BЈ/B and C forms than the wild-type sequence (7). The importance of a P1Ј acidic residue for SKI-1 activity was not studied before.…”

Section: Discussionmentioning

confidence: 99%

“…The signal peptidase cleavage generates an A form (amino acids ) that is subsequently autocatalytically cleaved in the endoplasmic reticulum (ER) at two alternate BЈ and B sites: RKVF 133 2 (SKI-1-(134 -1052)) and RKVFRSLK 137 2 (SKI-1-(138 -1052)), respectively. The latter products are then transported to the cis/medial Golgi whereupon they are further autocatalytically processed into a C-form at RRLL 186 2, generating SKI-1-(187-1052) (5,7,35,37).…”

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confidence: 99%

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Development of Protein-based Inhibitors of the Proprotein of Convertase SKI-1/S1P

Pullikotil

Vincent

Nichol

et al. 2004

Journal of Biological Chemistry

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Processing of membrane-bound transcription factors such as sterol regulatory element-binding proteins (SREBPs) and the ER-stress response factor ATF6, and glycoproteins of some hemorrhagic fever viruses are initiated by the proprotein convertase SKI-1/S1P. So far, no cellular protein-based inhibitor of the hydrophobicamino acid specific SKI-1 is known. The prosegment of the basic-amino acid specific convertases (e.g. furin and PC5) or ␣ 1 -PDX, a variant of ␣ 1 -antitrypsin (␣ 1 -AT) exhibiting an RIPR 358 sequence at the reactive site loop, were shown to potently inhibit these secretory proteinases. Accordingly, we tested the SKI-1-inhibitory potential of various point mutants of either the 198 amino acid preprosegment of SKI-1-(1-198) or ␣ 1 -AT. Transient transfections data showed that, out of numerous mutants studied, the R134E prosegment mutant or the ␣ 1 -AT reactive site loop variants RRVL 358 , RRYL 358 and RRIL 358 are the best specific cellular inhibitors of SKI-1. The observed inhibition of the processing of endogenous SREBP-2, exogenous ATF6 and a PDGF-A (RRLL 86 ) variant were >55% and reach ϳ80% in stable transfectants. We also show that SKI-1 forms SDS-stable complexes with these ␣ 1 -AT variants, but not with wild-type ␣ 1 -AT or ␣ 1 -PDX. Finally, these inhibitors were also shown to affect the processing and stability of the Crimean-Congo hemorrhagic fever virus glycoprotein.Proteins and peptides that are biologically active are often generated by intracellular limited proteolysis of inactive precursors. The mammalian proprotein convertases (PCs) 1 of the secretory pathway are calcium-dependent serine proteinases related to bacterial subtilisin that cleave various precursors at the general consensus motif (K/R)(X) n (K/R)2, where n ϭ 0, 2, 4, or 6 and X is any amino acid (1-3). The PC family counts seven basic amino acid-specific kexin-like convertases: furin, PC1/3, PC2, PC4, PACE4, PC5/6, and PC7/LPC (4). The eighth member is the recently discovered pyrolysin-like SKI-1/S1P that cleaves at the consensus motif (R/K)X(hydrophobic)Z2, where Z is variable (5), while the last member (6, 7) NARC-1 cleaves the sequence VFAQ 153 2 in its prosegment (8, 9). 2 More PCs contain an N-terminal signal sequence, followed by a prosegment, a catalytic domain and a P-domain. In addition, PCs possess a C-terminal segment that varies between the different members. The critical role of PCs in the proteolytic maturation of multiple proproteins, their implication in various pathologies (1, 10, 11), and their unidentified specific and/or redundant functions, make them attractive targets for the development of potent and selective inhibitors. The various successful approaches include: active site-directed chloromethyl ketone inhibitors (12, 13), reversible peptide-based inhibitors (14 -17), plant derivatives (18), and several engineered variants of protein-based inhibitors that possess a furin-like motif. These include ␣2-macroglobulin (␣2-MF) (19), ␣ 1 -antitrypsin (␣ 1 -AT) Portland (␣ 1 -PDX) (20 -22), proteinase i...

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“…This protease, called SKI-1, was found to process substrates along the secretory pathway, preferentially in the ER compartment, unlike the other PPCs that cleave protein precursors after their transit to the trans-Golgi network. The proposed consensus sequence for SKI-1 is (R/K)X-(hydrophobic)-Z2, where Z is any amino acid, preferentially Leu, Thr, Lys, or Phe but excluding Val, Pro, Glu, Asp, or Cys (25)(26)(27). Because the sequence KRKVNLK 108 has an aliphatic residue in the P2 position, but not Arg or Lys in position P4, the cleavage in the human thyroid cell cannot be attributable to SKI-1 but must be due to a unknown endoprotease.…”

Section: Discussionmentioning

confidence: 99%

Endoproteolytic Cleavage of Human Thyroperoxidase

Fourn¹,

Ferrand

Franc

2005

Journal of Biological Chemistry

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Human thyroperoxidase (hTPO), the key enzyme involved in thyroid hormone synthesis, is synthesized in the form of a 933-amino acid polypeptide that subsequently undergoes posttranslational modifications such as N-and O-glycosylation and heme fixation. In the present study, it was established that the N-terminal part of hTPO is cleaved during the maturation of the enzyme. In the first set of experiments performed in this study, Chines hamster ovary (CHO) cells transfected with hTPO cDNA generated four different species after deglycosylation, namely a 98-kDa species, which corresponds to the fulllength deglycosylated hTPO, and two 94-kDa and one 92-kDa species, which were truncated in the N-terminal parts. The three latter forms were detected only at the cell surface. A proprotein convertase inhibitor prevented these cleavages, and experiments using monensin and brefeldin A showed that they occurred in a post-endoplasmic reticulum compartment. Site-directed mutagenesis studies were performed in which Arg 65 was identified as one of the cleavage sites. In the second part of the study, hTPO from human thyroid glands was purified using a monoclonal antibody recognizing the folded form of hTPO. Amino acid determination showed that the Nterminal part of this protein begins at Thr 109 . This cleavage process differs from that observed in CHO cells. The fact that this hTPO was endoglucosaminidase H-sensitive indicated that the cleavage of the propeptide occurs in the endoplasmic reticulum. To analyze the role of the hTPO prosequence, cDNAs with and without prosequence (Cys 15 -Lys 108 ) were transfected into CHO cells. hTPO propeptide deletion drastically decreased the proportion of the folded hTPO form, and under these conditions the cell surface activity disappeared completely. These results strongly suggest that the prosequence plays a crucial role as an intramolecular chaperone, facilitating the folding of hTPO.Thyroperoxidase (TPO) 1 is the main enzyme involved in thyroid hormone synthesis; it catalyzes both the iodination of thyroglobulin and the coupling of some of the iodotyrosyl residues required for the formation of thyroid hormones. The fulllength 3048-bp transcript of human TPO (hTPO) codes for a protein consisting of 933 amino acids. Several other transcripts in which various exons are spliced out have been described (1-3). Human TPO is a type I membrane-bound glycoprotein containing a heme prosthetic group (for a review, see Refs. 4 and 5). The large extracytoplasmic domain, which is oriented toward the follicular lumen, contains the catalytic site and four potential N-glycosylation sites. Although TPO catalyzes the process of hormonosynthesis at the apical cell surface of the thyrocyte, only a small fraction of this enzyme is to be found at this location, and most of it is located in the endoplasmic reticulum (ER) (6). After transfecting the full hTPO cDNA into CHO cells, a similar pattern of distribution between ER and the cell surface was obtained (7), and only 15-20% of the hTPO molecules were able to a...

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Biosynthesis and Cellular Trafficking of the Convertase SKI-1/S1P

Cited by 108 publications

References 46 publications

Site-1 protease is required for cartilage development in zebrafish

Site-1 protease is required for cartilage development in zebrafish

Development of Protein-based Inhibitors of the Proprotein of Convertase SKI-1/S1P

Endoproteolytic Cleavage of Human Thyroperoxidase

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