KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake.

KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. From our previous structural studies, it seemed unlikely that KP4 acts by forming channels in the target cell membrane. Instead, KP4 was proposed to act by blocking fungal calcium channels, as KP4 was shown to inhibit voltage-gated calcium channels in rat neuronal cells, and its effects on fungal cells were abrogated by exogenously added calcium. Here, we extend these studies and demonstrate that KP4 acts in a reversible manner on the cell membrane and does not kill the cells, but rather inhibits cell division. This action is mimicked by EGTA and is abrogated specifically by low concentrations of calcium or non-specifically by high ionic strength buffers. We also demonstrate that KP4 affects (45)Ca uptake in U. maydis. Finally, we show that cAMP and a cAMP analogue, N 6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, both abrogate KP4 effects. These results suggest that KP4 may inhibit cell growth and division by blocking calcium-regulated signal transduction pathways.

Isolation of rat dihydrofolate reductase gene and characterization of recombinant enzyme.

While assays of many antifolate inhibitors for dihydrofolate reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we report the isolation of the rat DHFR gene through screening of a rat liver cDNA library. The rat liver DHFR gene has an open reading frame of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat enzyme with those from other species indicate a high level of conservation at the primary sequence level and more so for the amino acid residues comprising the active site of the enzyme. Expression of the rat DHFR gene in bacteria produced a recombinant protein with high enzymatic activity. The recombinant protein also paralleled the human enzyme with respect to the inhibition by most of the antifolates tested with PT652 and PT653 showing a reversal in their patterns. Our results indicated that rat DHFR can be used as a model to study antifolate compounds as potential drug candidates. However, variations between rat and human DHFR enzymes, coupled with unique features in the inhibitors, could lead to the observed differences in enzyme sensitivity and selectivity.

The H1 double-stranded RNA genome of Ustilago maydis virus-H1 encodes a polyprotein that contains structural motifs for capsid polypeptide, papain-like protease, and RNA-dependent RNA polymerase.

The Ustilago maydis viral (UmV) genome consists of three distinct size groups of double-stranded RNA (dsRNA) segments: H (heavy), M (medium), and L (light). The H segments have been suggested to encode all essential viral proteins, but without any molecular evidences. As a preliminary step to understand viral genomic organization and the molecular mechanism governing gene expression in UmV, we determined the complete nucleotide sequence of the H1 dsRNA genome in P1 viral killer subtype. The H1 dsRNA genome (designated UmV-H1) contained a single open reading frame that encodes a polyprotein of 1820 residues, which is predicted to be autocatalytically processed by a viral papain-like protease to generate viral proteins. The amino-terminal region is the capsid polypeptide with a predicted molecular mass of 79.9 kDa. The carboxy-terminal region is the RNA-dependent RNA polymerase (RDRP) that has a high sequence homology to those of the totiviruses. The H2 dsRNA also encodes a distinct RDRP, suggesting that UmV is a complex virus system like the Saccharomyces cerevisiae viruses ScV-L1 and -La.

Salivary histatin 5 and human neutrophil defensin 1 kill Candida albicans via shared pathways.

Salivary histatins are a family of basic histidine-rich proteins in which therapeutic potential as drugs against oral candidiasis is apparent, considering their potent in vitro antifungal activity and lack of toxicity to humans. Histatin 5 (Hst 5) kills the fungal pathogen Candida albicans via a mechanism that involves binding to specific sites on the yeast cell membrane and subsequent release of cellular ATP in the absence of cytolysis. We explored the killing pathway activated by Hst 5 and compared it to those activated by other antifungal agents. The candidacidal activity of human neutrophil defensin 1 (HNP-1) shared very similar features to Hst 5 cytotoxic action with respect to active concentrations and magnitude of induction of nonlytic ATP efflux, depletion of intracellular ATP pools, and inhibitor profile. Hst 5 and HNP-1 are basic proteins of about 3 kDa; however, they have unique primary sequences and solution structures that cannot explain how these two molecules act so similarly on C. albicans to induce cell death. Our finding that HNP-1 prevented Hst 5 binding to the candidal Hst 5 binding protein suggests that the basis for the overlapping actions of these two naturally occurring antimicrobial proteins may involve interactions with shared yeast components.

Liposomes as formulation excipients for protein pharmaceuticals: a model protein study.

PURPOSE: The advent of recombinant DNA technology has made possible the pharmaceutical use of a wide range of proteins and peptides. However, the complex structure of proteins renders them susceptible to physical instabilities such as denaturation, aggregation and precipitation. We tested the hypothesis that partial unfolding and exposure of hydrophobic domains leads to physical instability, and investigated approaches to stabilize protein formulations. METHODS: KP6 beta, an 81 amino acid killer toxin from Ustilago maydis, was used as a model protean. Circular dichroism and fluorescence spectroscopy were used to study the temperature dependent folding/ unfolding characteristics of KP6 beta. ANS (1,8 anilinonaphthalene sulfonate), a fluorescent probe that partitions into hydrophobic domains, was used to detect exposure of hydrophobic domains. RESULTS: As the temperature was elevated, near-UV CD indicated progressive loss of KP6 beta tertiary structure, while far-UV CD indicated retention of secondary structure. Increasing exposure of hydrophobic domains was observed, as indicated by the penetration of ANS. At elevated temperatures (60 degrees C), KP6 beta3 conserved most secondary structural features. However, tertiary structure was disordered, suggesting the existence of a partially folded, structured intermediate state. Liposomes bound to partially unfolded structures and prevented the formation of aggregates. CONCLUSIONS: Partial unfolding resulted in increased exposure of hydrophobic domains and aggregation of KP6 beta, but with preservation of secondary structure. Liposomes interacted with the structured intermediate state, stabilizing the protein against aggregation. These results suggest a general formulation strategy for proteins, in which partially unfolded structures are stabilized by formulation excipients that act as molecular chaperones to avoid physical instability.

Kinetics of ribosomal pausing during programmed -1 translational frameshifting.

In the Saccharomyces cerevisiae double-stranded RNA virus, programmed -1 ribosomal frameshifting is responsible for translation of the second open reading frame of the essential viral RNA. A typical slippery site and downstream pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site. The translational intermediate associated with a ribosome paused at this position is detected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes paused at the slippery site, and the fraction of paused ribosomes that frameshift are estimated. About 10% of ribosomes pause at the slippery site in vitro, and some 60% of these continue in the -1 frame. Ribosomes that continue in the -1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro. Altering the rate of translational initiation alters the rate of frameshifting in vivo. Our in vitro and in vivo experiments can best be interpreted to mean that there are three methods by which ribosomes pass the frameshift site, only one of which results in frameshifting.

Structure of Ustilago maydis killer toxin KP6 alpha-subunit. A multimeric assembly with a central pore.

Ustilago maydis is a fungal pathogen of maize, some strains of which secrete killer toxins. The toxins are encoded by double-stranded RNA viruses in the cell cytoplasm. The U. maydis killer toxin KP6 contains two polypeptide chains, alpha and beta, having 79 and 81 amino acids, respectively, both of which are necessary for its killer activity. The crystal structure of the alpha-subunit of KP6 (KP6alpha) has been determined at 1.80-A resolution. KP6alpha forms a single domain structure that has an overall shape of an ellipsoid with dimensions 40 A x 26 A x 21 A and belongs to the alpha/beta-sandwich family. The tertiary structure consists of a four-stranded antiparallel beta-sheet, a pair of antiparallel alpha-helices, a short strand along one edge of the sheet, and a short N-terminal helix. Although the fold is reminiscent of toxins of similar size, the topology of KP6alpha is distinctly different in that the alpha/beta-sandwich motif has two right-handed betaalphabeta split crossovers. Monomers of KP6alpha assemble through crystallographic symmetries, forming a hexamer with a central pore lined by hydrophobic N-terminal helices. The central pore could play an important role in the mechanism of the killing action of the toxin.

Functions of conserved motifs in the RNA-dependent RNA polymerase of a yeast double-stranded RNA virus.

At least eight conserved motifs are visible in the totivirus RNA-dependent RNA polymerase (RDRP). We have systematically altered each of these in the Saccharomyces cerevisiae double-stranded RNA virus ScVL1 by substituting the conserved motifs from a giardiavirus. The results help define the conserved regions of the RDRP involved in polymerase function and those essential for other reasons.

In vitro selection of packaging sites in a double-stranded RNA virus.

The Saccharomyces cerevisiae double-stranded RNA virus ScVL1 recognizes a small sequence in the viral plus strand for both packaging and replication. Viral particles will bind to this viral binding sequence (VBS) with high affinity in vitro. An in vitro selection procedure has been used to optimize binding, and the sequences isolated have been analyzed for packaging and replication in vivo. The selected sequence consists of a stem with a bulged A residue topped by a loop of several bases. Four residues of the 18 bases are absolutely conserved for tight binding. These all fall in regions that appear to be single stranded. Eight more residues have preferred identities, and six of these are in the stem. The VBS is similar to the R17 bacteriophage coat protein binding site. Packaging and replication require tight binding to viral particles.

The Ustilago maydis virally encoded KP1 killer toxin.

Some strains of the plant-pathogenic fungus Ustilago maydis secrete toxins (killer toxins) that are lethal to susceptible strains of the same fungus. There are three well-characterized killer toxins in U. maydis-KP1, KP4, and KP6-which are secreted by the P1, P4, and P6 subtypes, respectively. These killer toxins are small polypeptides encoded by segments of an endogenous, persistent double-stranded RNA (dsRNA) virus in each U. maydis subtype. In P4 and P6, the M2 dsRNA segment encodes the toxin. In this work, the KP1 killer toxin was purified for internal amino acid sequence analysis, and P1M2 was identified as the KP1 toxin-encoding segment by sequence analysis of cDNA clones. The KP1 toxin is a monomer with a predicted molecular weight of 13.4kDa and does not have extensive sequence similarity with other viral anti-fungal toxins. The P1M2 segment is different from the P4 and P6 toxin-encoding dsRNA segments in that the 3' non-coding region of its plus strand has no sequence homology to the 3' ends of the plus strands of P1M1, P4M2, or P6M2.

A second double-stranded RNA virus from yeast.

Two double-stranded RNA viruses exist as permanent persistent infections of the yeast Saccharomyces cerevisiae: ScVL1 and ScVLa. Both belong to the Totiviridae, which include a number of fungal and protozoan double-stranded RNA viruses. Although ScVL1 and ScVLa share the same genomic organization and mode of expression and coexist in the same cells, they show no evidence of recombination: with one limited exception, sequence conservation is detectable only in regions conserved in all totiviruses. Both have two open reading frames on their single essential RNAs: cap (encoding a capsid polypeptide) and pol (encoding an RNA-dependent RNA polymerase). The ScVLa virus, like ScVL1, appears to express its Pol domain by a -1 translational frameshift.

High-level secretion of a virally encoded anti-fungal toxin in transgenic tobacco plants.

Ustilago maydis killer toxins are small polypeptides (7-14 kDa) which kill susceptible cells of closely related fungal species. The KP4 toxin is a single polypeptide subunit with a molecular weight of 11.1 kDa. In this work, a transgenic tobacco plant was constructed which secretes the KP4 toxin at a high level. The KP4 toxin expressed in this transgenic plant was of the same size and specificity as the authentic Ustilago KP4 toxin. The expression level was at least 500 times higher than that of the KP6 toxin expressed in plants. Transgenic crop plants producing the KP4 toxin could be rendered resistant to KP4-susceptible fungal pathogens.

Interference with replication of two double-stranded RNA viruses by production of N-terminal fragments of capsid polypeptides.

It is possible to interfere with the replication of a number of plant RNA viruses by systemic production of viral capsid polypeptides or RNA-dependent RNA polymerases, or by production of untranslatable portions of viral plus strands or minus strands. Interference can occur by a number of mechanisms. We have discovered that the Saccharomyces cerevisiae double-stranded RNA viruses ScVL1 and ScVLa, which exist as permanent persistent infections of their host cells, can be cured very efficiently by production of N-terminal fragments of their capsid polypeptides. These totiviruses produce only two polypeptides: a capsid polypeptide (Cap) and a Cap-Pol fusion polypeptide with RNA-dependent RNA polymerase activity. Three types of interference can be detected: interference due to overproduction of both Cap and Cap-Pol, interference due to overproduction of Cap (and consequent distortion of the Cap to Cap-Pol ratio), and interference due to negative complementation by N-terminal fragments of Cap. Some N-terminal fragments of Cap appear to be incorporated into viral particles, but only in the presence of a complete Cap protein. We postulate that incorporation of N-terminal fragments of Cap results in the formation of defective particles.

Processing and secretion of a virally encoded antifungal toxin in transgenic tobacco plants: evidence for a Kex2p pathway in plants.

Ustilago maydis is a fungal pathogen of maize. Some strains of U. maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U. maydis. We show here that one of these toxins, the KP6 killer toxin, is synthesized by transgenic tobacco plants containing the viral toxin cDNA under the control of a cauliflower mosaic virus promoter. The two components of the KP6 toxin, designated alpha and beta, with activity and specificity identical to those found in toxin secreted by U. maydis cells, were isolated from the intercellular fluid of the transgenic tobacco plants. The beta polypeptide from tobacco was identical in size and N-terminal sequence to the U. maydis KP6 beta polypeptide. Processing of the KP6 preprotoxin in U. maydis requires a subtilisin-like processing protease, Kex2p, which is present in both animal and fungal cells and is required for processing of (among other things) small secreted polypeptide hormones and secreted toxins. Our findings present evidence for Kex2p-like processing activity in plants. The systemic production of this viral killer toxin in crop plants may provide a new method of engineering biological control of fungal pathogens in crop plants.

Packaging in a yeast double-stranded RNA virus.

The yeast virus ScV-L1 has only two genes, cap and pol, which encode the capsid polypeptide and the viral polymerase, respectively. The second gene is translated only as a cap-pol fusion protein. This fusion protein is responsible for recognition of a specific small stem and loop region of the viral plus strands, of 19 to 31 bases in length, ensuring packaging specificity. We have used a related virus, ScV-La, which has about 29% codon identity with ScV-L1 in the most conserved region of the pol gene, to map the region in pol that is responsible for packaging L1. Characterization of a number of chimeric viral proteins that recognize L1 but have the La capsid region delimits the region necessary for recognition of L1 to a 76- to 82-codon portion of pol. In addition, we show that overproduction of the La capsid polypeptide results in curing of the ScV-La virus, analogous to the production of plants resistant to RNA viruses by virtue of systemic production of viral coat protein.

Structure and heterologous expression of the Ustilago maydis viral toxin KP4.

Killer toxins are polypeptides secreted by some fungal species that kill sensitive cells of the same or related species. In the best-characterized cases, they function by creating new pores in the cell membrane and disrupting ion fluxes. Immunity or resistance to the toxins is conferred by the preprotoxins (or products thereof) or by nuclear resistance genes. In several cases, the toxins are encoded by one or more genomic segments of resident double-stranded RNA viruses. The known toxins are composed of one to three polypeptides, usually present as multimers. We have further characterized the KP4 killer toxin from the maize smut fungus Ustilago maydis. This toxin is also encoded by a single viral double-stranded RNA but differs from other known killer toxins in several respects: it has no N-linked glycosylation either in the precursor or in the mature polypeptide, it is the first killer toxin demonstrated to be a single polypeptide, and it is not processed by any of the known secretory proteinases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.

A closely related group of RNA-dependent RNA polymerases from double-stranded RNA viruses.

Probably one of the first proteinaceous enzymes was an RNA-dependent RNA polymerase (RDRP). Although there are several conserved motifs present in the RDRPs of most positive and double-stranded RNA (dsRNA) viruses, the RDRPs of the dsRNA viruses show no detectable sequence similarity outside the conserved motifs. There is now, however, a group of dsRNA viruses of lower eucaryotes whose RDRPs are detectably similar. The origin of this sequence similarity appears to be common descent from one or more noninfectious viruses of a progenitor cell, an origin that predates the differentiation of protozoans and fungi. The cause of this preservation of sequence appears to be constraints placed on the RDRP by the life-style of these viruses--the maintenance of a stable, persistent, noninfectious state.

RNA structural requirements for RNA binding, replication, and packaging in the yeast double-stranded RNA virus.

Several sites of interaction with viral proteins have been mapped in the plus strand of the Saccharomyces cerevisiae double-stranded RNA virus, ScV. These include a site necessary and sufficient for viral particle binding to plus strands (VBS) and a site necessary and sufficient for interference with replication of viral plus strands (INS). We show that the INS and VBS are identical and that they are necessary and sufficient for packaging. One of the viral RNAs has two adjacent VBS, which have additive INS activity. The second VBS has similar affinity for viral particles as the first but its complex with particles exhibits faster dissociation. Binding to the two sites in the viral RNA is independent but equivalent. This may mean two recognition sites per particle. This is what would be expected for two molecules of the protein thought to be responsible for sequence specific recognition, the cap-pol fusion polypeptide, per particle. Comparison of the secondary structures of several binding sites, as determined by site-directed mutagenesis and by ribonuclease mapping of single- and double-stranded regions, reveals a requirement for a specific loop sequence and two stem regions, in which there is no sequence specificity. Contrary to what was described in previous work, a bulge loop, but not a "bulged" A residue is necessary for binding of the second VBS. A minimal region of 30 bases is enough to cause both packaging and replication.

A family of Ustilago maydis expression vectors: new selectable markers and promoters.

The cDNA expression system for Ustilago maydis has been expanded to include different selectable markers and promoters. These new elements allow the simultaneous expression of two genes in the same transformant.