A cleavable cytolysin-neuropeptide Y bioconjugate enables specific drug delivery and demonstrates intracellular mode of action.

Myxobacterial tubulysins are promising chemotherapeutics inhibiting microtubule polymerization, however, high unspecific toxicity so far prevents their application in therapy. For selective cancer cell targeting, here the coupling of a synthetic cytolysin to the hY1-receptor preferring peptide [F(7),P(34)]-neuropeptide Y (NPY) using a labile disulfide linker is described. Since hY1-receptors are overexpressed in breast tumors and internalize rapidly, this system has high potential as peptide-drug shuttle system. Molecular characterization of the cytolysin-[F(7),P(34)]-NPY bioconjugate revealed potent receptor activation and receptor-selective internalization, while viability studies verified toxicity. Triple SILAC studies comparing free cytolysin with the bioconjugate demonstrated an intracellular mechanism of action regardless of the delivery pathway. Treatments resulted in a regulation of proteins implemented in cell cycle arrest confirming the tubulysin-like effect of the cytolysin. Thus, the cytolysin-peptide bioconjugate fused by a cleavable linker enables a receptor-specific delivery as well as a potent intracellular drug-release with high cytotoxic activity.

Two motifs with different function regulate the anterograde transport of the adiponectin receptor 1.

The anterograde trafficking of GPCR has been described as a tightly controlled process involving specific amino acid sequences that mediate the receptor transport. In this study, we investigated whether the cell surface delivery of the adiponectin receptor 1, a newly identified class of heptahelix receptors different from G protein-coupled receptors, is regulated. Sequential N-terminal deletion revealed that the export of the AdipoR1 from the endoplasmic reticulum (ER) is controlled by distinct parts of the receptor N-terminus. Strong evidence is provided that the ER exit is mediated by two specific sequences, a F(X)(3)F(X)(3)F and a D(X)(3)LL motif. Disruption of these motifs led to a substantial accumulation of the AdipoR1 in the ER. Mutation of similar motifs in the AdipoR1 C-terminus did not result in aberrant receptor localization, suggesting that these motifs are sequence and position specific to the AdipoR1 N-terminus. Further analysis of the regulation mechanism identified an interaction with the chaperone BiP and additionally, strong evidence is provided that both motifs exert different biological function in the AdipoR1 ER export. In conclusion, our data demonstrate that the receptor transport shares similar ER exit motifs although AdipoR are structurally different from GPCR. However, since even two specific sequences are identified, the anterograde trafficking of the AdipoR1 seems to be regulated in a more complex manner.

Molecular mechanisms of signal transduction via adiponectin and adiponectin receptors.

The adipocytokine adiponectin and its receptor (AdipoR) comprise a new receptor-ligand system that is involved in a variety of clinically important morbidities such as obesity, type 2 diabetes and cardiovascular diseases. Adiponectin exerts a multitude of beneficial and tissue specific effects depending on its unique, tightly regulated multimerization behavior. Post-translational modifications are essential for the multimer assembly before secretion and protein stability in the circulation. AdipoR1 and 2 have been discovered as a new class of heptahelix receptors structurally and functionally distinct from G-protein-coupled receptors. Both AdipoRs bind adiponectin and the downstream signaling of both AdipoRs is mediated mainly by phosphorylation of AMPK and activation of peroxisome proliferator-activated receptor α, which influence the lipid and glucose metabolism of skeletal muscle and liver cells as well as inflammatory processes and vascular endothelial integrity. Several intracellular binding partners of the AdipoR N-terminus such as APPL1, CK2ß; and ERp46 have been identified and shown to control receptor signaling. Adiponectin has also been reported to modulate the dimerization and internalization of AdipoRs, which provides new insights into the molecular characteristics of this unusual receptor. The understanding of the functional mechanisms of adiponectin signal transduction is critical to benefit from the full therapeutic potential of the adiponectin-AdipoR system.

Dimerization of adiponectin receptor 1 is inhibited by adiponectin.

AdipoR1 and AdipoR2 are newly discovered members of the huge family of seven-transmembrane receptors, but both receptors are structurally and functionally different from G-protein-coupled receptors. Little is known about the oligomerization of the AdipoRs. Here, we show the presence of endogenous AdipoR1 dimers in various cell lines and human muscle tissue. To directly follow and localize the dimerization, we applied bimolecular fluorescence complementation (BiFC) in combination with flow cytometry. We visualized and quantified AdipoR1 homodimers in HEK293 cells. Moreover, we identified a GxxxG dimerization motif in the fifth transmembrane domain of the AdipoR1. By mutating both glycine residues to phenylalanine or glutamic acid, we were able to modulate the dimerization of AdipoR1, implicating a role for the GxxxG motif in AdipoR1 dimerization. Furthermore, we tested whether the AdipoR1 ligand adiponectin had any influence on receptor dimerization. Interestingly, we found that adiponectin decreases the receptor dimerization in a concentration-dependent manner. This effect is mainly mediated by segments of the collagen-like domain of full-length adiponectin. Accordingly, this is the first direct read-out signal of adiponectin at the AdipoR1 receptor, which revealed the involvement of specific amino acids of both the receptor and the ligand to modulate the quaternary structure of the AdipoR1.

Protein kinase CK2 interacts with adiponectin receptor 1 and participates in adiponectin signaling.

Adiponectin is an adipokine with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Its effects on energy homeostasis, glucose and lipid metabolism are mediated by two ubiquitously expressed seven-transmembrane receptors, AdipoR1 and -R2. With the exception of APPL1 and RACK1, no intracellular binding partners of adiponectin receptors are reported and thus signaling pathways downstream of these receptors remain largely unknown. To incorporate adiponectins protective potential in drug development it is essential to understand adiponectin signaling cascades in detail. A yeast two-hybrid approach employing AdipoR1s cytoplasmatic N-terminus led to the identification of the regulatory subunit of protein kinase CK2. We confirmed the interaction in co-immunoprecipitation, ELISA experiments and co-localization analysis in mammalian cells. Furthermore we could localize the interaction site in an N-terminal basic region close to the transmembrane domain. In adiponectin stimulation experiments of C2C12 mouse myotubes and MCF7 cells incorporating CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole (DMAT) we found a modulator role of CK2 in adiponectin signaling. Accordingly we identified the regulatory subunit of protein kinase CK2 as a novel intracellular partner of AdipoR1 and have strong evidence of CK2 as an effector molecule in adiponectin signaling. Since CK2 is involved in signaling cascades of other adipokines and hormones, e.g. leptin and insulin, our findings suggest a possible key function in crosstalk between adiponectin and insulin signaling pathways and could provide further insight into the anti-diabetic effects of adiponectin.