Role of Yeast Insulin-Degrading Enzyme Homologs in Propheromone Processing and Bud Site Selection

Adames, Neil R.; Blundell, Kelly; Ashby, Matthew N.; Boone, Charles

doi:10.1126/science.270.5235.464

Cited by 128 publications

(168 citation statements)

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“…FLN is a member of the M16 family of metalloproteases, enzymes character-ized by an inverted active site motif, HXXEH, where the histidines coordinate a catalytic zinc ion [8]. Other M16 family members are also oligoendopeptidases whose substrates include physiologically important molecules such as insulin [9], ␣ factor [10,11], somatostatin [12], and transforming growth factor ␣ [13]. Chelators such as EDTA and 1,10-phenanthroline readily inhibit FLN [6].…”

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confidence: 99%

Plasmodium falciparum falcilysin: an unprocessed food vacuole enzyme

Murata

Goldberg

2003

Molecular and Biochemical Parasitology

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Five hundred million infections and nearly two million deaths each year are attributed to the protozoan Plasmodium falciparum, a causative agent of human malaria. With the increasing prevalence of drug resistant strains, there is an urgent need to identify new drug targets. Examination of the parasite's unique metabolic pathways, such as hemoglobin degradation, provides candidates for chemotherapeutic development. Intraerythrocytic development of the parasite is dependent upon degradation of red blood cell hemoglobin. A semi-ordered pathway of proteases mediates this catabolism, which occurs in the acidic organelle called the food vacuole. The aspartic proteases, plasmepsins I and II, are proposed to be responsible for initial cleavage of hemoglobin in a conserved hinge region of the alpha chain [1,2]. Plasmepsins, and a family of cysteine proteases, falcipain-2 and 3, then carry out further degradation of the denatured globin [2][3][4][5]. The resulting small globin peptides serve as substrates for falcilysin (FLN) [6].FLN was first identified in 1999 when analysis of degraded globin fragments from the food vacuole revealed several peptides with cleavage sites that could not be attributed to the known proteases [7]. Specifically, a protease preferring to cleave substrates at polar or charged residues was implicated; this stands in contrast to the other hemoglobin-degrading proteases, which favor cleavage at hydrophobic residues. Purification of the native enzyme from food vacuoles revealed that it is a monomeric enzyme with a molecular weight of 130,000, by far the largest hemoglobin catabolic protease. FLN is a member of the M16 family of metalloproteases, enzymes character- * Corresponding author.

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confidence: 99%

Plasmodium falciparum falcilysin: an unprocessed food vacuole enzyme

Murata

Goldberg

2003

Molecular and Biochemical Parasitology

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“…The M16 family is divided into subfamilies A and B. Subfamily B includes the mitochondrial processing peptidase (MPP) and the chloroplast stromal processing peptidase; both enzymes are involved in removing N-terminal targeting peptides (13)(14)(15). Falcilysin belongs to subfamily A whose members are oligoendopeptidases involved in processing biologically important peptides such as yeast ␣ factor (16,17), insulin (18), and transforming growth factor ␣ (19). All of the subfamily A members are large enzymes (greater than 100 kDa) with the catalytic machinery located in the N terminus.…”

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confidence: 99%

Plasmodium falciparum Falcilysin

Murata

Goldberg

2003

Journal of Biological Chemistry

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The malaria parasite Plasmodium falciparum degrades host cell hemoglobin within an acidic food vacuole. The metalloprotease falcilysin has previously been identified as an important component of this catabolic process. Using random peptide substrate analysis, we confirm that recombinant falcilysin is highly active at acidic pH, consistent with its role in hemoglobin degradation. Unexpectedly, the enzyme is also robustly active at neutral pH, but with a substantially different substrate specificity. Imaging studies confirm the location of falcilysin in the food vacuole and reveal association with vesicular structures elsewhere within the parasite. These data suggest that falcilysin may have an expanded role beyond globin catabolism and may function as two different proteases in its two locations.Plasmodium falciparum is the causative agent of the deadliest form of human malaria. Each year 500 million infections and up to 2 million deaths are attributed to Plasmodium parasites (1). This disease is a major public health issue in many areas of the world where the increasing prevalence of drugresistant strains underscores the need to identify new drug targets. Examination of the parasite's unique metabolic pathways, such as hemoglobin degradation, provides numerous attractive candidates for chemotherapeutic development.Plasmodium infects and develops within human erythrocytes where hemoglobin is degraded in a semi-ordered cascade of proteases to provide energy and nutrients to the growing parasite (2). Hemoglobin degradation occurs in the unique acidic environment of the food vacuole. A family of aspartic proteases, the plasmepsins, mediates the initial cleavage within a conserved hinge region of the hemoglobin alpha chains (3, 4). The denatured globin is further degraded by plasmepsins in concert with a family of cysteine proteases, falcipain-2 and -3 (2, 4 -7). The resulting globin peptides serve as substrates for the zinc metalloprotease falcilysin (FLN).1 FLN prefers to degrade small globin peptides up to 20 amino acids in length at a pH optimum of 5.2 (8), consistent with the acidic environment of the food vacuole (9, 10). The temporal expression pattern of FLN is similar to that of other globinases yet differs by the absence of proteolytic processing during biosynthesis (11).FLN is a member of the M16 family of the clan ME zinc metalloproteases. This family is characterized by an inverted active site motif of HXXEH (12), where the two histidines and a glutamic acid residue located 75-84 residues C-terminal to this motif are involved in coordinating the catalytic zinc ion. The M16 family is divided into subfamilies A and B. Subfamily B includes the mitochondrial processing peptidase (MPP) and the chloroplast stromal processing peptidase; both enzymes are involved in removing N-terminal targeting peptides (13-15). Falcilysin belongs to subfamily A whose members are oligoendopeptidases involved in processing biologically important peptides such as yeast ␣ factor (16, 17), insulin (18), and transforming growth fact...

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“…Although a-factor is absent in the culture medium of a stel4 mutant, as has been seen previously (Sapperstein et al, 1994), AFRP is present (Figure 3, lane 6). Thus, unmethylated AFRP can apparently be exported, whereas unmethylated a-factor cannot be (Sapperstein et al, 1994 Adames et al, 1995). In these mutants the amount of mature a-factor (M) that is generated is either greatly diminished or abolished, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Third, unlike mature a-factor, the export of AFRP is STE6 independent, suggesting that AFRP may use a novel transporter for its exit from the cell. Thus, these results suggest the existence of novel proteolytic processing and export components specifically dedicated Adames et al (1995) to the production of AFRP and distinct from those components known to mediate the biogenesis of a-factor.…”

Section: Introductionmentioning

confidence: 92%

“…The first cleavage occurs within the N-terminal extension (between T7 and A8 of the a-factor precursor); the second cleavage occurs at the junction of the N-terminal extension and mature a-factor (between N21 and Y22) to generate mature bioactive a-factor (Chen et al, 1997; see also Figure 13). These cleavages are mediated by the zinc metalloproteases Ste24p and Axllp, respectively (Adames et al, 1995;Fujimura-Kamada et al, 1997). Once formed, mature a-factor is exported from the cell.…”

Section: Introductionmentioning

confidence: 99%

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A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.

Chen

Choi

Wang

et al. 1997

MBoC

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Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl-and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axll) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-independent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.

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Role of Yeast Insulin-Degrading Enzyme Homologs in Propheromone Processing and Bud Site Selection

Cited by 128 publications

References 43 publications

Plasmodium falciparum falcilysin: an unprocessed food vacuole enzyme

Plasmodium falciparum falcilysin: an unprocessed food vacuole enzyme

Plasmodium falciparum Falcilysin

A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.

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