An affinity-directed protein missile system for targeted proteolysis

Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernández, Alejandro; Sapkota, Gopal P.

doi:10.1098/rsob.160255

Cited by 75 publications

(106 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…albumin) and the therapeutic target, thus resulting in increased serum half‐lives (Tijink et al ., ; Vosjan et al ., ). In addition, the stability and small size of Nbs make them ideal candidates for targeting intracellular proteins and show great potential as intrabodies (Lobato and Rabbitts, ; Blanco‐Toribio et al ., ; Bouchet et al ., ; Van Impe et al ., ; Bethuyne et al ., ; Fulcher et al ., ; Moutel et al ., ). Other applications of Nbs have been recently reviewed (Bruce et al ., ).…”

Section: Introductionmentioning

confidence: 97%

Escherichia coli surface display for the selection of nanobodies

Salema

Fernández

2017

Microbial Biotechnology

View full text Add to dashboard Cite

SummaryNanobodies (Nbs) are the smallest functional antibody fragments known in nature and have multiple applications in biomedicine or environmental monitoring. Nbs are derived from the variable segment of camelid heavy chain‐only antibodies, known as VHH. For selection, libraries of VHH gene segments from naïve, immunized animals or of synthetic origin have been traditionally cloned in E. coli phage display or yeast display systems, and clones binding the target antigen recovered, usually from plastic surfaces with the immobilized antigen (phage display) or using fluorescence‐activated cell sorting (FACS; yeast display). This review briefly describes these conventional approaches and focuses on the distinct properties of an E. coli display system developed in our laboratory, which combines the benefits of both phage display and yeast display systems. We demonstrate that E. coli display using an N‐terminal domain of intimin is an effective platform for the surface display of VHH libraries enabling selection of high‐affinity Nbs by magnetic cell sorting and direct selection on live mammalian cells displaying the target antigen on their surface. Flow cytometry analysis of E. coli bacteria displaying the Nbs on their surface allows monitoring of the selection process, facilitates screening, characterization of antigen‐binding clones, specificity, ligand competition and estimation of the equilibrium dissociation constant (KD).

show abstract

Section: Introductionmentioning

confidence: 97%

Escherichia coli surface display for the selection of nanobodies

Salema

Fernández

2017

Microbial Biotechnology

View full text Add to dashboard Cite

show abstract

“…Control and VHL-aGFP. 16 AdPROM cell lines were generated as described previously [22,23], using retroviral-based infections to deliver the AdPROM constructs to the FAM83D GFP/GFP U2OS cells.…”

Section: Generation Of Control and Vhl-agfp16 Adprom Cell Linesmentioning

confidence: 99%

FAM 83D directs protein kinase CK 1α to the mitotic spindle for proper spindle positioning

Fulcher

Mei

et al. 2019

EMBO Reports

Self Cite

View full text Add to dashboard Cite

The concerted action of many protein kinases helps orchestrate the error‐free progression through mitosis of mammalian cells. The roles and regulation of some prominent mitotic kinases, such as cyclin‐dependent kinases, are well established. However, these and other known mitotic kinases alone cannot account for the extent of protein phosphorylation that has been reported during mammalian mitosis. Here we demonstrate that CK1α, of the casein kinase 1 family of protein kinases, localises to the spindle and is required for proper spindle positioning and timely cell division. CK1α is recruited to the spindle by FAM83D, and cells devoid of FAM83D , or those harbouring CK1α‐binding‐deficient FAM83D F283A/F283A knockin mutations, display pronounced spindle positioning defects, and a prolonged mitosis. Restoring FAM83D at the endogenous locus in FAM83D − / − cells, or artificially delivering CK1α to the spindle in FAM83D F283A/F283A cells, rescues these defects. These findings implicate CK1α as new mitotic kinase that orchestrates the kinetics and orientation of cell division.

show abstract

“…This AdPROM system was engineered with the CUL2 substrate receptor VHL tethered to aGFP [12]. The efficacy of this proteolytic AdPROM suggested that this technology could be applied to achieve degradation of endogenous proteins by simply replacing the aGFP with small polypeptide binders that specifically recognize endogenous target proteins.…”

Section: Introductionmentioning

confidence: 99%

Targeting endogenous proteins for degradation through the affinity-directed protein missile system

et al. 2017

Self Cite

View full text Add to dashboard Cite

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins.

show abstract

An affinity-directed protein missile system for targeted proteolysis

Cited by 75 publications

References 40 publications

Escherichia coli surface display for the selection of nanobodies

Escherichia coli surface display for the selection of nanobodies

FAM 83D directs protein kinase CK 1α to the mitotic spindle for proper spindle positioning

Targeting endogenous proteins for degradation through the affinity-directed protein missile system

Contact Info

Product

Resources

About