High circulating cholesterol is associated with hypercholesterolemia, atherosclerosis, and stroke. However, the relation between cholesterol and tumorigenesis/metastasis is controversial. The ...proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein cholesterol homeostasis by targeting the low-density lipoprotein receptor (LDLR) for degradation. PCSK9 is mostly expressed in liver, which is one of the most common sites for metastatic disease. To reveal the function of PCSK9 and also evaluate the impact of cholesterol in liver metastasis development, B16F1 melanoma cells were injected into wild-type (WT) and
Pcsk9
-/-
mice to induce liver metastasis. On chow diet,
Pcsk9
-/-
mice harbored two-fold less liver metastases than WT mice. This decrease is related to low cholesterol levels in
Pcsk9
-/-
mice, as the protection was lost after normalizing
Pcsk9
-/-
cholesterol levels by a 2-week high cholesterol diet. Furthermore, a prolongation of this diet strongly increased metastasis in both genotypes, suggesting that high cholesterol levels promote metastatic progression. The protective effect of the PCSK9 deficiency is also associated with increased apoptosis in liver stroma and metastases. Tumor necrosis factor.α (TNFα) mRNA and protein were, respectively, higher in liver stroma and plasma of injected mice, likely increasing the apoptotic TNFα signaling. Furthermore, the anti-apoptotic factor B-cell lymphoma 2 was downregulated. TNFα regulation is LDLR-independent, as its mRNA level was similarly upregulated in mice lacking both PCSK9 and LDLR. Our findings show that PCSK9 deficiency reduces liver metastasis by its ability to lower cholesterol levels and by possibly enhancing TNFα-mediated apoptosis.
Recombinant adenoviruses were used for the expression of human amyloid precursor protein (APP) of Alzheimer's disease in primary cultures of rat cortical neurons and astrocytes. The catabolic ...pathways of human APP were studied 3 to 4 days after infection, when the equilibrium of APP production was reached. Although the expression of human wild type APP (WtAPP) by rat neurons induced the production of both extracellular and intraneuronal amyloid peptide (Aβ), Aβ was not detected in the culture medium of rat astrocytes producing human WtAPP. Because a low β-secretase activity was previously reported in rodent astrocytes, we wondered whether modifications of the APP amino acid sequence at the β-secretase clipping site would modify the astrocytic production of Aβ. Interestingly, rat astrocytes produced high amounts of Aβ after expression of human APP carrying a double amino acid substitution responsible for Alzheimer's disease in a large Swedish family (SwAPP). In both rat cortical neurons and astrocytes, the β-secretase cleavage of the human SwAPP occurred very early in the secretion process in a cellular compartment in which a different sorting of SwAPP and WtAPP seems unlikely. These results suggest that human WtAPP and SwAPP could be processed by different β-secretase activities.
PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor ...proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes. PUBLICATION ABSTRACT
Effects of the Prosegment and pH on the Activity of PCSK9 Benjannet, Suzanne; Luna Saavedra, Yascara Grisel; Hamelin, Josée ...
Journal of biological chemistry/The Journal of biological chemistry,
12/2010, Letnik:
285, Številka:
52
Journal Article
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PCSK9, a target for the treatment of dyslipidemia, enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes, up-regulating LDL-cholesterol levels. Whereas the targeting and ...degradation of the PCSK9-LDLR complex are under scrutiny, the roles of the N- and C-terminal domains of PCSK9 are unknown. Although autocatalytic zymogen processing of PCSK9 occurs at Gln152↓, here we show that human PCSK9 can be further cleaved in its N-terminal prosegment at Arg46↓ by an endogenous enzyme of insect High Five cells and by a cellular mammalian protease, yielding an ∼4-fold enhanced activity. Removal of the prosegment acidic stretch resulted in ∼3-fold higher binding to LDLR in vitro, in ≥4-fold increased activity on cellular LDLR, and faster cellular internalization in endosome/lysosome-like compartments. Finally, swapping the acidic stretch of PCSK9 with a similar one found in the glycosylphosphatidylinositol-anchored heparin-binding protein 1 does not impair PCSK9 autoprocessing, secretion, or activity and confirmed that the acidic stretch acts as an inhibitor of PCSK9 function. We also show that upon short exposure to pH values 6.5 to 5.5, an ∼2.5-fold increase in PCSK9 activity on total and cell surface LDLR occurs, and PCSK9 undergoes a second cleavage at Arg248, generating a two-chain PCSK9-ΔN248. At pH values below 5.5, PCSK9 dissociates from its prosegment and loses its activity. This pH-dependent activation of PCSK9 represents a novel pathway to further activate PCSK9 in acidic endosomes. These data enhance our understanding of the functional role of the acidic prosegment and on the effect of pH in the regulation of PCSK9 activity.
Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the γ-secretase ...cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a γ-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous γ-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Aβ secretion. Deletion of the cytoplasmic sequence of APP (APPΔC) inhibits both APP endocytosis and Aβ production. When APPΔC and PS1 are coexpressed in CHO cells, Aβ is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Aβ without endocytosis of APP by CHO cells.
Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species ...essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases
The ovine Doppel-encoding gene transcription unit (TU) and proximal flanking regions were cloned from a bacterial artificial chromosome (BAC) and sequenced. The 4586 bp TU is composed of two exons ...and one intron. When compared with its human counterpart, beside the open reading frame and part of the 3′ untranslated sequence, significant regions of homology were found within the intron and the proximal 5′ flanking regions. Sequence analysis of the BAC clone revealed that the ovine
Prnd gene is located around 52 kb downstream of the
Prnp locus. Expression of the sheep and murine
Prnd genes was observed in all tissues analyzed of transgenic mice bearing the BAC insert, with a noticeable highest expression level observed in the testis, with no associated noticeable phenotype. The present data suggest that, in contrast to ovine
Prnp, ovine
Prnd expression in transgenic mice has no obvious influence on conferred susceptibility to scrapie.
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