Tenecin 3 is a glycine-rich, antifungal protein of 78 residues isolated from the insect Tenebrio molitor larva. As an initial step towards understanding the antifungal mechanism of tenecin 3, we examined how this protein interacts with the pathogenic fungus Candida albicans to exert its antifungal action. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential of C. albicans by the initial interaction. Fluorescence confocal microscopy and flow cytometric analysis revealed that tenecin 3 is rapidly internalized into the cytoplasmic space in energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic states. These results suggest that the internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal cellular process. The internalized tenecin 3 is dispersed in the cytoplasm, and the loss of cell viability occurs after this internalization. [14]. These studies have shown that the formation of transmembrane pores or ion channels on the cellular membranes cause the leakage of essential metabolites. The disruption of the microbial cell structure is the major killing mechanism for these cationic, antimicrobial proteins. This mechanism is also generally applicable to the antifungal action mechanism of these proteins, and even to the antiviral mechanism [15]. Other well known antifungal mechanisms of plant antifungal proteins, such as chitinases [16] [19], include the disruption of the cell wall structure and interference in cell wall synthesis. However, some antimicrobial proteins, including the cysteine-rich plant defensin Rs-AFP 2 [20], the insect antifungal protein drosomycin [5], and the glycine-rich insect antifungal protein AFP [21], show no growth inhibition for Gram negative and Gram positive bacteria. It has been proposed that the channel formation through direct protein-lipid interaction is not a probable antifungal mechanism for these proteins although their action mechanisms have not yet been fully elucidated [22]. There is a high sequence homology between Rs-AFP 2 and drosomycin, while AFP has no significant homology with these proteins [21]. We previously isolated tenecin 3 as an antifungal protein from the insect Tenebrio molitor and found that it had no antibacterial activity [23]. As tenecin 3 shares 37.3% identity with AFP from Sarcophaga peregrina, it can be categorized into an antifungal protein group with AFP and another insect antifungal protein, holotricin 3, from Holotricia diomphalia [24]. Tenecin 3 is also a glycine-rich protein, as are AFP and holotricin 3. However, little is known about the action mechanism of AFP and its homologs. The binding of AFP to Candia albicans was thought to be the cause of some damage to the cells even though this binding is weak and dependent on ionic environments [21]. Tenecin 3 consists of 78 amino acids and has distinctive features in its primary Abbreviations: AFP, ant...
Recurrent reports about protease-sensitive sites in the junction of the preS and S region of the hepatitis B virus large surface protein have raised the question about a possible biological role of S protein-depleted, independent preS protein fragments in the virus life cycle. In the present study, this question was addressed by exogenous introduction of fluorescence-labeled recombinant preS proteins into permeabilized HepG2 cells. While maltose-binding proteins (MBP) were evenly distributed throughout the cytoplasm, MBP-preS fusion proteins selectively accumulated in the nucleus. Using truncated preS proteins, the effective domain for this nuclear accumulation was localized around the preS2 region. The mode of this action differs from conventional nuclear translocation mechanism in its energy- and mediator-independency and in that it is not saturated regardless of the increase of preS protein concentration. The biological meaning of this phenomenon has to be further studied. However, in regard to hepatitis B virus infection, this observation might provide a clue for unveiling the still poorly characterized events after initial internalization of the virus, which might make use of the nuclear translocation effect of the preS2 region to facilitate the infection.
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