The chemistry and enzymology of the type I signal peptidases

Dalbey, RE; Lively, MO; Bron, Sierd; Dijl, van Jan Maarten

doi:10.1002/pro.5560060601

Cited by 256 publications

(242 citation statements)

References 104 publications

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“…Since the only known ER peptidase in the classical secretory pathway involved in N-terminal processing is the SPC, the parasite SPC could possibly have an additional role in signal peptide cleavage and also be responsible for PEXEL cleavage. If the parasite SPC is indeed responsible for PEXEL cleavage, then the substrate specificity of this novel ER peptidase would have to be able to accommodate both PEXEL cleavage (RxL↓) along with classical signal peptide cleavage (AxA↓) [34,35]. Analysis of the P. falciparum genome reveals homologues of the SPC21 catalytic subunit (PlasmoDB ID: MAL13P1.167, GenBank accession no.…”

Section: Dissection Of the Pexel Motifmentioning

confidence: 99%

N-terminal processing of proteins exported by malaria parasites

Chang

Falick

Carlton

et al. 2008

Molecular and Biochemical Parasitology

171

177

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Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signalmediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this Nterminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence.

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Section: Dissection Of the Pexel Motifmentioning

confidence: 99%

N-terminal processing of proteins exported by malaria parasites

Chang

Falick

Carlton

et al. 2008

Molecular and Biochemical Parasitology

171

177

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“…The recent crystal structure (27) of the catalytic domain of SPase I (28 -29) has revealed that most of the amino acids that are strictly conserved in Boxes B, C, D, and E of the signal peptidase family (30) are found to be located near the active site (see Fig. 1A).…”

mentioning

confidence: 99%

The Role of the Conserved Box E Residues in the Active Site of the Escherichia coli Type I Signal Peptidase

Klenotic¹,

Carlos²,

Schuenemann³

et al. 2000

Journal of Biological Chemistry

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Type I signal peptidases are integral membrane proteins that function to remove signal peptides from secreted and membrane proteins. These enzymes carry out catalysis using a serine/lysine dyad instead of the prototypical serine/histidine/aspartic acid triad found in most serine proteases. Site-directed scanning mutagenesis was used to obtain a qualitative assessment of which residues in the fifth conserved region, Box E, of the Escherichia coli signal peptidase I are critical for maintaining a functional enzyme. First, we find that there is no requirement for activity for a salt bridge between the invariant Asp-273 and the Arg-146 residues. In addition, we show that the conserved Ser-278 is required for optimal activity as well as conserved salt bridge partners Asp-280 and Arg-282. Finally, Gly-272 is essential for signal peptidase I activity, consistent with it being located within van der Waals proximity to Ser-278 and general base Lys-145 side-chain atoms. We propose that replacement of the hydrogen side chain of Gly-272 with a methyl group results in steric crowding, perturbation of the active site conformation, and specifically, disruption of the Ser-90/Lys-145 hydrogen bond. A refined model is proposed for the catalytic dyad mechanism of signal peptidase I in which the general base Lys-145 is positioned by Ser-278, which in turn is held in place by Asp-280.The majority of proteins exported from a cell are made with a signal or leader peptide. These peptides are responsible for targeting proteins to the cytoplasmic membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes (1-3). The protein is then translocated across the membrane, and the N-terminal peptide is removed from the pre-protein by a type I signal peptidase.SPase I 1 has been isolated from Gram-negative (4 -6) and Gram-positive (7, 8) bacteria as well as from several eukaryotic organisms (9 -12). These endopeptidases are membrane-bound and specific for the region within the signal peptide immediately preceding the cleavage site. The substrate protein is cleaved during or after the protein is transported across the membrane bilayer. Cleavage occurs by way of nucleophilic attack by a catalytic serine O␥ on the peptide bond between the presequence and the mature region of the pre-protein.The best-studied enzyme of this family is SPase I, or leader peptidase, of Escherichia coli. It has been cloned (13), sequenced (14), and purified (15, 16). Its substrate specificity has been characterized; small-uncharged residues at the P1 (Ϫ1) and P3 (Ϫ3) positions of the substrate are required for cleavage (17)(18)(19). SPase I utilizes a serine/lysine dyad mechanism to perform its enzymatic function rather than the canonical catalytic triad found in most serine proteases (20 -23). To date, only a few other enzymes, such as LexA (24), UmuD (25), and the Tsp protease (26) have been identified with this unusual active site mechanism. Additional analysis is required to provide insight into other residues near this dyad in the SPase family and th...

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“…However, with the exception of certain ␤-lactam compounds (56), most signal peptidases are insensitive to the commonly used protease inhibitors (57). Translocation into canine microsomes and processing of in vitro translated bovine prepro-PTH to pro-PTH were not inhibited by MG132 (data not shown).…”

Section: Discussionmentioning

confidence: 95%

Biosynthesis and Secretion of Parathyroid Hormone Are Sensitive to Proteasome Inhibitors in Dispersed Bovine Parathyroid Cells

Sakwe

Engström

Larsson

et al. 2002

Journal of Biological Chemistry

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Preproparathyroid hormone (prepro-PTH) is one of the proteins abundantly synthesized by parathyroid chief cells; yet under normal growth conditions, little or no prepro-PTH can be detected in these cells. Although this may be attributed to effective cotranslational translocation and proteolytic processing, proteasome-mediated degradation of PTH precursors may be important in the regulation of the levels of these precursors and hence PTH secretion. The effects of N-acetyl-Leu-Leunorleucinal, N-acetyl-Leu-Leu-methional, carbobenzoxy-Leu-Leu-leucinal (MG132), benzyloxycarbonyl-IleGlu(t-butyl)-Ala-leucinal (proteasome inhibitor I), and lactacystin on the biosynthesis and secretion of PTH were examined in dispersed bovine parathyroid cells. We demonstrate that treatment of these cells with proteasome inhibitors caused the accumulation of prepro-PTH and pro-PTH. Compared with mock-treated cells, the processing of pro-PTH to PTH was delayed, and the secretion of intact PTH decreased in proteasome inhibitor-treated cells. Relieving the inhibition of the proteasome by chasing MG132-treated cells in medium without the inhibitor led to the rapid disappearance of the accumulated prepro-PTH, and the rate of PTH secretion was restored to levels comparable to those in mocktreated cells. Furthermore, overexpression of the Hsp70 family of molecular chaperones was observed in proteasome inhibitor-treated cells, and we show that PTH/PTH precursors interact with these molecular chaperones. These data suggest the involvement of parathyroid cell proteasomes in the quality control of PTH biosynthesis.The calcium concentration in mammalian body fluids is tightly regulated predominantly by the actions of parathyroid hormone (PTH) 1 on bone, kidney, and intestine (1-4 ] o ) secretion of PTH. However, the signaling pathway(s) leading to the rapid and specific changes in PTH secretion remains largely unknown.The primary precursor prepro-PTH is translocated into the endoplasmic reticulum (ER) and cleaved to pro-PTH within 1 min. Pro-PTH then transits the ER and attains the trans-Golgi network within 20 min, where it is cleaved to mature PTH. Depending on the needs and predominantly on [Ca 2ϩ ] o , mature PTH is packaged into either secretory granules for exocytosis or storage granules. Secretion of de novo synthesized PTH is believed to occur within 30 min of prepro-PTH formation (6) and constitutes the bulk of secreted PTH (7-9). At the posttranscriptional level, acute changes in [Ca 2ϩ ] o (Ͻ24 h) do not significantly affect the rate of PTH biosynthesis. However, sustained or chronic hypocalcemia and hypercalcemia affect the stability and hence the levels of PTH mRNA (10 -14, 44).Intracellular proteolysis of mature bioactive PTH has been reported to be one of the mechanisms by which parathyroid cells regulate the amount of hormone available for secretion in response to changes in [Ca 2ϩ ] o (15,16). This PTH metabolism is now known to be mediated by calpains (17) and cathepsins B and D (18 -21). Inhibition of these PTH-degrading ac...

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The chemistry and enzymology of the type I signal peptidases

Cited by 256 publications

References 104 publications

N-terminal processing of proteins exported by malaria parasites

N-terminal processing of proteins exported by malaria parasites

The Role of the Conserved Box E Residues in the Active Site of the Escherichia coli Type I Signal Peptidase

Biosynthesis and Secretion of Parathyroid Hormone Are Sensitive to Proteasome Inhibitors in Dispersed Bovine Parathyroid Cells

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