Synthetic approaches to protein phosphorylation.

Reversible protein phosphorylation is critically important in biology and medicine. Hundreds of thousands of sites of protein phosphorylation have been discovered but our understanding of the functions of the vast majority of these post-translational modifications is lacking. This review describes several chemical and biochemical methods that are under development and in current use to install phospho-amino acids and their mimics site-specifically into proteins. The relative merits of total chemical synthesis, semisynthesis, and nonsense suppression strategies for studying protein phosphorylation are discussed in terms of technical simplicity, scope, and versatility.

Internalization and intracellular trafficking of a PTD-conjugated anti-fibrotic peptide, AZX100, in human dermal keloid fibroblasts.

A challenge in advanced drug delivery is selectively traversing the plasma membrane, a barrier that prohibits the intracellular delivery of most peptide and nucleic acid-based therapeutics. A variety of short amino acid sequences termed protein transduction domains (PTDs) first identified in viral proteins have been utilized for over 20 years to deliver proteins nondestructively into cells, however, the mechanisms by which this occurs are varied and cell-specific. Here we describe the results of live cell imaging experiments with AZX100, a cell-permeable anti-fibrotic peptide bearing an "enhanced" PTD (PTD4). We monitored fluorescently labeled AZX100 upon cell surface binding and subsequent intracellular trafficking in the presence of cellular process inhibitors and various well-defined fluorescently labeled cargos. We conclude that AZX100 enters cells via caveolae rapidly, in a manner that is independent of glycoconjugates, actin/microtubule polymerization, dynamins, multiple GTPases, and clathrin, but is associated with lipid rafts as revealed by methyl-beta-cylodextrin. AZX100 treatment increases the expression of phospho-caveolin (Y14), a critical effector of focal adhesion dynamics, suggesting a mechanistic link between caveolin-1 phosphorylation and actin cytoskeleton dynamics. Our results reveal novel and interesting properties of PTD4 and offer new insight into the cellular mechanisms facilitating an advanced drug delivery tool.

A possible role of insulin-like growth factor-II C-peptide in regulating the function of steroidogenic cells in adult frog adrenal glands.

The sole structural determinant for the differential ability of the insulin-like growth factors (IGF-I and IGF-II) to induce autophosphorylation of specific insulin receptor (IR) tyrosine residues and activate downstream signaling molecules is the C domain. The IR is structurally related to the type I insulin-like growth factor receptor (IGF-IR). This study aimed to identify the presence of IGF receptors by which the IGF-II C-peptide could mediate its effects in the frog (Rana ridibunda) adrenal glands and to observe whether injection of IGF-II C-peptide affects the function of adrenal steroidogenic cells using light and transmission electron microscopy and by the evaluation of the immunoreactivity of steroidogenic acute regulatory protein (StAR). After IGF-II C-peptide injection, there was a reduction of StAR protein immunoreactivity levels, an accumulation of large lipid droplets in close contact with each other, and an induction of proliferation of the steroidogenic cells. These results indicate a possible role of IGF-II C-peptide in steroidogenic cell function and in induction of steroidogenesis. The detection in this study of IGF-I receptor (IGF-IR) immunoreactivity in frog adrenal glands also indicates that the metabolic and mitogenic effects of IGF-II C-peptide in these glands may occur via the IGF-IR.

Phospholemman modulates Na+/Ca2+ exchange in adult rat cardiac myocytes.

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca(2+) homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na(+)/Ca(2+) exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold (P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na(+) out:1 Ca(2+) in) was significantly (P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca(2+)](o), the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na(+)/Ca(2+) exchange.

Visualization of IP(3) dynamics reveals a novel AMPA receptor-triggered IP(3) production pathway mediated by voltage-dependent Ca(2+) influx in Purkinje cells.

IP(3) signaling in Purkinje cells is involved in the regulation of cell functions including LTD. We have used a GFP-tagged pleckstrin homology domain to visualize IP(3) dynamics in Purkinje cells. Surprisingly, IP(3) production was observed in response not only to mGluR activation, but also to AMPA receptor activation in Purkinje cells in culture. AMPA-induced IP(3) production was mediated by depolarization-induced Ca(2+) influx because it was mimicked by depolarization and was blocked by inhibition of the P-type Ca(2+) channel. Furthermore, trains of complex spikes, elicited by climbing fiber stimulation (1 Hz), induced IP(3) production in Purkinje cells in cerebellar slices. These results revealed a novel IP(3) signaling pathway in Purkinje cells that can be elicited by synaptic inputs from climbing fibers.

Effects of cadmium chloride on the paracellular barrier function of intestinal epithelial cell lines.

In the present study we characterized the functional and structural disruption of the paracellular barrier of intestinal epithelium in vitro in relation to cytotoxicity after apical Cd2+ exposure. For that purpose filter-grown Caco-2 and IEC-18 cells were apically exposed to 5 to 100 microM CdCl2 for 4 or 14 h. It was found that the effects of Cd2+ on the epithelial barrier were concentration- and time-dependent. The first detected effects of Cd2+ in Caco-2 cells after 4 h exposure were a decrease in transepithelial electrical resistance, increased permeabilities of mannitol and PEG-4000, and changes in intercellular localization of ZO-1, occludin, and e-cadherin. The effects were far more pronounced after prolonged exposure. The disruption of the paracellular barrier by 5 to 30 microM Cd2+ was detected without a significant loss of viability of the Caco-2 cells. In the IEC-18 cells, Cd2+ concentrations affecting the barrier (50 and 100 microM) also affected cell viability. In both cell lines the effects on the cell layers continued to develop after removal of extracellular Cd2+. This correlated with the cellular retention of Cd2+, which was high for the 12 h following 4 h accumulation. This study showed that the decreased epithelial barrier function of intestinal epithelial cells is accompanied by tight junction disruption. It is concluded that Cd2+ causes increased paracellular permeability by disruption of junctional function and structure. The initial junctional effects of Cd2+ suggest that Cd2+ increases its own bioavailability by causing disruption of the intestinal paracellular barrier.

Diffusion of proteins across the nuclear envelope of HeLa cells.

We describe an experimental system to study nucleocytoplasmic diffusion of proteins in living HeLa cells. To localize proteins to the nucleus, substrates were created that contain a nuclear localization sequence fused to Aequorea victoria green fluorescent protein (GFP). Transiently and stably transfected HeLa cells were used for these assays. A protein of 29-kDa molecular mass that harbors GFP and the bipartite Xenopus nucleoplasmin nuclear localization sequence (NLS) accumulates efficiently in nuclei of HeLa cells. However, in the absence of active facilitated nuclear import, the reporter protein exits the nucleus and equilibrates between nucleus and cytoplasm. We define different conditions that promote the diffusion of small nuclear proteins across the nuclear envelope of mammalian culture cells. Our results set the stage to analyze the competence of nuclear pore complexes for nucleocytoplasmic diffusion of macromolecules in living cells.

Nuclear import of DNA in digitonin-permeabilized cells.

DNA can enter intact mammalian nuclei with varying degrees of efficiency in both transfected and microinjected cells, yet very little is known about the mechanism by which it crosses the nuclear membrane. Nucleocytoplasmic transport of fluorescently labeled DNA was studied using a digitonin-permeabilized cell system. DNA accumulated in the nucleus with a punctate staining pattern in over 80% of the permeabilized HeLa cells. Nuclear localization of the labeled DNA was energy dependent and occurred through the nuclear pore, but did not require the addition of soluble cytoplasmic protein factors necessary for protein import.

Metal (Fe3+) affinity chromatography: differential adsorption of tau phosphoproteins.

Tau is a neuronal cytoskeletal protein consisting of a group of isoforms with apparent molecular masses ranging from 45 to 62 kDa. Tau purified from brain exists in multiple phosphorylated forms and abnormally phosphorylated tau appears to play an important role in the neuropathology of Alzheimer's disease. To separate the differentially phosphorylated populations of tau, a chromatographic technique using ferric ions adsorbed onto iminodiacetic acid substituted Sepharose was developed. Several distinct populations of tau were isolated based on the phosphorylation state. These preparations can be used for further investigation of how each specific phosphorylation state modulates the metabolism and function of tau.