Devis Sinani

Distinct Mechanisms for Ctr1-mediated Copper and Cisplatin Transport

The Ctr1 family of integral membrane proteins is necessary for high affinity copper uptake in eukaryotes. Ctr1 is also involved in cellular accumulation of cisplatin, a platinum-based anticancer drug. Although the physiological role of Ctr1 has been revealed, the mechanism of action of Ctr1 remains to be elucidated. To gain a better understanding of Ctr1-mediated copper and cisplatin transport, we have monitored molecular dynamics and transport activities of yeast Saccharomyces cerevisiae Ctr1 and its mutant alleles. Co-expression of functional Ctr1 monomers fused with either cyan or yellow fluorescent protein resulted in fluorescence resonance energy transfer (FRET), which is consistent with multimer assembly of Ctr1. Copper near the Km value of Ctr1 enhanced FRET in a manner that correlated with cellular copper transport. In vitro cross-linking of Ctr1 confirmed that copper-induced FRET reflects conformational changes within pre-existing Ctr1 complexes. FRET assays in membrane-disrupted cells and protein extracts showed that intact cell structure is necessary for Ctr1 activity. Despite Ctr1-dependent cellular accumulation, cisplatin did not change Ctr1 FRET nor did it attenuate copper-induced FRET. A Ctr1 allele defective in copper transport enhanced cellular cisplatin accumulation. N-terminal methionine-rich motifs that are dispensable for copper transport play a critical role for cisplatin uptake. Taken together, our data reveal functional roles for structural remodeling of the Ctr1 multimeric complex in copper transport and suggest distinct mechanisms employed by Ctr1 for copper and cisplatin transport.

Copper Transport Activity of Yeast Ctr1 Is Down-regulated via Its C Terminus in Response to Excess Copper

Copper is an essential yet toxic trace element. The Ctr1 family of proteins plays a critical role for copper uptake in eukaryotes. However, the mechanisms of action of Ctr1 are largely unknown. Our previous data demonstrated that copper transport induces conformational changes in the cytosolic C terminus of the yeast Saccharomyces cerevisiae Ctr1. To define the physiological significance of this molecular event and gain better insights into the mechanism of Ctr1-mediated copper uptake, we have characterized the functional roles of the Ctr1 C terminus. A Ctr1 mutant lacking the entire C-terminal cytosolic tail is functional in high affinity copper uptake; however, yeast cells expressing this mutant are extremely sensitive to excess copper. Toxic copper uptake is not attributed to elevated expression or distinct subcellular localization of this mutant as compared with wild type Ctr1. Further characterization of the function of Ctr1 containing deletions or site-directed mutations at the C terminus indicates a structural role for the C terminus in controlling Ctr1 activities. In response to excess copper, Ctr1-mediated copper transport is rapidly blocked in a C terminus-dependent mechanism associated with direct binding of copper. We propose that conformational changes in the cytosolic tail of yeast Ctr1 by copper sensing within this domain lead to the inhibition of Ctr1-mediated copper transport. These data suggest a new regulatory mechanism by which yeast cells maintain homeostatic copper acquisition.

A Cadmium-transporting P1B-type ATPase in Yeast Saccharomyces cerevisiae

Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense mutation in a predicted ATP-binding residue conserved in P1B-type ATPases. Consistent with a previous report that identifies an equivalent mutation in a copper-transporting P1B-type ATPase of a Wilson disease patient, the PCA1 allele found in laboratory yeast strains is nonfunctional. Overexpression or deletion of the functional allele in yeast demonstrates that PCA1 is a cadmium efflux pump. Cadmium as well as copper and silver, but not other metals examined, dramatically increase PCA1 protein expression through post-transcriptional regulation and promote subcellular localization to the plasma membrane. Our study has revealed a novel metal detoxification mechanism in yeast mediated by a P1B-type ATPase that is unique in structure, substrate specificity, and mode of regulation.

Regulation of the latency–reactivation cycle by products encoded by the bovine herpesvirus 1 (BHV-1) latency-related gene

Like other α-herpesvirinae subfamily members, the primary site for bovine herpesvirus 1 (BHV-1) latency is ganglionic sensory neurons. Periodically BHV-1 reactivates from latency, virus is shed, and consequently virus transmission occurs. Transcription from the latency-related (LR) gene is readily detected in neurons of trigeminal ganglia (TG) of calves or rabbits latently infected with BHV-1. Two micro-RNAs and a transcript encompassing a small open reading frame (ORF-E) located within the LR promoter can also be detected in TG of latently infected calves. A BHV-1 mutant that contains stop codons near the beginning of the first open reading frame (ORF2) within the major LR transcript (LR mutant virus) has been characterized. The LR mutant virus does not express ORF2, a reading frame that lacks an initiating ATG (reading frame B), and has reduced expression of ORF1 during productive infection. The LR mutant virus does not reactivate from latency following dexamethasone treatment suggesting that LR protein expression regulates the latency–reactivation cycle. Higher levels of apoptosis occur in TG neurons of calves infected with the LR mutant viruses when compared to wild-type BHV-1 indicating that the anti-apoptotic properties of the LR gene is necessary for the latency–reactivation cycle. ORF2 inhibits apoptosis and regulates certain viral promoters, in part, because it interacts with three cellular transcription factors (C/EBP-alpha, Notch1, and Notch3). Although ORF2 is important for the latency–reactivation cycle, we predict that other LR gene products play a supportive role during life-long latency in cattle.

Cellular Transcription Factors Induced in Trigeminal Ganglia during Dexamethasone-Induced Reactivation from Latency Stimulate Bovine Herpesvirus 1 Productive Infection and Certain Viral Promoters

ABSTRACT Bovine herpesvirus 1 (BHV-1), an alphaherpesvirinae subfamily member, establishes latency in sensory neurons. Elevated corticosteroid levels, due to stress, reproducibly triggers reactivation from latency in the field. A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves consistently induces reactivation from latency. Lytic cycle viral gene expression is detected in sensory neurons within 6 h after DEX treatment of latently infected calves. These observations suggested that DEX stimulated expression of cellular genes leads to lytic cycle viral gene expression and productive infection. In this study, a commercially available assay—Bovine Gene Chip—was used to compare cellular gene expression in the trigeminal ganglia (TG) of calves latently infected with BHV-1 versus DEX-treated animals. Relative to TG prepared from latently infected calves, 11 cellular genes were induced more than 10-fold 3 h after DEX treatment. Pentraxin three, a regulator of innate immunity and neurodegeneration, was stimulated 35- to 63-fold after 3 or 6 h of DEX treatment. Two transcription factors, promyelocytic leukemia zinc finger (PLZF) and Slug were induced more than 15-fold 3 h after DEX treatment. PLZF or Slug stimulated productive infection 20- or 5-fold, respectively, and Slug stimulated the late glycoprotein C promoter more than 10-fold. Additional DEX-induced transcription factors also stimulated productive infection and certain viral promoters. These studies suggest that DEX-inducible cellular transcription factors and/or signaling pathways stimulate lytic cycle viral gene expression, which subsequently leads to successful reactivation from latency in a small subset of latently infected neurons.

A bovine herpesvirus 1 protein expressed in latently infected neurons (ORF2) promotes neurite sprouting in the presence of activated Notch1 or Notch3

ABSTRACT Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can lead to life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia, but stress can induce reactivation from latency. The latency-related (LR) RNA is the only viral transcript abundantly expressed in latently infected sensory neurons. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) is not reactivated from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 inhibits apoptosis in transiently transfected cells, suggesting that it plays a crucial role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and inhibits its ability to trans activate certain viral promoters. Notch3 RNA and protein levels are increased during reactivation from latency, suggesting that Notch may promote reactivation. Activated Notch signaling interferes with neuronal differentiation, in part because neurite and axon generation is blocked. In this study, we demonstrated that ORF2 promotes neurite formation in mouse neuroblastoma cells overexpressing Notch1 or Notch3. ORF2 also interfered with Notch-mediated trans activation of the promoter that regulates the expression of Hairy Enhancer of Split 5, an inhibitor of neurite formation. Additional studies provided evidence that ORF2 promotes the degradation of Notch3, but not that of Notch1, in a proteasome-dependent manner. In summary, these studies suggest that ORF2 promotes a mature neuronal phenotype that enhances the survival of infected neurons and consequently increases the pool of latently infected neurons.

A Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 Interacts with Notch1 and Notch3

ABSTRACT Like other A lphaherpesvirinae subfamily members, bovine herpesvirus 1 (BHV-1) establishes latency in sensory neurons. The latency-related RNA (LR-RNA) is abundantly expressed in latently infected sensory neurons. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) does not reactivate from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 is not the only viral product expressed during latency, but it is important for the latency reactivation cycle because it inhibits apoptosis. In this study, a yeast 2-hybrid screen revealed that ORF2 interacted with two cellular transcription factors, Notch1 and Notch3. These interactions were confirmed in mouse neuroblastoma cells by confocal microscopy and in an in vitro “pulldown” assay. During reactivation from latency, Notch3 RNA levels in trigeminal ganglia were higher than those during latency, suggesting that Notch family members promote reactivation from latency or that reactivation promotes Notch expression. A plasmid expressing the Notch1 intercellular domain (ICD) stimulated productive infection and promoters that encode the viral transcription factor bICP0. The Notch3 ICD did not stimulate productive infection as efficiently as the Notch1 ICD and had no effect on bICP0 promoter activity. Plasmids expressing the Notch1 ICD or the Notch3 ICD trans-activated a late promoter encoding glycoprotein C. ORF2 reduced the trans-activation potential of Notch1 and Notch3, suggesting that ORF2 interfered with the trans-activation potential of Notch. These studies provide evidence that ORF2, in addition to inhibiting apoptosis, has the potential to promote establishment and maintenance of latency by sequestering cellular transcription factors.

Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans-Activation of the bICP0 Promoter

ABSTRACT Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can result in life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia. Periodically, reactivation from latency occurs, resulting in virus transmission. The latency-related (LR) RNA is abundantly expressed in latently infected sensory neurons, suggesting that LR gene products regulate the latency-reactivation cycle. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) does not reactivate from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 inhibits apoptosis in transiently transfected cells, suggesting that it plays an important role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and consequently inhibits their ability to trans-activate the bICP0 early and glycoprotein C promoters. In this study, we identified ORF2 sequences that were necessary for inhibiting cold shock-induced apoptosis or Notch1-mediated trans-activation of the bICP0 early promoter and stimulation of productive infection. Relative to ORF2 sequences necessary for inhibiting apoptosis, distinct domains in ORF2 were important for interfering with Notch1-mediated trans-activation. Five consensus protein kinase A and/or protein kinase C phosphorylation sites within ORF2 regulate the steady-state levels of ORF2 in transfected cells. A nuclear localization signal in ORF2 was necessary for inhibiting Notch1-mediated trans-activation but not apoptosis. In summary, ORF2 has more than one functional domain that regulates its stability and functional properties.

Stress-Induced Cellular Transcription Factors Expressed in Trigeminal Ganglionic Neurons Stimulate the Herpes Simplex Virus 1 ICP0 Promoter

ABSTRACT Alphaherpesvirinae family members can reactivate from latency following stress. The synthetic corticosteroid dexamethasone induces certain cellular transcription factors in murine and bovine trigeminal ganglionic neurons. Three dexamethasone-induced transcription factors, Krüppel-like factor 15, Slug, and SPDEF, stimulated the herpes simplex virus type 1-infected cell protein 0 (ICP0) promoter more than 150-fold. Conversely, other viral promoters (VP16 and ICP4) were not strongly stimulated, suggesting that the ICP0 promoter is preferentially activated by dexamethasone-simulated stress.

ICP27 protein encoded by bovine herpesvirus type 1 (bICP27) interferes with promoter activity of the bovine genes encoding beta interferon 1 (IFN-β1) and IFN-β3

Bovine herpes virus 1 (BHV-1) infection leads to upper respiratory tract infections, conjunctivitis, and the infection predisposes cattle to secondary bacterial infections. The infected cell protein 0 (bICP0) encoded by BHV-1 suppresses antiviral innate immune signaling by interfering with expression of interferon beta (IFN-β). In contrast to humans or mice, cattle contain three IFN-β genes that have distinct transcriptional promoters. We previously cloned and characterized all three bovine IFN-β promoters. In this study, we provide evidence that bICP27; a viral early protein that shuttles between the nucleus and cytoplasm inhibits transcriptional activity of two bovine IFN-β gene promoters (IFN-β1 and IFN-β3). Conversely, the BHV-1 infected cell protein 0 (bICP0) early promoter was not inhibited by bICP27. C-terminal mutants lacking the bICP27 zinc RING finger-like motif did not efficiently inhibit IFN-β3 promoter activity but inhibited IFN-β1 promoter activity as efficiently as wild type bICP27. An N-terminal mutant lacking the nuclear localization signal (NLS) and nucleolar localization signal (NoLS) was localized to the cytoplasm and this mutant had no effect on IFN-β promoter activity. In summary, these studies provided evidence that bICP27 inhibited IFN-β1 and IFN-β3 promoter activity in transiently transfected cells.