Deamidation, but not truncation, decreases the urea stability of a lens structural protein, betaB1-crystallin

Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type betaB1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (betaB1(DeltaN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens betaB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.     

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.     

Identification of a flower-specific cDNA,<Emphasis Type="Italic">RsPCP1</Emphasis>, encoding putative pollen coat protein from radish

To better understand the molecular control of floral development, we identified a flower-specific cDNA,RsPCPI, from Korean radish (Raphanus sativus). Based on nucleotide sequence analysis, this clone contains an open reading frame of 65 amino acids and shares 91% identity with a pollen coat protein from cabbage (Brassica oleracea). Southern analysis revealed thatRsPCPI is present as a single-copy gene or a member of a small gene family in the radish genome. BecauseRsPCPI mRNA was present exclusively in mature floral buds but not in young floral buds or in vegetative tissues, we propose that this gene is anther-specific.     

G protein-linked effector and second messenger systems involved in melatonin signal transduction

The isolation and characterization of multiple melatonin receptors in a wide range of tissues and cells signifies the functional diversity of melatonin. In different cellular environments, melatonin can regulate distinct second messengers or even positively or negatively regulate the same signal transduction pathway. The capacity by which melatonin receptors modulate the activities of various effector molecules is determined by the complement of signaling components present in any particular cell type. The specific interactions between many signaling molecules have been discerned in an increasing number of cellular systems and this information is being used to explain the physiological actions of melatonin. This review will attempt to summarize recent research by many groups that has revealed numerous subtleties of the melatonin-coupled signaling pathways.     

In vitro activities of native and designed peptide antibiotics against drug sensitive and resistant tumor cell lines

In order to develop peptide agents with reduced length and enhanced tumoricidal activity, we have designed gaegurin 6 (GGN6) derivatives through deletions and/or substitutions of amino acids. The deletion of hydrophobic amino terminal region completely abolished antitumor activity whereas the deletion of carboxy terminal region had little influence on antitumor activity. Antitumor activity of the PTP peptides did not correlate with antibacterial activity. PTP7, the most potent derivative, was found to have comparable antitumor activity to GGN6 in spite of reduced number of amino acids which is about half the size of gaegurin 6; furthermore, it showed little cytotoxicity on PBMCs and RBCs. GGN6 and PTP7 also showed equivalent cytotoxicity against drug sensitive (MCF-7) and multidrug-resistant cell lines (MCF-7/DOX). Plasma membrane blebbing and DNA fragmentation of peptide-treated tumor cells indicated that the peptides could induce apoptosis in tumor cells. These results suggest that GGN6 and its derivatives can be developed as new anticancer agents and may provide a new strategy for overcoming MDR which is a major problem in cancer therapy.     

Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases

When cells are stimulated by mitogens, extracellular signal-regulated kinase (ERK) is activated by phosphorylation of its regulatory threonine (Thr) and tyrosine (Tyr) residues. The inactivation of ERK may occur by phosphatase-mediated removal of the phosphates from these Tyr, Thr or both residues together. In this study, antibodies that selectively recognize all combinations of phosphorylation of the regulatory Thr and Tyr residues of ERK were developed, and used to study the inactivation of ERK upon mitogenic stimulation. We found that inactivation of ERK in the early stages of mitogenic stimulation involves separate Thr and Tyr phosphatases which operate differently in the nucleus and in the cytoplasm. Thus, ERK is differentially regulated in various subcellular compartments to secure proper length and strength of activation, which eventually determine the physiological outcome of many external signals.