Abstract

In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/−, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.

Details

Title
Restoring functional neurofibromin by protein transduction
Author
Mellert, K 1 ; Lechner, S 2 ; Lüdeke, M 3 ; Lamla, M 4 ; Möller, P 5 ; Kemkemer, R 6 ; Scheffzek, K 2 ; Kaufmann, D 7 

 Institute of Pathology, University of Ulm, Ulm, Germany; Institute of Human Genetics, University of Ulm, Ulm, Germany 
 Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria 
 Institute of Human Genetics, University of Ulm, Ulm, Germany 
 Institute of Organic Chemistry III, Ulm University, Ulm, Germany 
 Institute of Pathology, University of Ulm, Ulm, Germany 
 Department of New Materials and Biosystems, Max Planck Institute for Medical Research, Stuttgart, Germany; Reutlingen University, Applied Chemistry, Reutlingen, Germany 
 Institute of Human Genetics, University of Ulm, Ulm, Germany; Reutlingen University, Applied Chemistry, Reutlingen, Germany 
Pages
1-9
Publication year
2018
Publication date
Apr 2018
Publisher
Nature Publishing Group
e-ISSN
20452322
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2027027262
Copyright
© 2018. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.