A femtomol range FRET biosensor reports exceedingly low levels of cell surface furin: implications for the processing of anthrax protective antigen
Authors: Yingxiao Wang
Alex Y Strongin
Sergey A Shiryaev
Albert G Remacle
Katarzyna Gawlik
Mingxing Ouyang
Vladislav S Golubkov
Journal: PLoS ONE (2010)
Discipline: Multidisciplinary
Keywords: Furin; endoproteinase; proproteins; biologically active proteins; Golgi compartment
Affiliations: Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.

Abstract:  Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins. Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax. Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR / STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors. 

Retracted: Yes

PubPeer comments:
 Figure 7, right panel. * Red boxes. The  IP lane looks unexpectedly similar to the  TCL lane. * Blue arrows: sharp vertical transition lines appear to be visible between some of the lanes. ![file] , 
 Here is a Forensically rendition of Figure 7, for those who might not immediately see the resemblance between the lanes. ![file] , 
 Figure 10A. * Blue boxes and arrows: In the U251 lanes, lane 2 and 4 look unexpectedly similar * Green boxes and arrows: In the LoVo:furWT lanes, lane 1 and 3 look unexpectedly similar, although one lane is stretched sideways a bit differently and in mirror image. ![file] , 
 Retraction, May 2022: https: journals.plos.org plosone article?id=10.1371 journal.pone.0269294  Following the publication of this article [1], concerns were raised regarding the results presented in multiple figures. Specifically, * The following data appear similar despite representing different conditions: u25cb Fig 7 (right panel), lanes 1 and 3. u25cb Fig 10A, lanes 2 and 4, and lanes 5 and 7 flipped horizontally. * There appear to be vertical discontinuities in the following figures when colour levels are adjusted: u25cb Fig 1, between lanes 1u20132. u25cb Fig 3 (lower left panel), between lanes 1u20132. u25cb Fig 7 (right panel), between lanes 2u20133, 3u20134 and 6u20137. u25cb Fig 9 (right panel), between lanes 1u20132. u25cb Fig 10A, between lanes 4u20135. The corresponding author stated that the raw image data underlying the above figures are no longer available. They acknowledged that the presented results are merged from multiple experiments, and stated that splice lines are clearly visible and were intentional. In relation to the concerns in Figs 7 and 10, the corresponding author acknowledged that the data in the lanes listed above appear similar, but asserted that they are not identical. In the absence of raw image data, the editors remain concerned about the integrity of these figures. In light of the concerns affecting multiple figure panels that question the integrity of these data, the PLOS ONE Editors retract this article. AYS did not agree with the retraction and stands by the articleu2019s findings. All other authors either did not respond directly or could not be reached. ,