Subtilisin-like Processing Enzyme Research Articles

The kunitz domain protein BLI-5 plays a functionally conserved role in cuticle formation in a diverse range of nematodes

The cuticle of parasitic nematodes performs many critical functions and is essential for proper development and for protection from the host immune response. The biosynthesis, assembly, modification and turnover of this exoskeleton have been most extensively studied in the free-living nematode, Caenorhabditis elegans, where it represents a complex multi-step process involving a whole suite of enzymes. The biosynthesis of the cuticle has an additional level of complexity, as many of the enzymes also require additional proteins to aid their activation and selective inhibition. Blister-5 (BLI-5) represents a protein with a kunitz-type serine protease interacting domain and is involved in cuticle collagen biosynthesis in C. elegans, through its interaction with subtilisin-like processing enzymes (such as BLI-4). Mutation of the bli-5 gene causes blistering of the collagenous adult cuticle. Homologues of BLI-5 have been identified in several parasitic species that span different nematode clades. In this study, we molecularly and biochemically characterize BLI-5 homologues from the clade V nematodes C. elegans and Haemonchus contortus and from the clade III filarial nematode Brugia malayi. The nematode BLI-5 orthologues possess a shared domain structure and perform similar in vitro and in vivo functions, performing important proteolytic enzyme functions. The results demonstrate that the bli-5 genes from these diverse parasitic nematodes are able to complement a C. elegansbli-5 mutant and thereby support the use of the C. elegans model system to examine gene function in the experimentally less-amenable parasitic species.

The Proprotein Convertase SKI-1/S1P: ALTERNATE TRANSLATION AND SUBCELLULAR LOCALIZATION

Subtilisin kexin isozyme-1 (SKI-1) represents the first mammalian member of secretory subtilisin-like processing enzymes that cleaves after nonbasic residues. It is synthesized as an inactive precursor that undergoes three sequential autocatalytic processing steps of its N-terminal prosegment and an ectodomain shedding at a site near the transmembrane domain. The various cellular functions of SKI-1 emphasize the need to understand the sites of its activation and shedding. We have previously shown that SKI-1 undergoes autocatalytic shedding at the sequence KHQKLL(953) downward arrow, resulting in a membrane-bound stump called St-1 (amino acids 954-1052). However, little is known about the cellular localization of SKI-1 or its shed forms. In the present study, we have further identified a smaller C-terminal fragment St-2 generated closer to the transmembrane domain. By sequencing and mass spectrometric analysis, the start site and the molecular mass of St-2 were determined. Site-directed mutagenesis revealed the critical amino acid involved in this novel process. Mutation of Met(990) to M990A, M990I, and M990L failed to generate St-2, suggesting an internal alternate translation event at Met(990), as confirmed by an in vitro transcription/translation assay. Confocal microscopy defined the subcellular localization of SKI-1 and its fragments. The data show that most of membrane-bound SKI-1 and its stumps St-1 and St-2 localize to the Golgi and can enter the endosomal/lysosomal compartments but do not sort to the cell surface. Deletion studies showed that the transmembrane domain of SKI-1 determines its trafficking. Finally, rSt-1 and rSt-2 seem to affect the processing of ATF6 by SKI-1, but cellular stress does not regulate the production of St-2.

Disordered cell integrity signaling caused by disruption of the kexB gene in Aspergillus oryzae.

We isolated the kexB gene, which encodes a subtilisin-like processing enzyme, from a filamentous fungus, Aspergillus oryzae. To examine the physiological role of kexB in A. oryzae, we constructed a kexB disruptant (DeltakexB), which formed shrunken colonies with poor generation of conidia on Czapek-Dox (CD) agar plates and hyperbranched mycelia in CD liquid medium. The phenotypes of the DeltakexB strain were restored under high osmolarity in both solid and liquid culture conditions. We found that transcription of the mpkA gene, which encodes a putative mitogen-activated protein kinase involved in cell integrity signaling, was significantly higher in DeltakexB cells than in wild-type cells. The DeltakexB cells also contained higher levels of transcripts for cell wall-related genes encoding beta-1,3-glucanosyltransferase and chitin synthases, which is presumably attributable to cell integrity signaling through the increased gene expression of mpkA. As expected, constitutively increased levels of phosphorylated MpkA were observed in DeltakexB cells on the CD plate culture. High osmotic stress greatly downregulated the increased levels of both transcripts of mpkA and the phosphorylated form of MpkA in DeltakexB cells, concomitantly suppressing the morphological defects. These results suggest that the upregulation of transcription levels of mpkA and cell wall biogenesis genes in the DeltakexB strain is autoregulated by phosphorylated MpkA as the active form through cell integrity signaling. We think that KexB is required for precise proteolytic processing of sensor proteins in the cell integrity pathway or of cell wall-related enzymes under transcriptional control by the pathway and that the KexB defect thus induces disordered cell integrity signaling.

Expression of the proprotein convertases PC1 and PC2 mRNAs in thyrotropin releasing hormone neurons of the rat paraventricular nucleus of hypothalamus

PC1 and PC2 are subtilisin-like processing enzymes capable of cleaving thyrotropin releasing hormone (TRH) precursor (pro-TRH) at paired basic residues in vitro. In the paraventricular nucleus of the hypothalamus (PVN), pro-TRH is synthesized to control adenohypophysial thyrotropin and prolactin release. Biochemical and immunological approaches have shown that in the hypothalamus, pro-TRH is extensively cleaved at pairs of basic amino acids. We quantified, by two different approaches, in situ hybridization (ISH) on consecutive cryostat sections or double label ISH, the proportion of PVN TRH neurons containing either PC1 or PC2 mRNAs. Both techniques gave similar results: PC2 mRNA was present in 60–70% of TRH neurons, and PC1 mRNA in 37–46%. Values were similar in the anterior and medial parts of the parvocellular PVN. TRH neurons containing either PC1 or PC2 mRNA were found throughout the areas containing TRH cells without any evidence of anatomical segregation. These results suggest a biochemical heterogeneity in PVN TRH biosynthetic machinery.

Regional distribution of neuropeptide processing endopeptidases in adult rat brain

Many peptide hormone and neuropeptide precursors undergo post-translational processing at mono- and/or dibasic residues. An enzymatic activity capable of processing prodynorphin at a monobasic processing site designated ‘dynorphin converting enzyme’ has been previously reported in rat brain and bovine pituitary. In this study the distribution of dynorphin converting enzyme activity in ten regions of rat brain has been compared with the distribution of subtilisin-like processing enzymes and with the immuno-reactive dynorphin peptides. The distribution of dynorphin converting enzyme activity generally matches the distribution of immuno-reactive dynorphin B-13 in most but not all brain regions. The regions that are known to have a relatively large number of immuno-reactive dynorphin-neurons also contain high levels of dynorphin converting enzyme activity. The distribution of dynorphin converting enzyme activity does not match the distribution of subtilisin-like processing enzyme or carboxypeptidase E activities. Taken together the data support the possibility that the dynorphin converting enzyme is involved in the maturation of dynorphin, as well as other neuropeptides, and peptide hormones.

Occurrence of a furin-like prohormone processing enzyme in Aplysia neuroendocrine bag cells

1. 1. Strong evidence is accumulating that the endoproteases which process prohormones at dibasic residue cleavage sites are members of a subtilisin-related class of proteases. 2. 2. Using the polymerase chain reaction (PCR), we have isolated and characterized an Aplysia californica neuroendocrine bag cell cDNA product (270 base pairs) that encodes a sequence which is highly homologous to the subtilisin-related class of processing proteases that includes yeast Kex2, human/mouse/ Drosophila furin, human PC2, and mouse PC1/PC3 and PC2. 3. 3. The characterized cDNA PCR product showed the highest degree of residue identity with the furin-related group of proteins (human/mouse furin 71%; Drosophila furin 63%). 4. 4. These results establish that Aplysia contain a subtilisin-like gene and suggest that the expression of this gene may play a role in processing Aplysia precursor proteins in the bag cells and likely also in the exocrine atrial gland. 5. 5. Furthermore, the Aplysia nucleotide sequence results, together with available sequence information from human, mouse, and Drosophila furins, provide reasonable evidence that the furin-like enzymes may represent a separate subclass of the subtilisin-like processing enzymes.