Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9.

Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9's C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.

The Multi-Leu peptide inhibitor discriminates between PACE4 and furin and exhibits antiproliferative effects on prostate cancer cells.

The proprotein convertases (PCs) play an important role in protein precursor activation through processing at paired basic residues. However, significant substrate cleavage redundancy has been reported between PCs. The question remains whether specific PC inhibitors can be designed. This study describes the identification of the sequence LLLLRVKR, named Multi-Leu (ML)-peptide, that displayed a 20-fold selectivity on PACE4 over furin, two enzymes with similar structural characteristics. We have previously demonstrated that PACE4 plays an important role in prostate cancer and could be a druggable target. The present study demonstrates that the ML-peptide significantly reduced the proliferation of DU145 and LNCaP prostate cancer-derived cell lines and induced G0/G1 cell cycle arrest. However, the ML-peptide must enter the cell to inhibit proliferation. It is concluded that peptide-based inhibitors can yield specific PC inhibitors and that the ML-peptide is an important lead compound that could potentially have applications in prostate cancer.

Molecular Validation of PACE4 as a Target in Prostate Cancer.

Prostate cancer remains the single most prevalent cancer in men. Standard therapies are still limited and include androgen ablation that initially causes tumor regression. However, tumor cells eventually relapse and develop into a hormone-refractory prostate cancer. One of the current challenges in this disease is to define new therapeutic targets, which have been virtually unchanged in the past 30 years. Recent studies have suggested that the family of enzymes known as the proprotein convertases (PCs) is involved in various types of cancers and their progression. The present study examined PC expression in prostate cancer and validates one PC, namely PACE4, as a target. The evidence includes the observed high expression of PACE4 in all different clinical stages of human prostate tumor tissues. Gene silencing studies targeting PACE4 in the DU145 prostate cancer cell line produced cells (cell line 4-2) with slower proliferation rates, reduced clonogenic activity, and inability to grow as xenografts in nude mice. Gene expression and proteomic profiling of the 4-2 cell line reveals an increased expression of known cancer-related genes (e.g., GJA1, CD44, IGFBP6) that are downregulated in prostate cancer. Similarly, cancer genes whose expression is decreased in the 4-2 cell line were upregulated in prostate cancer (e.g., MUC1, IL6). The direct role of PACE4 in prostate cancer is most likely through the upregulated processing of growth factors or through the aberrant processing of growth factors leading to sustained cancer progression, suggesting that PACE4 holds a central role in prostate cancer.

Proteolytic processing of angiopoietin-like protein 4 by proprotein convertases modulates its inhibitory effects on lipoprotein lipase activity.

Angiopoietin-like protein 4 (ANGPTL4) has been associated with a variety of diseases. It is known as an endogenous inhibitor of lipoprotein lipase (LPL), and it modulates lipid deposition and energy homeostasis. ANGPTL4 is cleaved by unidentified protease(s), and the biological importance of this cleavage event is not fully understood with respect to its inhibitory effect on LPL activity. Here, we show that ANGPTL4 appears on the cell surface as the full-length form, where it can be released by heparin treatment in culture and in vivo. ANGPTL4 protein is then proteolytically cleaved into several forms by proprotein convertases (PCs). Several PCs, including furin, PC5/6, paired basic amino acid-cleaving enzyme 4, and PC7, are able to cleave human ANGPTL4 at a consensus site. PC-specific inhibitors block the processing of ANGPTL4. Blockage of ANGPTL4 cleavage reduces its inhibitory effects on LPL activity and decreases its ability to raise plasma triglyceride levels. In summary, the cleavage of ANGPTL4 by these PCs modulates its inhibitory effect on LPL activity.

Selective and potent furin inhibitors protect cells from anthrax without significant toxicity.

Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions.

Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics.

Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analogue peptidic inhibitors, we could identify highly potent furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a K(i) value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Molecular modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.

Regulation of C/EBPdelta-dependent transactivation by histone deacetylases in intestinal epithelial cells.

The C/EBPdelta transcription factor is involved in the positive regulation of the intestinal epithelial cell acute phase response. C/EBPdelta regulation by histone deacetylases (HDACs) during the course of inflammation remains to be determined. Our aim was to examine the effect of HDACs on C/EBPdelta-dependent regulation of haptoglobin, an acute phase protein induced in intestinal epithelial cells in response to pro-inflammatory cytokines. HDAC1, HDAC3, and HDAC4 were expressed in intestinal epithelial cells, as determined by Western blot. GST pull-down assays showed specific HDAC1 interactions with the transcriptional activation and the b-ZIP C/EBPdelta domains, while the co-repressor mSin3A interacts with the C-terminal domain. Immunoprecipitation assays confirmed the interaction between HDAC1 and the N-terminal C/EBPdelta amino acid 36-164 domain. HDAC1 overexpression decreased C/EBPdelta transcriptional activity of the haptoglobin promoter, as assessed by transient transfection and luciferase assays. Chromatin immunoprecipitation analysis showed a displacement of HDAC1 from the haptoglobin promoter in response to inflammatory stimuli and an increased acetylation of histone H3 and H4. HDAC1 silencing by shRNA expression increased both basal and IL-1beta-induced haptoglobin mRNA levels in epithelial intestinal cells. Our results suggest that interactions between C/EBPs and HDAC1 negatively regulate C/EBPdelta-dependent haptoglobin expression in intestinal epithelial cells.

Outcomes of Clostridium difficile-associated disease treated with metronidazole or vancomycin before and after the emergence of NAP1/027.

OBJECTIVE: To reassess the comparative efficacy of vancomycin versus metronidazole in the treatment of Clostridium difficile-associated disease (CDAD) after the emergence in 2003 of the hypervirulent NAP1/027 strain. METHODS: A retrospective cohort study was conducted in a tertiary-care Canadian hospital among 1,616 patients treated initially with metronidazole (N=1,360), vancomycin (N=219), or both (N=37), between 1991 and 2006, and followed for 60 days after diagnosis. Primary outcome was severe/complicated CDAD (SC-CDAD) defined as any of: (a) death within 30 days, (b) septic shock, (c) megacolon, (d) perforation, or (e) emergency colectomy. Adjusted odds ratios (AOR) and their 95% confidence intervals (CI) were calculated, stratifying into pre-epidemic (1991-2002) and epidemic (2003-2006) periods. Secondary outcome was recurrence within 60 days. RESULTS: Risk factors for SC-CDAD were the same in both periods: age>or=65 yr, male sex, immunosuppression, hospital acquisition, tube feeding, short duration of diarrhea, fever, elevated leukocytosis, or creatinine. Adjusting for confounders and using metronidazole therapy as baseline, vancomycin therapy was associated with a lower probability of developing SC-CDAD in 1991-2002 (AOR 0.21, 95% CI 0.05-0.99, P=0.048) but not during 2003-2006 (AOR 0.90, 95% CI 0.53-1.55, P=0.71). For both metronidazole and vancomycin, risk of recurrence increased in 2003-2004 but decreased in 2005-2006. CONCLUSIONS: Loss of superiority of vancomycin over metronidazole coincided with the emergence of NAP1/027. Toxin hyperproduction by NAP1/027 might be such that the disease follows its natural course. Novel therapeutic approaches are needed. The higher risk of recurrence in 2003-2004 probably reflected reinfections rather than relapses.

Management and outcomes of a first recurrence of Clostridium difficile-associated disease in Quebec, Canada.

BACKGROUND: During an epidemic of Clostridium difficile-associated disease (CDAD) caused by a strain that is a hyper-producer of toxins A and B, the frequency of a first recurrence after metronidazole treatment of the initial episode doubled in 2003-2004, compared with 1991-2002. METHODS: To examine whether administration of metronidazole as treatment for a first recurrence of CDAD remained appropriate, we reviewed data for patients with CDAD diagnosed in a hospital in Quebec, Canada, during 1991-2005, who experienced a first recurrence. The frequency of a second recurrence within 60 days after the first one was measured using Kaplan-Meier analysis. Cox regression was used for multivariate analysis. RESULTS: A total of 463 patients had a first recurrence of CDAD, of whom 154 (33.3%) experienced a second recurrence. Independent predictors of a second recurrence were age and duration of hospitalization after the first recurrence; this latter finding suggested that many such episodes were reinfections rather than relapses. Neither choice of treatment drug (metronidazole or vancomycin) nor use of the same drug for treatment of first recurrence, as had been used during the initial episode, was associated with increased risk of a second recurrence. However, 51 patients (11.0%) developed at least 1 complication (i.e., shock, need for colectomy, megacolon, perforation, or death within 30 days) during the first recurrence. Older age, a high leukocyte count, and renal failure at first recurrence were strongly associated with a complicated CDAD. CONCLUSIONS: Metronidazole is not inferior to vancomycin for treatment of patients with a first recurrence of CDAD, but the risk of complications with any treatment of CDAD may be higher than has previously been documented.