Candida albicans is the most common cause of hematogenously disseminated and oropharyngeal candidiasis. Both of these diseases are characterized by fungal invasion of host cells. Previously, we have found that C. albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the fungal surface protein and host cell receptors that mediate this process. We found that the C. albicans Als3 is required for the organism to be endocytosed by human umbilical vein endothelial cells and two different human oral epithelial lines. Affinity purification experiments with wild-type and an als3Δ/als3Δ mutant strain of C. albicans demonstrated that Als3 was required for C. albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. Furthermore, latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not. Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin–cadherin binding. Therefore, Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. These results uncover the first known fungal invasin and provide evidence that C. albicans Als3 is a molecular mimic of human cadherins.

The human fungal pathogen Candida albicans colonizes and invades a wide range of host tissues. Adherence to host constituents plays an important role in this process. Two members of the C. albicans Als protein family (Als1p and Als5p) have been found to mediate adherence; however, the functions of other members of this family are unknown. In this study, members of the ALS gene family were cloned and expressed in Saccharomyces cerevisiae to characterize their individual functions. Distinct Als proteins conferred distinct adherence profiles to diverse host substrates. Using chimeric Als5p-Als6p constructs, the regions mediating substratespecific adherence were localized to the N-terminal domains in Als proteins. Interestingly, a subset of Als proteins also mediated endothelial cell invasion, a previously unknown function of this family. Consistent with these results, homology modeling revealed that Als members contain anti-parallel ␤-sheet motifs interposed by extended regions, homologous to adhesins or invasins of the immunoglobulin superfamily. This finding was confirmed using circular dichroism and Fourier transform infrared spectrometric analysis of the N-terminal domain of Als1p. Specific regions of amino acid hypervariability were found among the N-terminal domains of Als proteins, and energy-based models predicted similarities and differences in the N-terminal domains that probably govern the diverse function of Als family members. Collectively, these results indicate that the structural and functional diversity within the Als family provides C. albicans with an array of cell wall proteins capable of recognizing and interacting with a wide range of host constituents during infection.

Hammerhead is suitable for screening large databases of flexible molecules for binding to a protein of known structure. It correctly docks a variety of known flexible ligands, and it spends an average of only a few seconds on each compound during a screen. The approach is completely automated, from the elucidation of protein binding sites, through the docking of molecules, to the final selection of compounds for assay.

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9α-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9α-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9α-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.

We have examined the effects of a number of derivatives of ryanodine on K+ conduction in the Ca2+ release channel purified from sheep cardiac sarcoplasmic reticulum (SR). In a fashion comparable to that of ryanodine, the addition of nanomolar to micromolar quantities to the cytoplasmic face (the exact amount depending on the derivative) causes the channel to enter a state of reduced conductance that has a high open probability. However, the amplitude of that reduced conductance state varies between the different derivatives. In symmetrical 210 mM K+, ryanodine leads to a conductance state with an amplitude of 56.8 +/- 0.5% of control, ryanodol leads to a level of 69.4 +/- 0.6%, ester A ryanodine modifies to one of 61.5 +/- 1.4%, 9,21-dehydroryanodine to one of 58.3 +/- 0.3%, 9 beta,21beta-epoxyryanodine to one of 56.8 +/- 0.8%, 9-hydroxy-21-azidoryanodine to one of 56.3 +/- 0.4%, 10-pyrroleryanodol to one of 52.2 +/- 1.0%, 3-epiryanodine to one of 42.9 +/- 0.7%, CBZ glycyl ryanodine to one of 29.4 +/- 1.0%, 21-p-nitrobenzoyl-amino-9-hydroxyryanodine to one of 26.1 +/- 0.5%, beta-alanyl ryanodine to one of 14.3 +/- 0.5%, and guanidino-propionyl ryanodine to one of 5.8 +/- 0.1% (chord conductance at +60 mV, +/- SEM). For the majority of the derivatives the effect is irreversible within the lifetime of a single-channel experiment (up to 1 h). However, for four of the derivatives, typified by ryanodol, the effect is reversible, with dwell times in the substate lasting tens of seconds to minutes. The effect caused by ryanodol is dependent on transmembrane voltage, with modification more likely to occur and lasting longer at +60 than at -60 mV holding potential. The addition of concentrations of ryanodol insufficient to cause modification does not lead to an increase in single-channel open probability, such as has been reported for ryanodine. At concentrations of > or = 500 mu M, ryanodine after initial rapid modification of the channel leads to irreversible closure, generally within a minute. In contrast, comparable concentrations of beta-alanyl ryanodine do not cause such a phenomenon after modification, even after prolonged periods of recording (>5 min). The implications of these results for the site(s) of interaction with the channel protein and mechanism of the action of ryanodine are discussed. Changes in the structure of ryanodine can lead to specific changes in the electrophysiological consequences of the interaction of the alkaloid with the sheep cardiac SR Ca2+ release channel.

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Geranyl diphosphate synthase (GPPS) catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to form geranyl diphosphate. Geranyl diphosphate is the precursor of monoterpenes, a large family of natural occurring C 10 compounds predominately found in plants. Similar to plants but unique to animals, some bark beetle genera (Coleoptera: Scolytidae) produce monoterpenes that function in intraspecific chemical communication as aggregation and dispersion pheromones. The release of monoterpene aggregation pheromone mediates host colonization and mating. It has been debated whether these monoterpene pheromone components are derived de novo through the mevalonate pathway or result from simple modifications of dietary precursors. The data reported here provide conclusive evidence for de novo biosynthesis of monoterpene pheromone components from bark beetles. We describe GPPS in the midgut tissue of pheromone-producing male Ips pini. GPPS expression levels are regulated by juvenile hormone III, similar to other mevalonate pathway genes involved in pheromone biosynthesis. In addition, GPPS transcript is almost exclusively expressed in the anterior midgut of male I. pini, the site of aggregation pheromone biosynthesis. The recombinant enzyme was functionally expressed and produced geranyl diphosphate as its major product. The threedimensional model structure of GPPS shows that the insect enzyme has the sequence structural motifs common to E-isoprenyl diphosphate synthases.isoprenyl diphosphate synthase ͉ monoterpene biosynthesis ͉ ipsdienol

Mammalian platelets contain an array of antimicrobial peptides, termed platelet microbicidal proteins (PMPs). Human and rabbit PMPs include known chemokines, such as platelet factor-4 (hPF-4); PMP-1 is the rabbit orthologue of hPF-4. Chemokines that also exert direct antimicrobial activity have been termed kinocidins. A consensus peptide domain library representing mammalian PF-4 family members was analyzed to define structural domains contributing to antimicrobial activity against a panel of human pathogens. Secondary conformations were assessed by circular dichroism spectrometry, and molecular modeling was employed to investigate structural correlates of antimicrobial efficacy. Antimicrobial activity against isogenic peptide-susceptible or -resistant Staphylococcus aureus, Salmonella typhimurium, and Candida albicans strain pairs mapped to the C-terminal hemimer (38-74) and modular domains thereof (49-63 and 60-74). Increasing electrostatic charge and steric bulk were general correlates of efficacy. Structural data corroborated spatial distribution of charge, steric bulk and putative secondary structure with organism-specific efficacy. Microbicidal efficacies of the cPMP antimicrobial hemimer and C-terminal peptide (60-74) were retained in a complex human-blood biomatrix assay. Collectively, these results suggest that modular determinants arising from structural components acting independently and cooperatively govern the antimicrobial functions of PF-4 family kinocidins against specific target pathogens.