Processing of the -amyloid precursor protein (APP) by -and ␥-secretases generates the amyloidogenic peptide A, a major factor in the etiology of Alzheimer's disease. Following the recent identification of the -secretase -amyloid-converting enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant APP sw -secretase product C99. BACE is palmitoylated at three Cys residues within its transmembrane/cytosolic tail and is sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE is shed into the medium and that the extent of ectodomain shedding is palmitoylationdependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a decrease in the ␣-secretase product APPs␣. Although there is little increase in the generation of A by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one lacking the prosegment leads to enhanced A levels. These findings suggest that the shedding of BACE may play a role in the amyloidogenic processing of APP.Alzheimer's disease is a progressive degenerative disorder of the brain characterized by mental deterioration, memory loss, confusion, and disorientation. Among the cellular mechanisms contributing to this pathology are two types of fibrous protein deposition in the brain, intracellular neurofibrillary tangles consisting of polymerized tau protein, and abundant extracellular fibrils largely composed of -amyloid 1 (for reviews see Refs. 1-3). -Amyloid, also known as A, arises from proteolytic processing of the -amyloid precursor protein (APP) at the -and ␥-secretase cleavage sites. The cellular toxicity and amyloid-forming capacity of the two major forms of A (A 40 and especially A 42 ) have been well documented (1-3).An alternative, anti-amyloidogenic cleavage carried out by ␣-secretase(s) is located within the A peptide sequence of APP, thus precluding the formation of intact insoluble A. This cleavage by ␣-secretase within the (His-His-GlnLys2Leu-Val) sequence of APP is the major physiological route of APP maturation. The products of this reaction are a soluble 100 -120-kDa N-terminal fragment (APPs␣) and a C-terminal membrane-bound ϳ9-kDa segment (C83). In several recent reports, metalloproteinases such as ADAM9, -10, and -17 were shown to be involved in the ␣-secretase cleavage
Subtilisin kexin isozyme-1 (SKI-1)/site 1 protease is a mammalian subtilase composed of distinct functional domains. Among the major substrates of SKI-1 are the sterol regulatory element-binding proteins, regulating cholesterol and fatty acid homeostasis. Other substrates include the stress response factor activating transcription factor-6, the brain-derived neurotrophic factor, and the surface glycoproteins of highly infectious viruses belonging to the family of Arenaviridae. Domain deletion and/or point mutants were used to gauge the role of the various domains of SKI-1. Biosynthesis, cellular trafficking, and sterol regulatory element-binding protein-2 cleavage activity were used as diagnostic tools. Results revealed that Arg 130 and Arg 134 are critical for the autocatalytic primary processing of the prosegment and for the subsequent efficient exit of SKI-1 from the endoplasmic reticulum. Functional mapping of the growth factor cytokine receptor motif suggested a folding role within the endoplasmic reticulum. Microsequencing of the remaining membrane-bound stub following ectodomain shedding of SKI-1 localized the shedding site to KHQKLL 953 2. Site-directed mutagenesis, in vitro cleavage of a synthetic peptide containing the shedding site, and inhibitor studies favor an autocatalytic event occurring at a non-canonical SKI-1 recognition sequence, with P2 and P1 Leu being very critical. In conclusion, multiple domains ensuring optimal functional characteristics control SKI-1 activity and cellular trafficking.
Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum, carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans-acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 (carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO 2 -requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO 2 -enriched air. Therefore, growth inhibition without CO 2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.
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