Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into ␣ and ␤ families. Structures of several ␤-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys 1 -Cys 5 , Cys 2 -Cys 4 , and Cys 3 -Cys 6 . We found that human ␤-defensin 3 (hBD3), a recently described member of the growing ␤ family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys 1 -Cys 5 and of Cys 1 -Cys 6 , we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native ␤ pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by ␣-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human ␤-defensins and the design of novel peptide antibiotics.

A characteristic of integrins is their ability to transfer chemical and mechanical signals across the plasma membrane. Force generated by myosin II makes cells able to sense substrate stiffness and induce maturation of nascent adhesions into focal adhesions. In this paper, we present a comprehensive proteomic analysis of nascent and mature adhesions. The purification of integrin adhesion complexes combined with quantitative mass spectrometry enabled the identification and quantification of known and new adhesion-associated proteins. Furthermore, blocking adhesion maturation with the myosin II inhibitor blebbistatin markedly impaired the recruitment of LIM domain proteins to integrin adhesion sites. This suggests a common recruitment mechanism for a whole class of adhesion-associated proteins, involving myosin II and the zinc-finger-type LIM domain.

Azobenzene is a common light switch. Azobenzene, as ω‐amino acid derivative in peptides, exhibits redox properties that make it susceptible to reduction by thiols with concurrent thiol‐induced enhanced thermal Z‐to‐E isomerization rates.

Glucose-dependent insulinotropic polypeptide receptor (GIPR), a member of family B of the G-protein coupled receptors, is a potential therapeutic target for which discovery of nonpeptide ligands is highly desirable. Structure-activity relationship studies indicated that the N-terminal part of glucose-dependent insulinotropic polypeptide (GIP) is crucial for biological activity. Here, we aimed at identification of residues in the GIPR involved in functional interaction with N-terminal moiety of GIP. A homology model of the transmembrane core of GIPR was constructed, whereas a three-dimensional model of the complex formed between GIP and the N-terminal extracellular domain of GIPR was taken from the crystal structure. The latter complex was docked to the transmembrane domains of GIPR, allowing in silico identification of putative residues of the agonist binding/activation site. All mutants were expressed at the surface of human embryonic kidney 293 cells as indicated by flow cytometry and confocal microscopy analysis of fluorescent GIP binding. Mutation of residues Arg183, Arg190, Arg300, and Phe357 caused shifts of 76-, 71-, 42-, and 16-fold in the potency to induce cAMP formation, respectively. Further characterization of these mutants, including tests with alanine-substituted GIP analogs, were in agreement with interaction of Glu3 in GIP with Arg183 in GIPR. Furthermore, they strongly supported a binding mode of GIP to GIPR in which the N-terminal moiety of GIP was sited within transmembrane helices (TMH) 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5), and Phe357 (TMH6). These data represent an important step toward understanding activation of GIPR by GIP, which should facilitate the rational design of therapeutic agents.Glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) is a 42-residue hormone released by the enteroendocrine K cells lining the proximal duodenum (Jörnvall et al., 1981;Moody et al., 1984). GIP stimulates insulin secretion from pancreatic ␤-cells after ingestion of nutrients. The peptide has a very short half-life in the blood because it is vulnerable to degradation by the ubiquitous enzyme dipeptidyl peptidase IV (Mentlein et al., 1993). GIP, along with its sister incretin hormone glucagonlike peptide 1, has been shown to account for 50 to 70% of postprandial insulin secretion. The incretin effect is strictly glucose-dependent and is essential for the maintenance of glucose homeostasis. GIP further enhances its glucose-lowering effects by the inhibition of hepatic glucose production and the stimulation of proinsulin gene transcription and translation. Because of its hypoglycemic and hypolipidemic effects (Brown, 1974;Baggio and Drucker, 2007), GIP and its receptor (GIPR) are of high pharmacological interest, especially in identification and design of new molecules for the treatment of diabetes mellitus and obesity (Kieffer, 2003). The expression of GIPR in different organs and systems such as stoma...