Furin Is the Major Proprotein Convertase Required for KISS1-to-Kisspeptin Processing

<div><p>KISS1 is a broadly functional secreted proprotein that is then processed into small peptides, termed kisspeptins (KP). Since sequence analysis showed cleavage at KR or RR dibasic sites of the nascent protein, it was hypothesized that enzyme(s) belonging to the proprotein convertase family of proteases process KISS1 to generate KP. To this end, cell lines over-expressing KISS1 were treated with the proprotein convertase inhibitors, Dec-RVKR-CMK and α1-PDX, and KISS1 processing was completely inhibited. To identify the specific enzyme(s) responsible for KISS1 processing, mRNA expression was systematically analyzed for six proprotein convertases found in secretory pathways. Consistent expression of the three proteases – furin, PCSK5 and PCSK7 – were potentially implicated in KISS1 processing. However, shRNA-mediated knockdown of furin – but not PCSK5 or PCSK7 – blocked KISS1 processing. Thus, furin appears to be the essential enzyme for the generation of kisspeptins.</p></div

Mutation of putative PC sites in KISS1 blocks KP generation but does not abrogate metastasis suppressor function.

<p>A. Immunoblot showing KISS1 processing-deficient mutant (C1C2C3) which was mutated at the three putative furin processing sites (KISS1^{K57A/R67A/R124A}). C1C2C3 was not processed into KP [Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone-0084958-g001" target="_blank">Figure 1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone-0084958-g001" target="_blank">Figure 1B</a>]. * indicates C1C2C3 lane which was over-exposed to highlight lack of laddering below the full-length KISS1 band. B. Relative lung metastases (mean ± SEM) compared to C8161.9 (647±149 metastases). KISS1 and C1C2C3 transfected cells were significantly less metastatic.</p

KISS1 is secreted and processed into kisspeptins (KP) in multiple cell lines.

<p>A. (<i>Top panel</i>) Conditioned media collected from cultures of C8161.9, MelJuSo, MDA-MB-435, HeLa, COS7 and HEK293 cell lines which exogenously express KISS1. KP were immunoprecipitated with a monoclonal antibody detecting KISS1 (6a4.27), separated by Tris-tricine SDS-PAGE and then immunoblotted with anti-KISS1 antibody (1∶1000). (<i>Bottom panel</i>) Full-length KISS1 was detected in whole cell lysates using anti-KISS1 antibody as above (Mr∼15.9 kDa; Note: lower molecular mass bands were not detected from whole cell lysates as previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone.0084958-Nash2" target="_blank">[15]</a>). B. KISS1 processing into KP can be detected using immunoprecipitated KISS1/KP separated by Tris-tricine SDS-PAGE gel followed by Coomassie dye staining. KP bands were excised and analyzed by ESI-MS/MS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone.0084958.s001" target="_blank">Figure S1</a>]. KP sizes (approximate kDa) are predicted based upon the predicted PC cleavage sites in nascent KISS1 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone-0084958-g002" target="_blank">Figure 2A</a>). KP sizes and sequence were confirmed by ESI-MS/MS and are shown schematically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone.0084958.s001" target="_blank">Figure S1</a>.</p

RT-PCR analysis of the six members of PC family involved in the secretory pathway.

<p>C8161.9, MelJuSo, MDA-MB-435, MDA-MB-231, HeLa and HEK293 cells that process KISS1 to KP showed consistent expression of furin, PCSK5 and PCSK7 in all of the cell lines. <b>+</b>  =  PC mRNA detected <b>−</b>  =  PC mRNA not detected. Note: This depiction is solely qualitative, not quantitative.</p

Proprotein convertases are required for KISS1 processing.

<p>A. Amino acid sequence of KISS1 showing three predicted PC cleavage sites − RK, RR and KR in bold. Note: DYKDDDDK at position 68 is the FLAG epitope tag used for several of the studies described in this manuscript. B. Treatment of C8161.9, MelJuSo, HeLa, COS7 and HEK293 cells over-expressing KISS1 with the general PC inhibitor Dec-RVKR-CMK (100 µM) blocked KISS1 processing as detected by immunoprecipitation and immunoblot using anti-KISS1 antibody [See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone-0084958-g001" target="_blank">Figure 1</a> and Materials and Methods for details]. C. Over-expression of KISS1 and the biological PC inhibitor V5-tagged α1-PDX also resulted in loss of KISS1 processing into KP. Polypeptide sizes correspond to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone-0084958-g001" target="_blank">Figure 1</a>.</p

Furin is required for the generation of KP from KISS1.

<p>A, C. RT-PCR and immunoblot data showing reduced mRNA and protein levels of furin, PCSK5 and PCSK7 in HEK293 cells transduced with shRNA directed against each PC either individually or in combination. Bars in Panel C (by group; left to right)  =  furin, PCSK5 and PCSK7. B. shRNA-mediated knockdown of furin, PCSK5 and/or PCSK7 was in HEK293 cells over-expressing KISS1 show that cleavage of KISS1 is lost whenever furin is knocked down, but processing can still occur when PCSK5 or PCSK7 are knocked down. D. C8161.9, MelJuSo, HeLa and COS7 in which furin expression was knocked down by shRNA showed reduced or complete loss of KISS1 processing. KISS1 processing was observed in all cell lines when a scrambled control shRNA (Scr) was used. E. Restoration of furin activity in LoVo cells (which have no furin activity) by transduction of adenoviral particles with V5-tagged furin restored KISS1 processing to KP. LoVo cells transduced with an empty vector still do not show KISS1 cleavage into KP. Loading controls for V5-furin and KISS1 are shown using immunoblots with V5 and KISS1 antibodies. KISS1 and KP polypeptide sizes correspond to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone-0084958-g001" target="_blank">Figure 1</a>.</p

The toxicity and p53 knockdown efficiency of different hybrid nanoparticles.

<p>The toxicity and p53 knockdown efficiency of different hybrid nanoparticles.</p

Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma

<div><p>Objectives</p><p>The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318–1, a murine osteosarcoma cell line.</p><p>Methods</p><p>The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency.</p><p>Key findings</p><p>Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent.</p><p>Conclusions</p><p>This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.</p></div

Composition of different hybrid nanoparticles.

<p>Composition of different hybrid nanoparticles.</p

Identification of the formulation with the best knockdown efficiency by western blotting following nanoparticle transfection with different formulation (A. T1 and T2; B. T3-T8).

<p>318–1 cells were transfected with different formulations encapsulating either three different concentrations of p53 siRNA (<i>i</i>.<i>e</i>. 100, 125 and 150 pmol) (Fig 3A) or 150 pmol siRNA (Fig 3B). Representative western blotting results for p53, actin and vinculin are shown. This experiment was performed 3 independent times.</p