TY  - JOUR
A1  - Hagen, Sven
A1  - Mattay, Dinah
A1  - Raeuber, Christina
A1  - Mueller, Kristian M.
A1  - Arndt, Katja Maren
T1  - Characterization and inhibition of AF10-mediated interaction
JF  - Journal of peptide science
N2  - The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics. Copyright (c) 2014 European Peptide Society and John Wiley & Sons, Ltd.
KW  - protein-protein interaction
KW  - protein design and selection
KW  - protein engineering
KW  - coiled coil
KW  - leucine zipper
KW  - AF10
Y1  - 2014
U6  - https://doi.org/10.1002/psc.2626
SN  - 1075-2617
SN  - 1099-1387
VL  - 20
IS  - 6
SP  - 385
EP  - 397
PB  - Wiley-Blackwell
CY  - Hoboken
ER  - 
TY  - JOUR
A1  - Speck, Janina
A1  - Arndt, Katja Maren
A1  - Müller, Kristian M.
T1  - Efficient phage display of intracellularly folded proteins mediated by the TAT pathway
JF  - Protein engineering design & selection
N2  - Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.
KW  - g3p
KW  - phagemid display
KW  - protein design
KW  - protein engineering
KW  - selection
Y1  - 2011
U6  - https://doi.org/10.1093/protein/gzr001
SN  - 1741-0126
VL  - 24
IS  - 6
SP  - 473
EP  - 484
PB  - Oxford Univ. Press
CY  - Oxford
ER  -