Emerging Variants of Canine Enteric Coronavirus Associated with Outbreaks of Gastroenteric Disease.

A 2022 canine gastroenteritis outbreak in the United Kingdom was associated with circulation of a new canine enteric coronavirus closely related to a 2020 variant with an additional spike gene recombination. The variants are unrelated to canine enteric coronavirus-like viruses associated with human disease but represent a model for coronavirus population adaptation.

A reduced potential for lameness bacterial transmission by Lucilia sericata larvae and flies through metamorphosis.

Lameness in sheep is one of the most serious issues on farms in the UK and worldwide, affecting over 90% of all UK sheep flocks. Despite its severity and prevalence, there are knowledge gaps regarding transmission routes of bacterial pathogens associated with infectious lameness in sheep. As larvae of Lucilia sericata are commonly found on foot lesions on lame sheep, it was hypothesised that the flies or their larvae could harbour lameness associated bacteria. This study examined the gut contents of larvae obtained from the foot lesions of lame sheep and compared them to control larvae collected from infested cat food on the same farm. Of particular interest, were the presence of three different bacterial genera associated with lameness; Fusobacterium necrophorum, Dichelobacter nodosus and Treponema spp., for which viability was also investigated. Larvae were cultured In vitro and some allowed to metamorphose into flies before specific PCR assays were carried out on the gut contents. Results showed a significant association between the bacteria on the feet of the sheep and those within the larvae. Although the gut contents of all larvae found on sheep feet contained one or more of the lameness bacteria, none of the bacteria were recovered from the adult flies, suggesting a level of gut remodelling during metamorphosis. Interestingly, Treponema spp. and Fusobacterium spp. were viable when isolated from gut contents of larvae. Maintenance of infection from larvae to fly did not occur. However, it still remains important to control both disease and insect populations of farms to maintain animal welfare.

Edible flowers of Helichrysum italicum: Composition, nutritive value, and bioactivities.

Helichrysum italicum (H. italicum) is a halophyte shrub with bright yellow flowers with a strong curry-like aroma. The essential oils of H. italicum have been used in the production of cosmetics and pharmaceuticals, due to their antiallergic and anti-inflammatory properties. In the agri-food sector, H. italicum flowers can be used for seasoning and flavoring food, and as natural food preservatives. Here, we report on the composition, bioactive compounds, and nutritive value of H. italicum flowers. Flowers were mainly composed of carbohydrates (>80 % dry weight), followed by minerals (6.31 ± 0.95 % dw), protein (5.44 ± 0.35 % dw), and lipids (3.59 % ± 0.53 % dw). High percentages of Fe, Zn, Ca, and K were found in the flower material, along with a high content in antioxidants, polyphenols, and carotenoids, as corroborated by the nuclear magnetic resonance (NMR) data. Flowers were mainly composed of saturated fatty acids (SFAs) (54.50 ± 0.95 % of total FA), followed by polyunsaturated fatty acids (PUFAs) (37.73 ± 1.25 % of total FA) and monounsaturated fatty acids (MUFAs) (7.77 ± 0.34 %), as detected by gas chromatography mass spectrometry (GC-MS). The omega-6 PUFA linoleic acid (22.55 ± 0.76 % of total FA) was the most abundant fatty acid found. Flower extracts showed antimicrobial activity against Saccharomyces cerevisiae and Komagataella phaffii, as well as against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus) bacteria. H. italicum flower material was nontoxic to human intestinal Caco-2 model cells at concentrations up to 1.0 % w/v.

Hsp90 and phosphorylation of the Slt2(Mpk1) MAP kinase activation loop are essential for catalytic, but not non-catalytic, Slt2-mediated transcription in yeast.

In yeast, the Slt2(Mpk1) stress-activated protein kinase directs the activation of two transcription factors, Rlm1 and Swi4/Swi6, in response to cell wall stress. Rlm1 is activated through a phosphorylation by Slt2, whereas the Swi4/Swi6 activation is noncatalytic and triggered by the binding of phosphorylated forms of both Slt2 and a catalytically inactive pseudokinase (Mlp1). Previous studies have delineated a role for the molecular chaperone Hsp90 in the activation of Slt2, but the involvement of Hsp90 in these events of catalytic versus non-catalytic cell integrity signaling has remained elusive. In cells lacking Mlp1, the Hsp90 inhibitor radicicol was found to inhibit the Slt2-mediated catalytic activation of Rlm1, but not the noncatalytic activation of Swi4/Swi6. Mutation of residues in the TEY motif of the Slt2 activation loop strongly impacted both Hsp90 binding and Rlm1-mediated transcription. In contrast, many of these same mutations had only modest effects on Swi4/6 (Slt2-mediated, non-catalytic) transcription, although one that blocked both the Slt2:Hsp90 interaction and Rlm1-mediated transcription (E191G) triggered a hyperactivation of Swi4/6. Taken together, our results cement the importance of the Slt2 activation loop for both the binding of Hsp90 by Slt2 and the catalytic activation of cell integrity signaling.

UCS protein function is partially restored in the Saccharomyces cerevisiae she4 mutant with expression of the human UNC45-GC, but not UNC45-SM.

A dedicated UNC45, Cro1, She4 (UCS) domain-containing protein assists in the Hsp90-mediated folding of the myosin head. Only weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and striated muscle UNC45s; UNC45-GC and UNC45-SM, respectively). In vertebrates, UNC45-GC facilitates cytoskeletal functions, whereas the 55% identical UNC45-SM assists assembly of the contractile apparatus of cardiac and skeletal muscles. A Saccharomyces cerevisiae she4Δ mutant, totally lacking any UCS protein, was engineered to express as its sole Hsp90 either the Hsp90α or the Hsp90ß isoforms of human cytosolic Hsp90. A transient induction of the human UNC45-GC, but not UNC45-SM, could rescue the defective endocytosis in these she4Δ cells at 39 °C, irrespective of whether they possessed Hsp90α or Hsp90ß. UNC45-GC-mediated rescue of the localisation of a Myo5-green fluorescent protein (GFP) fusion to cortical patches at 39 °C was more efficient in the yeast containing Hsp90α, though this may relate to more efficient functioning of Hsp90α as compared to Hsp90ß in these strains. Furthermore, inducible expression of UNC45-GC, but not UNC45-SM, could partially rescue survival at a more extreme temperature (45 °C) that normally causes she4Δ mutant yeast cells to lyse. The results indicate that UCS protein function has been most conserved-yeast to man-in the UNC45-GC, not UNC45-SM. This may reflect UNC45-GC being the vertebrate UCS protein that assists formation of the actomyosin complexes needed for cytokinesis, cell morphological change, and organelle trafficking-events also facilitated by the myosins in yeast.

The crystal structure of the Sgt1-Skp1 complex: the link between Hsp90 and both SCF E3 ubiquitin ligases and kinetochores.

The essential cochaperone Sgt1 recruits Hsp90 chaperone activity to a range of cellular factors including SCF E3 ubiquitin ligases and the kinetochore in eukaryotes. In these pathways Sgt1 interacts with Skp1, a small protein that heterodimerizes with proteins containing the F-box motif. We have determined the crystal structure of the interacting domains of Saccharomyces cerevisiae Sgt1 and Skp1 at 2.8 Å resolution and validated the interface in the context of the full-length proteins in solution. The BTB/POZ domain of Skp1 associates with Sgt1 via the concave surface of its TPR domain using residues that are conserved in humans. Dimerization of yeast Sgt1 occurs via an insertion that is absent from monomeric human Sgt1. We identify point mutations that disrupt dimerization and Skp1 binding in vitro and find that the interaction with Skp1 is an essential function of Sgt1 in yeast. Our data provide a structural rationale for understanding the phenotypes of temperature-sensitive Sgt1 mutants and for linking Skp1-associated proteins to Hsp90-dependent pathways.

Mutation of the Ser18 phosphorylation site on the sole Saccharomyces cerevisiae UCS protein, She4, can compromise high-temperature survival.

Folding of the myosin head often requires the joint actions of Hsp90 and a dedicated UNC45, Cro1, She4 (UCS) domain-containing cochaperone protein. Relatively weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and smooth muscle UNC45s; UNC45-GC and UNC45-SM respectively). In vertebrates, UNC45-GC facilitates cytoskeletal function whereas the 55% identical UNC45-SM assists in the assembly of the contractile apparatus of cardiac and skeletal muscles. UNC45-SM, unlike UNC45-GC, shares with yeast She4 an IDSL sequence motif known to be a site of in vivo serine phosphorylation in yeast. Investigating this further, we found that both a non-phosphorylatable (S18A) and a phosphomimetic (S18E) mutant form of She4 could rescue the type 1 myosin localisation and endocytosis defects of the yeast she4Δ mutant at 39 °C. Nevertheless, at higher temperature (45 °C), only She4 (S18A), not She4(S18E), could substantially rescue the cell lysis defect of she4Δ mutant cells. In the yeast two-hybrid system, the non-phosphorylatable S18A and S251A mutant forms of She4 and UNC45-SM still displayed the stress-enhanced in vivo interaction with Hsp90 seen with the wild-type She4 and UNC45-SM. Such high-temperature enforcement to interaction was though lost with the phosphomimetic mutant forms (She4(S18E) and UNC45-SM (S251E)), an indication that phosphorylation might suppress these increases in She4/Hsp90 and UNC45-SM/Hsp90 interaction with stress.

Insights from yeast into whether the inhibition of heat shock transcription factor (Hsf1) by rapamycin can prevent the Hsf1 activation that results from treatment with an Hsp90 inhibitor.

UNLABELLED: In human cells TORC1 mTOR (target of rapamycin) protein kinase complex renders heat shock transcription factor 1 (Hsf1) competent for stress activation. In such cells, as well as in yeast, the selective TORC1 inhibitor rapamycin blocks this activation in contrast to Hsp90 inhibitors which potently activate Hsf1. Potentially therefore rapamycin could prevent the Hsf1 activation that frequently compromises the efficiency of Hsp90 inhibitor cancer drugs. Little synergy was found between the effects of rapamycin and the Hsp90 inhibitor radicicol on yeast growth. However certain rapamycin resistance mutations sensitised yeast to Hsp90 inhibitor treatment and an Hsp90 mutation that overactivates Hsf1 sensitised cells to rapamycin. Rapamycin inhibition of the yeast Hsf1 was abolished by this Hsp90 mutation, as well as with the loss of Ppt1, the Hsp90-interacting protein phosphatase that is the ortholog of mammalian PP5. Unexpectedly Hsf1 activation was found to have a requirement for the rapamycin binding immunophilin FKBP12 even in the absence of rapamycin, while TORC1 "bypass" strains revealed that the rapamycin inhibition of yeast Hsf1 is not exerted through two of the major downstream targets of TORC1, the protein phosphatase regulator Tap42 and the protein kinase Sch9--the latter the ortholog of human S6 protein kinase 1. SIGNIFICANCE: A problem with most of the Hsp90 inhibitor drugs now in cancer clinic trials is that they potently activate Hsf1. This leads to an induction of heat shock proteins, many of which have a "pro-survival" role in that they help to protect cells from apopotosis. As the activation of Hsf1 requires TORC1, inhibitors of mTOR kinase could potentially block this activation of Hsf1 and be of value when used in combination drug therapies with Hsp90 inhibitors. However many of the mechanistic details of the TORC1 regulation of Hsf1, as well as the interplay between cellular resistances to rapamycin and to Hsp90 inhibitors, still remain to be resolved.

Spotlight on the microbes that produce heat shock protein 90-targeting antibiotics.

Heat shock protein 90 (Hsp90) is a promising cancer drug target as a molecular chaperone critical for stabilization and activation of several of the oncoproteins that drive cancer progression. Its actions depend upon its essential ATPase, an activity fortuitously inhibited with a very high degree of selectivity by natural antibiotics: notably the actinomycete-derived benzoquinone ansamycins (e.g. geldanamycin) and certain fungal-derived resorcyclic acid lactones (e.g. radicicol). The molecular interactions made by these antibiotics when bound within the ADP/ATP-binding site of Hsp90 have served as templates for the development of several synthetic Hsp90 inhibitor drugs. Much attention now focuses on the clinical trials of these drugs. However, because microbes have evolved antibiotics to target Hsp90, it is probable that they often exploit Hsp90 inhibition when interacting with each other and with plants. Fungi known to produce Hsp90 inhibitors include mycoparasitic, as well as plant-pathogenic, endophytic and mycorrhizal species. The Hsp90 chaperone may, therefore, be a prominent target in establishing a number of mycoparasitic (interfungal), fungal pathogen-plant and symbiotic fungus-plant relationships. Furthermore the Hsp90 family proteins of the microbes that produce Hsp90 inhibitor antibiotics are able to reveal how drug resistance can arise by amino acid changes in the highly conserved ADP/ATP-binding site of Hsp90.

Features of the Streptomyces hygroscopicus HtpG reveal how partial geldanamycin resistance can arise with mutation to the ATP binding pocket of a eukaryotic Hsp90.

Much attention is focused on the benzoquinone ansamycins as anticancer agents, with several derivatives of the natural product geldanamycin (GdA) now in clinical trials. These drugs are selective inhibitors of Hsp90, a molecular chaperone vital for many of the activities that drive cancer progression. Mutational changes to their interaction site, the extremely conserved ATP binding site of Hsp90, would mostly be predicted to inactivate the chaperone. As a result, drug resistance should not arise readily this way. Nevertheless, Streptomyces hygroscopicus, the actinomycete that produces GdA, has evolved an Hsp90 family protein (HtpG) that lacks GdA binding. It is altered in certain of the highly conserved amino acids making contacts to this antibiotic in crystal structures of GdA bound to eukaryotic forms of Hsp90. Two of these amino acid changes, located on one side of the nucleotide-binding cleft, weakened GdA/Hsp90 binding and conferred partial GdA resistance when inserted into the endogenous Hsp90 of yeast cells. Crystal structures revealed their main effect to be a weakening of interactions with the C-12 methoxy group of the GdA ansamycin ring. This is the first study to demonstrate that partial GdA resistance is possible by mutation within the ATP binding pocket of Hsp90.

Mechanisms of Resistance to Hsp90 Inhibitor Drugs: A Complex Mosaic Emerges.

The molecular chaperone Hsp90 holds great promise as a cancer drug target, despite some of the initial clinical trials of Hsp90 inhibitor drugs having not lived up to expectation. Effective use of these drugs will benefit greatly from a much more detailed understanding of the factors that contribute to resistance, whether intrinsic or acquired. We review how cell culture studies have revealed a number of different mechanisms whereby cells can be rendered less susceptible to the effects of Hsp90 inhibitor treatment. A major influence is Hsp90 inhibition causing strong induction of the heat shock response, a stress response that increases cellular levels of prosurvival chaperones such as Hsp27 and Hsp70. Another problem seems to be that these inhibitors do not always access the Hsp90 proteins of the mitochondrion, forms of Hsp90 that-in cancer cells-are operating to suppress apoptosis. It should be possible to overcome these drawbacks through the appropriate drug redesign or with the combinatorial use of an Hsp90 inhibitor with a drug that targets either heat shock factor or the chaperone Hsp70. Still though, cells will often differ in the key antiapoptotic versus proapoptotic activities that are dependent on Hsp90, in the key steps in their apoptotic pathways responsive to Hsp90 inhibition or Hsp70 level, as well as the extents to which their survival is dependent on oncogenic tyrosine kinases that are clients of Hsp90. A systems approach will therefore often be required in order to establish the most prominent effects of Hsp90 inhibition in each type of cancer cell.

A simple yeast-based system for analyzing inhibitor resistance in the human cancer drug targets Hsp90alpha/beta.

Heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, is one of the most promising targets for cancer drug development. Whether any resistance to these Hsp90 inhibitor drugs could arise by Hsp90 mutation is still unknown. Yeast is readily engineered so that its essential Hsp90 function is provided by either isoform of the human cytosolic Hsp90, Hsp90alpha or Hsp90beta. However, its high intrinsic resistance to most drugs poses a major obstacle to the use of such Hsp90alpha- or Hsp90beta-expressing yeast cells as a model system to analyse whether drug resistance might arise by Hsp90 mutation. In order to overcome this problem, we have generated a strain that is both hypersensitive to Hsp90 inhibitors as it lacks multiple drug resistance genes, and in which different heterologous and mutant Hsp90s can be expressed by plasmid exchange. It is not rendered appreciably stress sensitive when made to express Hsp90alpha or Hsp90beta as its sole form of Hsp90. Should there be any development of resistance to the Hsp90 drugs now in cancer clinic trials, this system can provide a rapid initial test of whether any single nucleotide polymorphism appearing within the coding regions of Hsp90alpha or Hsp90beta could be a contributory factor in this resistance. We have used this strain to demonstrate that significant levels of resistance to the Hsp90 inhibitors radicicol and 17-allylamino-demethoxygeldanamycin (17-AAG) are generated as a result of the same single point mutation within the native Hsp90 of yeast (A107N), the human Hsp90alpha (A121N) and the human Hsp90beta (A116N).

The Hsp90/Cdc37p chaperone system is a determinant of molybdate resistance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae lacks enzymes that contain the molybdopterin co-factor and therefore any requirement for molybdenum as a trace mineral supplement. Instead, high molybdate levels are inhibitory to its growth. Low cellular levels of heat shock protein 90 (Hsp90), an essential chaperone, were found to enhance this sensitivity to molybdate. Certain Hsp90 point mutations and co-chaperone protein defects that partially compromise the function of the Hsp90/Cdc37p chaperone system also rendered S. cerevisiae hypersensitive to high molybdate levels. Sensitivity was especially apparent with mutations close to the Hsp90 nucleotide binding site, with the loss of the non-essential co-chaperone Sti1p (the equivalent of mammalian Hop), and with the abolition of residue Ser14 phosphorylation on the essential co-chaperone Cdc37p. While it remains to be proved that these effects reflect direct inhibition of the Hsp90 of the cell by the MoO(4) (2+) oxyanion in vivo; this possibility is suggested by molybdate sensitivity arising with a mutation in the Hsp90 nucleotide binding site that does not generate stress sensitivity or an impaired stress response. Molybdate sensitivity may therefore be a useful phenotype to score when studying mutations in this chaperone system.

Structural basis of the radicicol resistance displayed by a fungal hsp90.

Heat shock protein 90 (Hsp90) is a promising cancer drug target, as multiple oncogenic proteins are destabilized simultaneously when it loses its activity in tumor cells. Highly selective Hsp90 inhibitors, including the natural antibiotics geldanamycin (GdA) and radicicol (RAD), inactivate this essential molecular chaperone by occupying its nucleotide binding site. Often cancer drug therapy is compromised by the development of resistance, but a resistance to these Hsp90 inhibitors should not arise readily by mutation of those amino acids within Hsp90 that facilitate inhibitor binding, as these are required for the essential ATP binding/ATPase steps of the chaperone cycle and are tightly conserved. Despite this, the Hsp90 of a RAD-producing fungus is shown to possess an unusually low binding affinity for RAD but not GdA. Within its nucleotide binding site a normally conserved leucine is replaced by isoleucine, though the chaperone ATPase activity is not severely affected. Inserted into the Hsp90 of yeast, this conservative leucine to isoleucine substitution recreated this lowered affinity for RAD in vitro. It also generated a substantially enhanced resistance to RAD in vivo. Co-crystal structures reveal that the change to isoleucine is associated with a localized increase in the hydration of an Hsp90-bound RAD but not GdA. To the best of our knowledge, this is the first demonstration that it is possible for Hsp90 inhibitor resistance to arise by subtle alteration to the structure of Hsp90 itself.

Chaperone ligand-discrimination by the TPR-domain protein Tah1.

Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

Expressed as the sole Hsp90 of yeast, the alpha and beta isoforms of human Hsp90 differ with regard to their capacities for activation of certain client proteins, whereas only Hsp90beta generates sensitivity to the Hsp90 inhibitor radicicol.

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the activity of many of the most important regulatory proteins of eukaryotic cells (the Hsp90 'clients'). Vertebrates have two isoforms of cytosolic Hsp90, Hsp90alpha and Hsp90beta. Hsp90beta is expressed constitutively to a high level in most tissues and is generally more abundant than Hsp90alpha, whereas Hsp90alpha is stress-inducible and overexpressed in many cancerous cells. Expressed as the sole Hsp90 of yeast, human Hsp90alpha and Hsp90beta are both able to provide essential Hsp90 functions. Activations of certain Hsp90 clients (heat shock transcription factor, v-src) were more efficient with Hsp90alpha, rather than Hsp90beta, present in the yeast. In contrast, activation of certain other clients (glucocorticoid receptor; extracellular signal-regulated kinase-5 mitogen-activated protein kinase) was less affected by the human Hsp90 isoform present in these cells. Remarkably, whereas expression of Hsp90beta as the sole Hsp90 of yeast rendered cells highly sensitive to the Hsp90 inhibitor radicicol, comparable expression of Hsp90alpha did not. This raises the distinct possibility that, also for mammalian systems, alterations to the Hsp90alpha/Hsp90beta ratio (as with heat shock) might be a significant factor affecting cellular susceptibility to Hsp90 inhibitors.

Multiple dextranases from the yeast Lipomyces starkeyi.

The soil yeast Lipomyces starkeyi (NCYC 1436) secretes dextranase activity into the growth medium. Resolution of a dextranase-active protein fraction by SDS-PAGE produced three protein bands, of 66 kDa, 68 kDa and 78 kDa, and isoelectric focusing of the same fraction resulted in seven protein bands, of pIs 3.50, 3.85, 4.20, 4.80, 4.85, 5.00 and 5.30. Dextranase activity was demonstrated for all the isoelectric forms, and for the 78 kDa species in the presence of SDS. Amino acid compositions of the 66 kDa, 68 kDa and 78 kDa protein bands were determined, and the N-termini of the 66 kDa and 78 kDa protein bands were sequenced: the first two amino acids at the N-terminus of each protein were alanine and valine, respectively; an alanine-valine pair is seen early in the N-terminal coding sequences of the dextranases and the isopullulanase produced by the phylogenetically disparate organisms contributing to glycosyl hydrolase family 49.

In the yeast heat shock response, Hsf1-directed induction of Hsp90 facilitates the activation of the Slt2 (Mpk1) mitogen-activated protein kinase required for cell integrity.

Yeast is rendered temperature sensitive with loss of the C-terminal (CT) domain of heat shock transcription factor (Hsf1). This domain loss was found to abrogate heat stimulation of Slt2 (Mpk1), the mitogen-activated protein kinase that directs the reinforced cell integrity gene expression needed for high-temperature growth. In Hsf1 CT domain-deficient cells, Slt2 still undergoes Mkk1/2-directed dual-Thr/Tyr phosphorylation in response to the heat stimulation of cell integrity pathway signaling, but the low Hsp90 expression level suppresses any corresponding increase in Slt2 kinase activity due to Slt2 being a "client" of the Hsp90 chaperone. A non-Hsf1-directed Hsp90 overexpression restored the heat induction of Slt2 activity in these cells, as well as both Slt2-dependent (Rlm1, Swi4) and Slt2-independent (MBF) transcriptional activities. Their high-temperature growth was also rescued, not just by this Hsp90 overexpression but by osmotic stabilization, by the expression of a Slt2-independent form of the Rlm1 transcriptional regulator of cell integrity genes, and by a multicopy SLT2 gene vector. In providing the elevated Hsp90 needed for an efficient activation of Slt2, heat activation of Hsf1 indirectly facilitates (Slt2-directed) heat activation of yet another transcription factor (Rlm1). This provides an explanation as to why, in earlier transcript analysis compared to chromatin immunoprecipitation studies, many more genes of yeast displayed an Hsf1-dependent transcriptional activation by heat than bound Hsf1 directly. The levels of Hsp90 expression affecting transcription factor regulation by Hsp90 client protein kinases also provides a mechanistic model for how heat shock factor can influence the expression of several non-hsp genes in higher organisms.

Expressed in the yeast Saccharomyces cerevisiae, human ERK5 is a client of the Hsp90 chaperone that complements loss of the Slt2p (Mpk1p) cell integrity stress-activated protein kinase.

ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2Delta Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2Delta yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5.

Cloning and expression of Penicillium minioluteum dextranase in Saccharomyces cerevisiae and its exploitation as a reporter in the detection of mycotoxins.

A dextranase gene from Penicillium minioluteum (strain IMI068219) has been cloned, sequenced and expressed in Saccharomyces cerevisiae via fusion of the DNA segment encoding the mature dextranase protein with alpha-factor signal sequence, and insertion into the GAL1-controlled expression vector pYES2/CT. Galactose-induced expression yielded extracellular dextranase activity of 0.63 units/ml and cell-associated dextranase activity of 0.48 units/ml, after 24 h incubation. The dextranase construct was introduced into a strain of S. cerevisiae expressing the human cytochrome P450 3A4 (CYP3A4) and the cognate reductase, which was then used to develop a microplate toxicity bioassay. Toxicity was signalled as inhibition of dextranase activity, assayed fluorimetrically. This novel bioassay was assessed using six economically significant mycotoxins.