Functional analysis of human PACE4‐A and PACE4‐C isoforms: identification of a new PACE4‐CS isoform

Zhong, Mei; Benjannet, Suzanne; Lazure, Claude; Munzer, Scott; Seidah, Nabil G.

doi:10.1016/0014-5793(96)01059-9

Cited by 26 publications

(17 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…It is unclear why the endogenous mRNA species do not seem to give rise to an active protein product in the secretory pathway. The human cDNA expressed in RPE.40 cells encoded the PACE4A isoform, and it has been suggested that this isoform and the PACE4E isoform are the only catalytically active versions of PACE4 [23,40]. Our Northern analysis shows that the PACE4 mRNA large enough to encode either of these isoforms (the largest of the four mRNA species) is only weakly expressed in RPE.40 cells ; this low level of expression could account for our inability to detect PACE4 activity.…”

Section: Discussionmentioning

confidence: 62%

Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

Sucic

Moehring

Inocencio

et al. 1999

Biochemical Journal

View full text Add to dashboard Cite

PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 °C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.

show abstract

Section: Discussionmentioning

confidence: 62%

Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

Sucic

Moehring

Inocencio

et al. 1999

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…S4F-S4H). Among previously reported PACE4 isoforms, only isoform A (PACE4-FL) is known to be active or processed from pro-PACE4 to PACE4 (23). Enzymatic activity was thus tested using recombinant (r) PACE4-FL and rPACE4-altCT (S2; Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

PACE4 Undergoes an Oncogenic Alternative Splicing Switch in Cancer

et al. 2017

View full text Add to dashboard Cite

Inhibition of PACE4, a proprotein convertase that is overexpressed in prostate cancer, has been shown to block cancer progression in an androgen-independent manner. However, the basis for its overexpression and its growth-inhibitory effects are mitigated and uncertain. Here, we report that PACE4 pre-mRNA undergoes DNA methylation-sensitive alternative splicing of its terminal exon 3 0 untranslated region, generating an oncogenic, C-terminally modified isoform (PACE4-altCT). We found this isoform to be strongly expressed in prostate cancer cells, where it displayed an enhanced autoactivating process and a distinct intracellular routing that prevented its extracellular secretion. Together, these events led to a dramatic increase in processing of the progrowth differentiation factor pro-GDF15 as the first PACE4 substrate to be identified in prostate cancer. We detected robust expression of PACE4-altCT in other cancer types, suggesting that an oncogenic switch for this proenzyme may offer a therapeutic target not only in advanced prostate cancer but perhaps also more broadly in human cancer. Cancer Res; 77(24); 6863-79. Ó2017 AACR.

show abstract

“…When present during the preincubation as well as the pulse periods, the concentrations of tunicamycin and brefeldin A (BFA) were 5 and 2.5 g/ml, respectively. As described previously (24), cells were lysed, and the proteins therein, along with those in the culture media, were immunoprecipitated by incubation with either the rPC7 antiserum (see above) plus protein A-agarose or with lentil lectin-Sepharose (25). Following SDS-PAGE, the bands were visualized by exposure to x-ray film.…”

Section: Methodsmentioning

confidence: 99%

“…The labeled proteins were then run on an SDS-PAGE, after which the appropriate bands were eluted and analyzed on an Applied Biosystem gas-phase sequenator (model 470A) as described previously (12,25).…”

Section: Methodsmentioning

confidence: 99%

In Vitro Characterization of the Novel Proprotein Convertase PC7

Munzer¹,

Basak²,

Zhong³

et al. 1997

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Biochemical and enzymatic characterization of the novel proprotein convertase rat PC7 (rPC7) was carried out using vaccinia virus recombinants overexpressed in mammalian BSC40 cells. Pro-PC7 is synthesized as a glycosylated zymogen (101 kDa) and processed into mature rPC7 (89 kDa) in the endoplasmic reticulum. No endogenously produced soluble forms of this membrane-anchored protein were detected. A deletion mutant (65 kDa), truncated well beyond the expected Cterminal boundary of the P-domain, produced soluble rPC7 in the culture medium. Enzymatic activity assays of rPC7 using fluorogenic peptidyl substrates indicated that the pH optimum, Ca 2؉ dependence, and cleavage specificity of this enzyme are largely similar to those of furin. However, with some substrates, cleavage specificity more closely resembled that of yeast kexin, suggesting differential processing of proprotein substrates by this novel convertase. We examined the rPC7-and human furin-mediated cleavage of synthetic peptides containing the processing sites of three proteins known to colocalize in situ with rPC7. Whereas both enzymes correctly processed the pro-parathyroid hormone tridecapeptide and the pro-PC4 heptadecapeptide, neither enzyme cleaved a pro-epidermal growth factor hexadecapeptide. Thus, this study establishes that rPC7 is an enzymatically functional subtilisin/kexin-like serine proteinase with a cleavage specificity resembling that of hfurin. In addition, we have demonstrated that rPC7 can correctly process peptide precursors that contain the processing sites of at least two potential physiological substrates.

show abstract

Functional analysis of human PACE4‐A and PACE4‐C isoforms: identification of a new PACE4‐CS isoform

Cited by 26 publications

References 35 publications

Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

PACE4 Undergoes an Oncogenic Alternative Splicing Switch in Cancer

In Vitro Characterization of the Novel Proprotein Convertase PC7

Contact Info

Product

Resources

About