Signaling pathways underlying eosinophil cell motility revealed by using caged peptides

Abstract

Insights into structure-function relations of many proteins opens the possibility of engineering peptides to selectively interfere with a protein's activity. To facilitate the use of peptides as probes of cellular processes, we have developed caged peptides whose inf luence on specific proteins can be suddenly and uniformly changed by near-UV light. Two peptides are described which, on photolysis of a caging moiety, block the action of calcium-calmodulin or myosin light chain kinase (MLCK). The efficacy of theses peptides is demonstrated in vitro and in vivo by determining their effect before and after photolysis on activities of isolated enzymes and cellular functions known to depend on calcium-calmodulin and MLCK. These caged peptides each were injected into motile, polarized eosinophils, and when exposed to light promptly blocked cell locomotion in a similar manner. The results indicate that the action of calcium-calmodulin and MLCK, and by inference myosin II, are required for the ameboid locomotion of these cells. This methodology provides a powerful means for assessing the role of these and other proteins in a wide range of spatio-temporally complex functions in intact living cells.Several methods have been used in the past to probe the role of a protein in cell function, each with advantages as well as limitations. Organic compounds are available that can modulate the activity of proteins, but interpretation of effects often is complicated by their relatively slow onset and low selectivity for a specific protein. Impressive progress toward single protein specificity has been made with antisense (1) and homologous recombination (2) methods, which disrupt the expression, and thus the function of a specific protein. Interpretation of effects, or lack thereof, on a cellular function may be complicated, however, by compensatory pathways enhanced by the absence of the targeted protein. Peptides that bind to proteins with high affinity and high selectivity provide a means to rapidly and potently inhibit the activity of selected proteins, but peptides must be microinjected into cells, and microinjection itself can at least transiently alter cell function. It thus would be desirable to have a way to make a peptide that is initially inactive or ''caged'' because of a strategically placed photolabile moiety (3, 4). Such a peptide could be injected into a cell, and time allowed for it to distribute evenly and for normal cell function to be verified. The peptide's biological activity then could be unmasked by light-directed removal of the photolabile group. Each cell would be its own control, thereby diminishing effects of cell to cell variability, and active peptides could be produced rapidly (within milliseconds) and with good spatial resolution.We describe here the preparation and use of photoactivatable caged peptides targeted against calmodulin and myosin light chain kinase (MLCK). Because these two proteins are known to be essential in the control of smooth muscle contractility, the efficacy of the caged...

Multiple proteins mediate IQGAP1-stimulated cell migration

“…There is also compelling evidence that calmodulin participates in cell motility [19,30,32,37]. We previously documented that Ca 2+ /calmodulin attenuates the binding of IQGAP1 to Cdc42; no effect was observed with Ca 2+ -free calmodulin [7].…”

“…Ca 2+ and calmodulin have been reported to participate in cell migration [30][31][32]. Moreover, in the presence of Ca 2+ , calmodulin abrogates the binding of IQGAP1 to Cdc42 [7].…”

Caged compounds: photorelease technology for control of cellular chemistry and physiology

“…Effective interaction requires a 'resonance' between the light wave and a molecular electronic energy gap (that is, the ground state and an excited state, hence absorption is strictly quantized). Electrons oscillate at about 10 15 Hz, corresponding to wavelengths of about 200-700 nm, hence this is called the 'photochemical range' of light. If we think of light as a stream of particles, instead of an electromagnetic wave, then a photon becomes a 'reagent', and absorption is a bimolecular reaction that occurs by collision of particles.…”

“…Using solid-phase peptide synthesis, a caged tyrosine was incorporated into a small inhibitory peptide 15 . Other peptides have been caged using the same approach 16 , 52 , 53 , 54 .…”

Photochemical electrocyclic ring closure and leaving group expulsion from N-(9-oxothioxanthenyl)benzothiophene carboxamides

“…4 Carboxanilides 5 were found to release various leaving groups LG -that vary in basicity in essentially quantitative yields. Because of the potentially modular chromophore (ring A) and the ability of the putative zwitterionic intermediate to release relatively basic leaving groups such as thiolates 5,7 and 5 (1) 10 phenolates 8 , i.e., side-chain groups of cysteine and tyrosine residues in peptides and proteins, the photochemical electrocyclic ring closure approach to the generation of zwitterionic 15 intermediates appeared to be promising. In this paper we extend the absorption maximum of the chromophoric group to 385 nm by use of the thioxanthone chromophore as ring A, as shown for benzothiophene carboxamides 7-10.…”