Inhibition of intracellular proteolytic processing of soluble proproteins by an engineered <i>α</i>2-macroglobulin containing a furin recognition sequence in the bait region

Rompaey, Luc Van; Ayoubi, Torik; Ven, Willem Van de; Marynen, Peter

doi:10.1042/bj3260507

Cited by 42 publications

(26 citation statements)

References 63 publications

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“…Its reactive site was re-engineered by incorporating the SPC1 cleavage site, Arg-Xaa-Lys-Arg, in its reactive pocket, yielding a molecule of K a 1·1 10 7 M 1 (Lu et al 1993). Similar modifications of the bait region of the general protease inhibitor, 2 macroglobulin (Gly-Phe-Tyr-Glu686-Ser-Asp< Arg-Ser-Lys-Arg686-Ser-Leu), also led to the production of an intracellularly active SPC1 inhibitor (Van Rompaey et al 1997).…”

Section: Spc Inhibitionmentioning

confidence: 92%

Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications

Bergeron

Leduc²,

Day

2000

Journal of Molecular Endocrinology

169

121

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Limited proteolysis of most large protein precursors is carried out in vivo by the subtilisin-like pro-protein convertases. Many important biological processes such as peptide hormone synthesis, viral protein processing and receptor maturation involve proteolytic processing by these enzymes, making them potential targets for the development of novel therapeutic agents. However, the efficient development of such molecules requires a better understanding of the molecular mechanisms of proteolytic protein processing. Herein, we review the most recent findings on the molecular aspects of subtilisin-like convertase activity, such as the structural analysis of the proteases, the mechanisms of enzyme/substrate specificity, their interaction with other proteins such as 7B2, and the comparative tissue and cellular distribution of the enzymes and their substrates. These data are then used as a background for the review of the known biological functions of subtilisin-like pro-protein convertases, the reported clinical cases involving proteolytic processing defects and, finally, the ongoing development of new therapeutic inhibitor molecules based on this knowledge.

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Section: Spc Inhibitionmentioning

confidence: 92%

Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications

Bergeron

Leduc²,

Day

2000

Journal of Molecular Endocrinology

169

121

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“…Immunoprecipitates were resolved by SDS-PAGE (either 8 or 14% Tricine gels) and autoradiographed (21). All PC inhibitor proteins were cloned in pcDNA3 (Invitrogen), including those of ␣ 1 -PDX (8); the preprosegments of furin, PC7 (24), PC5 (25), and SKI-1 (26,27); and wild-type (␣ 2 M) and furin site-mutated (␣ 2 MG-F) ␣ 2 -macroglubulin (28). (24,25).…”

Section: Methodsmentioning

confidence: 99%

“…To determine whether a proprotein convertase(s) could carry out the processing of pro-BACE to BACE, we transiently coexpressed in HK293 cells the double-tagged [BACE F ] FG/V5 with an array of PC inhibitors including ␣ 1 -PDX (8,21); the preprosegments of furin, PC7 (24), PC5 (25), and SKI-1 (27); and the wild-type (␣ 2 M) and furin-inhibiting mutant (␣ 2 M-F) forms of ␣ 2 -macroglubulin (28). In addition, we prepared mutant forms of BACE in which the PC consensus cleavage site Arg residues in the prosegment were replaced by Ala at positions 42 or 45 (R42A or R45A, respectively).…”

Section: -Glumentioning

confidence: 99%

Post-translational Processing of β-Secretase (β-Amyloid-converting Enzyme) and Its Ectodomain Shedding

Benjannet

Elagöz

Wickham

et al. 2001

Journal of Biological Chemistry

279

134

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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

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“…Major limitations of these agents include either cell cytotoxicity and/or poor cellular permeability and targeting. Other approaches used recombinant-based inhibitors (19)(20)(21)(22). These strategies are based on the expression of proteins that contain a furin-like recognition sequence (RXXR) within the inhibitor binding region of either human a 1 -antitrypsin (22), a 2 -macroglobulin (21), proteinase-8 (20), or the turkey ovomucoid third domain (19).…”

Section: +mentioning

confidence: 99%

Human Carcinoma Cell Growth and Invasiveness Is Impaired by the Propeptide of the Ubiquitous Proprotein Convertase Furin

et al. 2005

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Furin, a potent proprotein convertase involved in activation of several cancer-related substrates, is synthesized as an inactive zymogen, thus minimizing the occurrence of premature enzymatic activity that would lead to inappropriate protein activation or degradation. This natural inhibitory mechanism is based on the presence of an inactivating prosegment at the NH 2 terminal of the zymogen. After initial autocatalytic cleavage, the prosegment remains tightly associated with the convertase until it reaches the trans-Golgi network where the dissociation of the prosegment and activation of furin occurs. We hypothesized that the inhibitory properties of the preprosegment of furin (ppFur) could be beneficial if ectopically expressed in tumor cells. Transfection of four human head and neck squamous cell carcinoma cell lines with the complete ppFur cDNA sequence (pIRES-EGFP-ppFur) or with the empty expression vector (pIRES-EGFP) was done. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, anchorage-independent growth in soft agar, and proliferation assays, as well as by investigating impairment of furin substrates processing. Following transfection of ppFur, a significant reduction in cell proliferation, tumorigenicity, and invasiveness was observed in vitro and in vivo. These biological changes are directly related to the inhibition of furin-mediated activation of crucial cancer-related substrates, such as membrane type 1 matrix metalloproteinase, transforming growth factor-B, insulin-like growth factor-1 receptor, and vascular endothelial growth factor-C. PpFur expression in head and neck squamous cell carcinoma cell lines showed a mechanistic link between furin inhibition, decreased substrate processing, cell proliferation, and invasive ability. These findings suggest that furin inhibition is a feasible approach to ameliorate and even abolish the malignant phenotype of various malignancies. (Cancer Res 2005; 65(10): 4162-71)

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Inhibition of intracellular proteolytic processing of soluble proproteins by an engineered α2-macroglobulin containing a furin recognition sequence in the bait region

Cited by 42 publications

References 63 publications

Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications

Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications

Post-translational Processing of β-Secretase (β-Amyloid-converting Enzyme) and Its Ectodomain Shedding

Human Carcinoma Cell Growth and Invasiveness Is Impaired by the Propeptide of the Ubiquitous Proprotein Convertase Furin

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