Yuri Cheburkin

AXL Is a Potential Target for Therapeutic Intervention in Breast Cancer Progression

Protein kinases play important roles in tumor development and progression. A variety of members of this family of signal transduction enzymes serve as targets for therapeutic intervention in cancer. We have identified the receptor tyrosine kinase (RTK) AXL as a potential mediator of motility and invasivity of breast cancer cells. AXL is expressed in most highly invasive breast cancer cells, but not in breast cancer cells of low invasivity. Ectopic expression of AXL was sufficient to confer a highly invasive phenotype to weakly invasive MCF7 breast cancer cells. Experimental inhibition of AXL signaling by a dominant-negative AXL mutant, an antibody against the extracellular domain of AXL, or short hairpin RNA knockdown of AXL decreased motility and invasivity of highly invasive breast cancer cells. To selectively interfere with cancer cell properties defining the rate of disease progression, we identified 3-quinolinecarbonitrile compounds, which displayed potent inhibitory activity against AXL and showed strong interference with motility and invasivity of breast cancer cells. Our findings validated the RTK AXL as a critical element in the signaling network that governs motility and invasivity of breast cancer cells, and allowed the identification of experimental anti-AXL small molecular inhibitors that represent lead substances for the development of antimetastatic breast cancer therapy.

Structural basis for Gas6–Axl signalling

Receptor tyrosine kinases of the Axl family are activated by the vitamin K‐dependent protein Gas6. Axl signalling plays important roles in cancer, spermatogenesis, immunity, and platelet function. The crystal structure at 3.3 Å resolution of a minimal human Gas6/Axl complex reveals an assembly of 2:2 stoichiometry, in which the two immunoglobulin‐like domains of the Axl ectodomain are crosslinked by the first laminin G‐like domain of Gas6, with no direct Axl/Axl or Gas6/Gas6 contacts. There are two distinct Gas6/Axl contacts of very different size, both featuring interactions between edge β‐strands. Structure‐based mutagenesis, protein binding assays and receptor activation experiments demonstrate that both the major and minor Gas6 binding sites are required for productive transmembrane signalling. Gas6‐mediated Axl dimerisation is likely to occur in two steps, with a high‐affinity 1:1 Gas6/Axl complex forming first. Only the minor Gas6 binding site is highly conserved in the other Axl family receptors, Sky/Tyro3 and Mer. Specificity at the major contact is suggested to result from the segregation of charged and apolar residues to opposite faces of the newly formed β‐sheet.

In Vivo Chemoenzymatic Control of N-Terminal Processing in Recombinant Human Epidermal Growth Factor

Protein synthesis initiates with Met in the cytosol of eukaryotes and formylmethionine (fMet) in prokaryotes and eukaryotic organelles. N-terminal methionine excision (NME) is the major source of N-terminal amino acid diversity in all three life kingdoms. The excision is dictated by the nature and bulkiness of the side-chain of the second amino acid (the penultimate residue) and is catalyzed by methionylaminopeptidases (MetAPs; EC 3.4.11.18). In bacteria, Lys, Arg, Leu, Phe and Ile protect the N-terminal Met from removal, whereas Met excision is promoted by having Gly, Ala, Pro, Cys, Ser, Thr or Val as the penultimate residues (Scheme 1A). NME is an irreversible cotranslational proteolysis, and is completed before the nascent polypeptide chains are fully synthesized. It was recently estimated that between 55 and 70% of the proteins of any given proteome undergo this Met excision. The canonical amino acid, Met is recognized, activated and charged onto its cognate tRNAs by methionyl-tRNA synthetase (MetRS). The N-formyl group from the initiator, methionine is enzymatically removed from the nascent polypeptide in bacteria. This is an essential prerequisite for the co-translational action of MetAP. E. coli MetRS exhibits remarkable flexibility in its substrate binding and tRNA charging—a fact that we and others have used for in vivo incorporation of a large number of Met analogues and Met-like amino acids (surrogates) into polypeptide sequences. The chemical diversification of Met side-chains that can be achieved in this way is quite impressive; it ranges from aliphatic, chalcogen and halogen-containing side-chains, to unsaturated chemical groups like alkenes or alkynes and other interesting bioorthogonal groups such as azides. 10] Previously, Tirrell and co-workers demonstrated nearly quantitative substitution of the Met residues in dihydrofolate reduct ACHTUNGTRENNUNGase by homopropargylglycine (Hpg). The initiator Met was also substituted, but the Hpg in the first position was not excised. Similarly, we globally replaced Met with trifluoromethionine in green fluorescent protein and discovered an efficient blockage of the N-terminal excision of trifluoro-Met when Ser was the penultimate residue. Based on these observations we reasoned that the incorporation of different Met analogues at the protein’s N-terminus would enable us to change the NME rules in recombinant proteins. We chose to study the effects of azidohomoalanine (Aha) and Hpg (Scheme 1B), whose chemically unique azido and alkyne sidechains, respectively, have recently gained great importance for bioorthogonal transformations. In order to test the catalytic efficiency of MetAP towards these analogues, we prepared a series of pentapeptides that contain Met, Hpg or Aha as N-terminal amino acids with Arg or Gly in the second position (penultimate residues). While Arg, which is a bulky penultimate residue, was expected to efficiently block excision of the N-terminal residues, Gly is the smallest amino acid and should support NME. Expectedly, the overnight incubation of Arg2 pentapeptides (Met1-Arg2-Gln3-Leu4-Phe5; Aha1-Arg2-Gln3-Leu4-Phe5; and Hpg1-Arg2-Gln3-Leu4-Phe5) with recombinant E. coli MetAP yielded no cleavage of Met, Aha or Hpg from the peptides (data not shown). That means that the excision was blocked completely by having Arg in the second position. On the other hand, the excision of Met, Aha and Hpg was observed in the case of Gly2 pentapeptides (Met1-Gly2-Gln3-Leu4-Phe5; Aha1Gly2-Gln3-Leu4-Phe5; and Hpg1-Gly2-Gln3-Leu4-Phe5). The canonical amino acid Met was fully cleaved after 10 min, but complete excision of Aha and Hpg was observed only after 100 and 250 min, respectively (Figure 1). Although Aha and Hpg are less efficiently processed than Met in Gly2 pentapepACHTUNGTRENNUNGtides, they are indeed substrates for E. coli MetAP. Nonetheless, the main differences are found in the excision kinetics. These allowed us to identify the following order of the in vitro cleavage efficiency in Gly2 pentapeptides: Met>Aha>Hpg. Interestingly, the Aha-pentapeptide was better processed by the E. coli MetAP than the Hpg-pentapeptide, but we observed the opposite with MetAP from Pyrococcus furiosus (unpub[a] L. Merkel, Dr. Yu. Cheburkin, Dr. B. Wiltschi, Dr. N. Budisa Max Planck Institute of Biochemistry, Molecular Biotechnology Am Klopferspitz 18, Martinsried (Germany) Fax: (+49)89-8578-2815 E-mail : budisa@biochem.mpg.de Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. A) The structure of recombinant human epidermal growth factor (hEGF) with a marked N-terminus, along with the simple NME rules when Gly or Arg are the penultimate residues. B) Chemical structures of methionine (Met) and its analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg).