A novel light-electricity sensing method for PCSK9 detection based on s-PdNFs with multifunctional property.

Amounts of studies show that proprotein convertase subtilisin/kexin type 9 (PCSK9) can increase the low-density lipoprotein cholesterol ((LDL-C), leading to the progression and development of atherosclerosis. Hence, design an effective method to detect serum PCSK9 is meaningful for the prevention, monitor and diagnosis of cardiovascular diseases. Here, we reported a dual-signal method for detecting PCSK9 using a signal label, sulphur-doped palladium nanoflowers (s-PdNFs), inspired by its multifunctional properties of quenching and catalysis, which would simultaneously achieve electrochemiluminescence (ECL) analysis and electrochemical detection. For the ECL analysis, s-PdNFs could effectively quench the ECL intensity of peroxydisulfate/oxygen (S2O82-/O2) system via ECL resonance energy transfer (ECL-RET). Importantly, the donor-acceptor pair (s-PdNFs-S2O82- pair) was firstly reported in the ECL-RET field. For the electrochemical detection, s-PdNFs with peroxidase-like activity, produce electric signals by catalyzing H2O2. Herein, a novel light-electricity dual signal immunosensor based on s-PdNFs was developed, and with a broad linear range of 5 fg mL-1 to 50 ng mL-1 (ECL channel) and 500 fg mL-1 to 50 ng mL-1 (electrochemical channel). Furthermore, the ECL channel and electrochemical channel can achieve the detection respectively which can meet different testing instruments. The two channels can also be combined to improve the accuracy of the detection. More importantly, the immunosensor realized the detection of PCSK9 in real serum samples demonstrated by good correlations with ELISA method. Our findings can promote the application of multifunctional materials in sensor and biomedicine field and provide a novel strategy for the detection of serum molecular.

Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels.

PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases plasma cholesterol levels by promoting LDL receptor degradation. Current antibody inhibitors block the interaction between PCSK9 and LDL receptors, significantly decrease plasma cholesterol levels, and provide beneficial clinical outcomes. To reduce the action of PCSK9 in plasma, a novel strategy that will produce a panel of non-native, conformationally-altered isomers of PCSK9 (X-PCSK9) to develop active immunotherapy targeting of native PCSK9 and inhibiting/blocking the interaction of PCSK9 with LDL receptor, thus decreasing plasma cholesterol levels is proposed. The authors used the scrambled disulfide bond technique to generate conformationally-altered isomers of the catalytic domain of mouse PCSK9. The focus was on the immune response of four X-isomers and their effects on plasma cholesterol and triglyceride levels in both C57BL/6J and Apoe-/- mice. The authors showed that the four immunogens produced significant immunogenicity against native PCSK9 to day 120 after immunization of C57BL/6J and Apoe-/- mice. This resulted in significantly decreased plasma cholesterol levels in C57BL/6J mice, and to a lesser degree in Apoe-/- mice. The X-PCSK9-B1 treated mice had increased LDL receptor mRNA and protein levels at day 120 after treatment. Thus, this study provides a new, potentially promising approach that uses long-term immunotherapy for a treatment of hypercholesterolemia.

Isolation and characterization of the circulating truncated form of PCSK9.

Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product. However, the circulating truncated form of PCSK9 has not been isolated and characterized. Utilizing antibodies which bind to either the catalytic domain or the C-terminal domain of PCSK9, the truncated PCSK9 was isolated from serum. MS was used to determine that this form of PCSK9 is a product of in vivo cleavage at Arg218 resulting in pyroglutamic acid formation of the nascent N terminus corresponding to Gln219 of intact PCSK9. We also determined that the truncated PCSK9 in serum lacked the N-terminal segment which contains amino acids critical for LDL receptor binding. A truncated PCSK9, expressed and purified from HEK293 cells with identical composition as the circulating truncated protein, was not active in inhibition of LDL uptake by HepG2 cells. These studies provide a definitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum.