Huntington's disease: Dancing in a dish

In a recent landmark paper, the Huntington''s disease (HD) iPSC Consortium reports on the establishment and characterization of a panel of iPSC lines from HD patients, and more importantly, the successful modeling of HD in vitro. In the same issue of Cell Stem Cell, An et al. reports on the successful targeted gene correction of HD in human iPSCs. Both advances are exciting, provide new resources for current and future HD research, and uncover new challenges to better understand and, most importantly, treat this devastating disease in the near future.Modeling human diseases using induced pluripotent stem cells (iPSCs) has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. Combined with advanced gene correction technology, human iPSCs hold great promise to provide patient-specific and mutation-free cells for potential cell replacement therapy. Huntington''s disease (HD) is an autosomal dominant neurodegenerative disorder, which causes motor dysfunction, psychiatric disturbances and cognitive impairment¹. HD is caused by an expanded cystosine adenine guanine (CAG) tri-nucleotide repeat encoding polyglutamine in the first exon of the Huntingtin (HTT) gene. To date, there is no effective therapy for preventing the onset or slowdown of this disorder. Preliminary clinical trials using fetal neural grafts had shown long-lasting functional benefits in patients². Though only effective in limited cases, these results suggest that cell-based therapy could be a potential treatment if a reliable and consistent cell source is available. For this purpose, an alternative cell source to overcome the logistical and biological hurdles of this disease had been actively explored in the past decade. With recent advancement in human iPSCs technology, HD patient-specific iPSCs coupled with an efficient directed cell differentiation protocol offers hope for an unlimited supply of autologous cells. Since HD is a monogenic disease, with a very well-established correlation between the number of CAG repeats and the age of disease onset, it provides an ideal target for iPSC-based gene correction that will allow for the production of disease-free cells for potential autologous cell therapy, and at the same time provide a much needed, valuable platform to further study the pathogenesis of the disease^3,4.This is in fact what has been recently accomplished in two reports published in Cell Stem Cell^5,6. The HD iPSC Consortium reports on the generation of HD patient-specific iPSC lines that showed CAG-repeat-expansion-associated phenotypes⁵. The study from An et al.⁶ reports on the successful targeted correction of expanded CAG repeat in HD patient iPSCs and the reversion of disease phenotypes.In the study reported from the HD iPSC Consortium, the authors generated 14 iPSC lines from HD patients and controls (listed in Open in a separate window

Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.Classically, transducing phages use the pac site-headful system for DNA packaging. Packaging is initiated on concatemeric post-replicative DNA by terminase cleavage at the sequence-specific pac site, a genome slightly longer than unit length is packaged, and packaging is completed by non-sequence-specific cleavage (reviewed in Rao and Feiss, 2008). Generalized transduction results from the initiation of packaging at pac site homologs in host chromosomal or plasmid DNA, and typically represents ∼1% of the total number of phage particles. In the alternative cos site mechanism packaging is also initiated on concatemeric post-replicative DNA by terminase cleavage at a sequence-specific (cos) site. Here, however, packaging is completed by terminase cleavage at the next cos site, generating a precise monomer with the cohesive termini used for subsequent circularization (Rao and Feiss, 2008). Although cos site homologs may exist in host DNA, it is exceedingly rare that two such sites would be appropriately spaced. Consequently, cos phages, of which lambda is the prototype, do not engage in generalized transduction. For this reason, cos-site phages have been preferred for possible phage therapy, since they would not introduce adventitious host DNA into target organisms.The Staphylococcus aureus pathogenicity islands (SaPIs) are the best-characterized members of the phage-inducible chromosomal island family of mobile genetic elements (MGEs; Novick et al., 2010). SaPIs are ∼15 kb mobile elements that encode virulence factors and are parasitic on specific temperate (helper) phages. Helper phage proteins are required to lift their repression (Tormo-Más et al., 2010, 2013), thereby initiating their excision, circularization and replication. Phage-induced lysis releases vast numbers of infectious SaPI particles, resulting in high frequencies of transfer. Most SaPI helper phages identified to date are pac phages, and many well-studied SaPIs are packaged by the headful mechanism (Ruzin et al., 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos sequences in their genomes, and can be efficiently packaged and transferred by cos phages to S. aureus strains at high frequencies (Quiles-Puchalt et al., 2014). Here we show that this transfer extends to non-aureus staphylococci and to Listeria monocytogenes.Since the pac phages transfer SaPIs to non-aureus staphylococci and to the Gram-positive pathogen Listeria monocytogenes (Maiques et al., 2007; Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and intergeneric transfer. We tested this with SaPIbov5, into which we had previously inserted a tetracycline resistance (tetM) marker to enable selection, and with lysogens of two helper cos phages, φ12 and φSLT, carrying SaPIbov5 (strains JP11010 and JP11194, respectively; Supplementary Table 1). The prophages in these strains were induced with mitomycin C, and the resulting lysates were adjusted to 1 μg ml⁻¹ DNase I and RNase A, filter sterilized (0.2 μm pore), and tested for SaPI transfer with tetracycline selection, as previously described (Ubeda et al., 2008). To test for trans-specific or trans-generic transduction, coagulase-negative staphylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, respectively, as previously described (Maiques et al., 2007; Chen and Novick, 2009). As shown in Figure 1 and Supplementary Table 2). In contrast, deletion of the SaPIbov5 cos site (strains JP11229 and JP11230) did not affect SaPI replication (Supplementary Figure 1), but completely eliminated SaPIbov5 transfer (Supplementary Table 2). The TerS protein is essential for φ12 and SaPIbov5 DNA packaging, but not for phage-mediated lysis (Quiles-Puchalt et al., 2014). As expected, this mutation abolished SaPIbov5 transfer (Open in a separate windowFigure 1(a) Map of SaPIbov5. Arrows represent the localization and orientation of ORFs greater than 50 amino acids in length. Rectangles represent the position of the ori (in purple) or cos (in red) sites. Positions of different primers described in the text are shown. (b) Amplimers generated for detection of SaPIbov5 in the different recipient strains. Supplementary Table 2 lists the sequence of the different primers used. The element was detected in S. epidermidis JP829 (Se-1), S. epidermidis JP830 (Se-2), L. monocytogenes SK1351 (Lm-1), L. monocytogenes EGDe (Lm-2), S. xylosus C2a (Sx) and S. aureus JP4226 (Sa).

Table 1

Intra- and intergeneric SaPIbov5 transfer^a

EU-LIFE revives funding debate: A group of mid-level life science research institutes is reopening the debate on how to fund research at the EU level calling for a stronger emphasis on excellence

EU-LIFE, which represents 10 European life science research institutes, has reopened the debate about how to fund research at the European level by calling for the budget of the European Research Council to be drastically increased.For more than a decade, European scientists have lobbied policy makers in Brussels to increase European Union (EU) funding for research and to spend the money they do provide more efficiently. This debate eventually led to the establishment of the European Research Council (ERC) in 2007, which provides significant grants and does so on the sole criterion of scientific excellence—something for which the scientific community pushed. As such, there seemed to be consensus about how to judge and fund science at the European level, including in the debate about the EU''s Horizon 2020 funding scheme—the EU''s framework for research and innovation—which will spend €80 billion over the next seven years (2014–2020). The conclusion seemed to be that the ERC should continue to support basic research on the basis of excellence, whereas other parts of the programme would focus on large cooperative projects, improving the competitiveness of Europe and meeting societal challenges such as climate change and public health.But a new body called EU-LIFE—set up in May 2013—has reopened the debate about how to fund science and is campaigning for a greater focus on rewarding excellence, even at the expense of funding projects on the grounds of fairness or to correct imbalances between EU member states. EU-LIFE was founded by 10 institutions including the Centre for Genomic Regulation (CRG; Barcelona, Spain), the Institut Curie (Paris, France) and the Max Delbrück Centre (Berlin, Germany), partly to provide a collective voice for mid-sized research institutes in the life sciences that might lack influence on their own (

The roads and bridges of science. Research infrastructures are key components of Europe's future research, but their funding is not guaranteed

Research infrastructures are a crucial component of modern biological research, but the EU has not yet figured out how to fund and maintain them.The development of recombinant gene technology in the 1970s heralded a new era of application-oriented research for molecular biology, with a huge economic impact. During the decades that have followed, biological research and development have become a major enterprise, with an increasing demand for sophisticated technologies, databases, tissue banks and other tools that range from microscopes and DNA sequencers to bioinformatics services and mutant collections. Biology has followed in the footsteps of physics and astronomy, which share costly instrumentation such as particle accelerators, observatories and satellites. A key difference is that biological research infrastructures are often distributed across several sites and are less costly to establish. Nevertheless, they are expensive to operate and maintain, and need long-term financial support.There is no doubt among scientists that research infrastructures are essential for biomedicine and the life sciencesThe European Union (EU) regards biomedical research as an important component of its future economic and social development as part of its ''Innovation Union'' strategy (EC, 2010), but the necessary creation and operation of research infrastructures is not keeping pace. European biologists have been highlighting the problem for years (van Dyck, 2005), to the effect that some pan-European infrastructures for biomedical research and the life sciences have been created, such as the European Bioinformatics Institute (EBI; Hinxton, UK). The European Commission (EC) also established the European Strategy Forum on Research Infrastructures (ESFRI) in 2002, to define the infrastructures required for international research (ESFRI, 2006, 2011). However, most of the planned projects for the biomedical and life sciences (ESFRI, 2011)

A new HCV mouse model on the block

The investigation of virus-induced liver disease and hepatocellular carcinoma needs small animal models modeling hepatitis C virus (HCV) infection and liver disease biology. A recent study published in Cell Research reports a novel mouse model which is permissive for chronic HCV infection and shows chronic liver injury including inflammation, steatosis and fibrosis.Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. The development of direct-acting antivirals has revolutionized treatment by offering cure¹. However, several hurdles remain. High costs limit treatment access in the majority of patients. Infection is often diagnosed at a late stage when advanced liver disease and cancer are established. Cure in advanced liver disease does not eliminate the risk of hepatocellular carcinoma (HCC). Re-infection remains possible and a vaccine is not available².To better understand the pathogenesis of virus-induced liver disease and HCC, a small animal model permissive for HCV infection and modeling liver disease biology is needed³. HCV infection is limited to humans and chimpanzees, predominantly due to distinct host-dependency factors and innate antiviral immune responses precluding cross-infection of other species⁴. Research efforts have focused on humanizing mice permissive to HCV infection. This has led to the development of conceptually three different types of mouse models.The human liver chimeric mouse is based on immune- and hepato-deficient mice repopulated with human hepatocytes. While the uPA-SCID⁵ and FRG⁶ models are extremely useful to study the viral life cycle and antivirals, the lack of an adaptive immune system and liver disease precludes the use for the study of liver disease biology and vaccine evaluation (7 based on modified Rag-2^−/− mice, activation of the overexpressed FK506-binding protein and caspase-8 fusion protein in the liver induces death of mouse hepatocytes and facilitates engraftment of human hepatocyte progenitor and CD34⁺ haematopoetic stem cells. While infected mice exhibit liver inflammation and fibrosis, this model appears to be limited with detection of virus only in the liver (8. Sustained and robust HCV infection for 90 days was achieved by crossing the 4hEF mice with mice knocked out for STAT1⁹. Furthermore, HCV infection in these mice elicited antiviral cellular and humoral immune responses. Although the animals were not reported to develop liver disease, this robust model represents a major breakthrough since it allows for studying HCV-induced immune responses and the preclinical evaluation of vaccine candidates in a small animal model ( Human liver chimeric uPA/SCID, FRGAFC8-huHSC/HepHumanized transgenic Rosa26-FlucC/O^TgReferences5,6^78,910Strain backgroundBalbCBalbCC57BL/6ICRConceptImmuno- and hepatodeficient mice repopulated with human hepatocytesImmuno- and hepatodeficient mice repopulated with human progenitor cellsHumanized for CD81, SR-BI, CLDN1 and OCLN; deficient in STAT1Humanized for CD81 and OCLN; Modified host-dependency factor and ISO expressionInoculumSerum, HCVccSerumHCVccSerum, HCVccChronic infection> 6 months3 months3 months> 12 monthsViral load: serum (copies/ml)10⁶ – 10⁷Not reported10⁴ – 10⁵10² – 10⁴Viral load: liver∼10^{6 *}10⁴ – 10^{5 *}10² – 10^{3 *}10⁴ – 10^{4 **}Adaptive immune systemAbsentHumanMouseMouseAnti-HCV B cell responsesAbsentNot reportedYesNot reportedAnti-HCV T cell responsesAbsentYesYesNot reportedEvidence for HCV associated human liver diseaseAbsentInflammation, fibrosisNot reportedInflammation, steatosis, fibrosisOpen in a separate windowCharacteristics of HCV infection, adaptive immune responses and occurrence of liver disease in HCV-permissive mouse models are listed. SR-BI, scavenger receptor class B type I; CLDN1, claudin-1; OCLN, occludin; HCVcc, cell culture-derived HCV.^*copies/μg total RNA;^**copies/mg liver tissue)Complementing these achievements, a recent study published in Cell Research by Chen et al.¹⁰ reports an immunocompetent animal model permissive for HCV infection and evidence for liver disease (10.In the previous report by Dorner et al.⁹, overexpressing human CD81 and OCLN in mice with STAT1 deficiency demonstrated sustained HCV infection for ∼90 days as against 12 months with ICR mice without obvious immune deficiency. To better understand the mechanisms for persistent infection in the new model, the C/OTg mice were backcrossed to C57BL/6 background to yield B6-C/OTg mice. Surprisingly, the B6-C/OTg mice did not support sustained HCV infection, indicating a potential functional role of genetic background in establishing chronic HCV infection¹⁰. Further investigations revealed significantly higher levels of apoE expression and progressive increase in miR-122 levels during the course of infection in C/OTg mice as compared to B6-C/OTg. In addition, the C/OTg mice showed transiently downregulated expression of anti-HCV interferon-stimulated genes (ISGs), namely ifi44 and Eif2ak2, unlike B6-C/OTg mice, in the first 2 weeks post infection. Furthermore, using transgenic technology the authors demonstrated that co-expression of both OCLN and CD81 was required for susceptibility to HCV infection. Based on these results, the authors conclude that the altered expression of defined host-dependency factors combined with different innate immune responses against HCV infection facilitates the establishment of HCV infection in this particular host background.Taken together, this study provides a novel immunocompetent HCV mouse model with evidence for HCV-associated liver diseases. The observation of liver disease in infected animals is interesting and of significant impact since it may allow the study of virus-induced liver injury including inflammation, steatosis and fibrosis ― an urgent unmet need in the field. Further studies are needed to study the causal relationship between HCV, inflammation and antiviral immune responses and liver disease in this model. A potential challenge could be the lower viral load compared to other models and human blood ― adaptation of viral strains to this model or further engineering of host-dependency factor expression in the mouse liver could overcome this limitation. Finally, a detailed characterization of antiviral immune responses may help to study whether this model will also be useful for vaccine development ― another challenge in HCV translational research.     

Association of Campylobacter jejuni Cj0859c Gene (fspA) Variants with Different C. jejuni Multilocus Sequence Types

Cj0859c variants fspA1 and fspA2 from 669 human, poultry, and bovine Campylobacter jejuni strains were associated with certain hosts and multilocus sequence typing (MLST) types. Among the human and poultry strains, fspA1 was significantly (P < 0.001) more common than fspA2. FspA2 amino acid sequences were the most diverse and were often truncated.Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and responsible for more than 90% of Campylobacter infections (7). Case-control studies have identified consumption or handling of raw and undercooked poultry meat, drinking unpasteurized milk, and swimming in natural water sources as risk factors for acquiring domestic campylobacteriosis in Finland (7, 9). Multilocus sequence typing (MLST) has been employed to study the molecular epidemiology of Campylobacter (4) and can contribute to virulotyping when combined with known virulence factors (5). FspA proteins are small, acidic, flagellum-secreted nonflagellar proteins of C. jejuni that are encoded by Cj0859c, which is expressed by a σ²⁸ promoter (8). Both FspA1 and FspA2 were shown to be immunogenic in mice and protected against disease after challenge with a homologous strain (1). However, FspA1 also protected against illness after challenge with a heterologous strain, whereas FspA2 failed to do the same at a significant level. Neither FspA1 nor FspA2 protected against colonization (1). On the other hand, FspA2 has been shown to induce apoptosis in INT407 cells, a feature not exhibited by FspA1 (8). Therefore, our aim was to study the distributions of fspA1 and fspA2 among MLST types of Finnish human, chicken, and bovine strains.In total, 367 human isolates, 183 chicken isolates, and 119 bovine isolates (n = 669) were included in the analyses (3). PCR primers for Cj0859c were used as described previously (8). Primer pgo6.13 (5′-TTGTTGCAGTTCCAGCATCGGT-3′) was designed to sequence fspA1. Fisher''s exact test or a chi-square test was used to assess the associations between sequence types (STs) and Cj0859c. The SignalP 3.0 server was used for prediction of signal peptides (2).The fspA1 and fspA2 variants were found in 62.6% and 37.4% of the strains, respectively. In 0.3% of the strains, neither isoform was found. Among the human and chicken strains, fspA1 was significantly more common, whereas fspA2 was significantly more frequent among the bovine isolates (Table ​(Table1).1). Among the MLST clonal complexes (CCs), fspA1 was associated with the ST-22, ST-45, ST-283, and ST-677 CCs and fspA2 was associated with the ST-21, ST-52, ST-61, ST-206, ST-692, and ST-1332 CCs and ST-58, ST-475, and ST-4001. Although strong CC associations of fspA1 and fspA2 were found, the ST-48 complex showed a heterogeneous distribution of fspA1 and fspA2. Most isolates carried fspA2, and ST-475 was associated with fspA2. On the contrary, ST-48 commonly carried fspA1 (Table ​(Table1).1). In our previous studies, ST-48 was found in human isolates only (6), while ST-475 was found in both human and bovine isolates (3, 6). The strict host associations and striking difference between fspA variants in human ST-48 isolates and human/bovine ST-475 isolates suggest that fspA could be important in host adaptation.

TABLE 1.

Percent distributions of fspA1 and fspA2 variants among 669 human, poultry, and bovine Campylobacter jejuni strains and their associations with hosts, STs, and CCs

Imprinted gene expression in hybrids: perturbed mechanisms and evolutionary implications

Diverse mechanisms contribute to the evolution of reproductive barriers, a process that is critical in speciation. Amongst these are alterations in gene products and in gene dosage that affect development and reproductive success in hybrid offspring. Because of its strict parent-of-origin dependence, genomic imprinting is thought to contribute to the aberrant phenotypes observed in interspecies hybrids in mammals and flowering plants, when the abnormalities depend on the directionality of the cross. In different groups of mammals, hybrid incompatibility has indeed been linked to loss of imprinting. Aberrant expression levels have been reported as well, including imprinted genes involved in development and growth. Recent studies in humans emphasize that genetic diversity within a species can readily perturb imprinted gene expression and phenotype as well. Despite novel insights into the underlying mechanisms, the full extent of imprinted gene perturbation still remains to be determined in the different hybrid systems. Here we review imprinted gene expression in intra- and interspecies hybrids and examine the evolutionary scenarios under which imprinting could contribute to hybrid incompatibilities. We discuss effects on development and reproduction and possible evolutionary implications.In many plants and animals, interspecific hybridization events yield offspring that are phenotypically different from either of the parent species. Such hybrids typically display developmental abnormalities and, in animals, often have reduced fertility or complete sterility, particularly in males. Hybrid incompatibilities arise because, although the parental species may be genetically similar, the genomes are still too divergent to sustain normal development, physiology and reproduction when mixed in the hybrid offspring (Wu and Ting, 2004). Extensive research has been performed on genetic incompatibilities in plant and animal hybrids (Ishikawa and Kinoshita, 2009; Johnson, 2010). Key loci have been mapped and characterized in experimental model species, providing important insights into the aberrant phenotypes such as male hybrid sterility (Maheshwari and Barbash, 2011).Phenotypic abnormalities in interspecies hybrids often differ greatly between the reciprocal crosses. The classic example of such an asymmetry is seen in reciprocal crosses between donkeys and horses, where both directions of the cross produce sterile offspring, but the gross phenotype of the progeny (that is, ‘mule'' versus ‘hinny'') depends on the direction of the cross. Horses and donkeys have a different chromosome number, but this cannot explain the differential hybrid phenotypes that depend on the direction of the cross (as the reciprocal crosses have the same autosomal karyotype). More than 50 years ago serum concentrations of a placental hormone were reported to be markedly higher in mule than in hinny conceptuses, suggestive of parental genome-specific gene expression (Allen, 1969).The North-American genus Peromyscus (‘deer mice'') has been studied extensively to explore hybrid incompatibilities in mammals (see Vrana et al., 1998). Also in interspecies hybrids in Mus (house mouse), between the sympatric species M. musculus and M. spretus, morphological differences are apparent between reciprocal hybrids (Zechner et al., 2004). These hybrid effects were observed in crosses between a mixed M. musculus domesticus strain and lab stocks of M. spretus. To be definitive about where the incompatibilities lie between M. musculus and M. spretus (or M. m. castaneus, see below), reciprocal crosses between several different wild-derived stocks (or wild caught animals) of M. musculus and M. spretus populations would be needed.

Table 1

Terminology and abbreviations

Widespread Distribution of Cell Defense against d-Aminoacyl-tRNAs

Several l-aminoacyl-tRNA synthetases can transfer a d-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme d-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively. Evidence was given that DTD1 homologs also exist in nearly all eukaryotes, whereas DTD2 homologs occur in plants. On the other hand, several bacteria, including most cyanobacteria, lack genes encoding a DTD1 homolog. Here we show that Synechocystis sp. PCC6803 produces a third type of deacylase (DTD3). Inactivation of the corresponding gene (dtd3) renders the growth of Synechocystis sp. hypersensitive to the presence of d-tyrosine. Based on the available genomes, DTD3-like proteins are predicted to occur in all cyanobacteria. Moreover, one or several dtd3-like genes can be recognized in all cellular types, arguing in favor of the nearubiquity of an enzymatic function involved in the defense of translational systems against invasion by d-amino acids.Although they are detected in various living organisms (reviewed in Ref. 1), d-amino acids are thought not to be incorporated into proteins, because of the stereospecificity of aminoacyl-tRNA synthetases and of the translational machinery, including EF-Tu and the ribosome (2). However, the discrimination between l- and d-amino acids by aminoacyl-tRNA synthetases is not equal to 100%. Significant d-aminoacylation of their cognate tRNAs by Escherichia coli tyrosyl-, tryptophanyl-, aspartyl-, lysyl-, and histidyl-tRNA synthetases has been characterized in vitro (3–9). Recently, using a bacterium, transfer of d-tyrosine onto tRNA^Tyr was shown to occur in vivo (10).With such misacylation reactions, the resulting d-aminoacyl-tRNAs form a pool of metabolically inactive molecules, at best. At worst, d-aminoacylated tRNAs infiltrate the protein synthesis machinery. Although the latter harmful possibility has not yet been firmly established, several cells were shown to possess a d-tyrosyl-tRNA deacylase, or DTD, that should help them counteract the accumulation of d-aminoacyl-tRNAs. This enzyme shows a broad specificity, being able to remove various d-aminoacyl moieties from the 3′-end of a tRNA (4–6, 11). Such a function makes the deacylase a member of the family of enzymes capable of editing in trans mis-aminoacylated tRNAs. This family includes several homologs of aminoacyl-tRNA synthetase editing domains (12), as well as peptidyl-tRNA hydrolase (13, 14).Two distinct deacylases have already been discovered. The first one, called DTD1, is predicted to occur in most bacteria and eukaryotes (see d-amino acids, including d-tyrosine (6). In fact, in an E. coli Δdtd strain grown in the presence of 2.4 mm d-tyrosine, as much as 40% of the cellular tRNA^Tyr pool becomes esterified with d-tyrosine (10).

TABLE 1

Distribution of DTD1 and DTD2 homologs in various phylogenetic groupsHomologs of DTD1 and DTD2 were searched for using a genomic Blast analysis against complete genomes in the NCBI Database (www.ncbi.nlm.nih.gov). Values in the table are number of species. For instance, E. coli is counted only once in γ-proteobacteria despite the fact that several E. coli strains have been sequenced.

Correlation of Fragile Histidine Triad (Fhit) Protein Structural Features with Effector Interactions and Biological Functions

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G₂/M or sub-G₁ fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or reduced in cancer cells because of rearrangement of the exquisitely DNA damage-sensitive fragile FHIT gene. Restoration of Fhit expression suppresses tumorigenicity of cancer cells of various types, and the ability to induce apoptosis in cancer cells in vitro is reduced by specific Fhit mutations (1, 2).Through studies of signal pathways affected by Fhit expression, by searches for Fhit protein effectors, and by in vitro analyses of Fhit activity, we and others have defined Fhit enzymatic activity in vitro (3), apoptotic activity in cells and tumors (4–6), and most recently identification of a Fhit protein complex that affects Fhit stability, mitochondrial localization, and interaction with ferredoxin reductase (Fdxr)⁵ (7). The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where Fhit interacts with Fdxr, which is responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit restoration in Fhit-deficient cancer cells increases production of intracellular reactive oxygen species (ROS), followed by increased apoptosis of cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, likely carrying oxidative DNA damage that contributes to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad (HIT) family of proteins, suggested that the protein product would hydrolyze diadenosine tetraphosphate or diadenosine triphosphate (Ap₃A) (8), and in vitro studies showed that Ap₃A was cleaved into ADP and AMP by Fhit. The catalytic histidine triad within Fhit was essential for catalytic activity (3), and a Fhit mutant that substituted Asn for His at the central histidine (H96N mutant) was catalytically inactive, although it bound substrate well (3). Early tumor suppression studies showed that cancer cells stably transfected with wild type (WT) or H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested the hypothesis that the Fhit-substrate complex sends the tumor suppression signal (9, 10). To test this hypothesis, a series of FHIT alleles was designed to reduce substrate-binding and/or hydrolytic rates and was characterized by quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in k_cat and increases as large as 30-fold in K_m. Mutants with 2–7-fold increases in K_m had significantly reduced apoptotic indices and the mutant with a 30-fold increase in K_m retained little apoptotic function. Thus, the proapoptotic function of Fhit, which is likely associated with tumor suppressor function, is limited by substrate binding and is unrelated to substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine phosphorylation by the Src family protein kinases, which can phosphorylate Tyr-114 of Fhit in vitro and in vivo (12). After co-expression of Fhit with the Elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated Fhit were purified, and enzyme kinetics studies showed that monophosphorylated Fhit exhibited monophasic kinetics with K_m and k_cat values ∼2- and ∼7-fold lower, respectively, than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics; one site had K_m and k_cat values ∼2- and ∼140-fold lower, respectively, than for unphosphorylated Fhit; the second site had a K_m ∼60-fold higher and a k_cat ∼6-fold lower than for unphosphorylated Fhit (13). Thus, it was possible that the alterations in K_m and k_cat values for phosphorylated forms of Fhit might favor formation and lifetime of the Fhit-Ap₃A complex and enhance tumor suppressor activity (see Fhit forms

---

Kinetic parameters

---

% Sub-G₁

---

Direct binding

---

Subcellular location

---

Co-IP in vivo

---

8-OHdG

---

Apoptosis

---

Tumor suppressor

---

K_m (mm)k_cat (s^–1)A549MKN74Hsp60FdxrHsp60Fdxr Fhit WT 1.6 +/– 0.19 2.7 +/– 0.95 43 24 Yes Yes Cyt & mito Yes Yes Yes Yes Yes Catalyt mutants    H96D Up 2-fold Down >2 × 10⁴ 29 NT NT NT Cyt & mito Yes Yes NT Yes NT    H96N Up 2-fold Down >5 × 10⁵ 31 14.4 NT NT Cyt & mito Yes Yes Yes Yes Yes Loop mutants    Y114A Up 23-fold Down 2-fold 3.7 NT NT NT Cyt +/– +/– +/– No No    Y114D NT NT 2.9 6 NT NT Cyt +/– +/– – No –/+    Y114E NT NT NT NT NT NT Cyt & mito –/+ –/+ – No NT    Y114F Up 5-fold Up 1.1-fold 11.5 3 NT NT Cyt & mito –/+ –/+ – No No    Y114W Up 5-fold Up 1.4-fold NT NT NT NT Cyt & mito –/+ – – NT NT    del113–117 Up 10-fold Down 38-fold 5 NT NT NT NT NT NT – No NT Other mutants    L25W Up 7-fold Down 4-fold 15 NT NT NT Cyt – – – NT –/+    I10W,L25W Up 32-fold Down 6-fold 11 NT NT NT NT NT NT NT NT NT    F5W Up 3.3 fold NT NT 5 NT NT NT NT NT +/– No NT Purified pFhit    pFhit Down 0.4-fold Down 7-fold NA NA –/+ Yes NA NA NA NA NA NA    ppFhit Down 0.4-fold Down > 100-fold NA NA –/+ Yes NA NA NA NA NA NA Up 60-fold Down 6-fold Open in a separate windowTo explore the in vivo importance of the Tyr-114 phosphorylation site and define Fhit-mediated signaling events, Semba et al. (14) compared the differential biological effects of Ad-FHIT WT and Ad-FHIT Tyr-114 mutant expression in human lung cancer cells. Caspase-dependent apoptosis was effectively induced only by WT Fhit protein. However, the biological significance of phosphorylation at Tyr-114 has been difficult to study because the endogenous phosphorylated forms have very short half-lives; activation of epidermal growth facto receptor family members induces Fhit phosphorylation by Src and proteasome degradation of phosphorylated Fhit (15).Although there are possible connections among the various pathways known to be altered in Fhit-deficient cells, apoptosis, DNA damage-response checkpoint activation, ROS production, and related biological effects of Fhit loss or overexpression, details of the pathway(s) leading from Fhit overexpression to cell death and tumor suppression have not been delineated. Now that a Fhit signaling complex has been identified, we set out to examine which structural features of Fhit protein might participate in individual steps of the pathway leading from Fhit overexpression through complex formation, subcellular localization, interaction with mitochondrial Fdxr, DNA damage induction, cell cycle changes, apoptosis, and ultimately tumor suppression. The underlying hypotheses were as follows: substrate-binding mutants would behave similarly to WT; nonsubstrate-binding mutants would be defective in some step of the pathway, perhaps complexing with heat shock proteins or Fdxr or perhaps induction of DNA damage; and Tyr-114 mutants, which also affect formation or stability of the enzyme-substrate complex, would also be defective in executing some step of the Fhit overexpression pathway to cell death. One goal was to identify specific mutants that exhibited deficiency in specific steps of the pathway, so that such mutants could be used to dissect each step in more detail. Using in vitro Fhit and Fhit-effector protein interactions, we aimed to determine the following: 1) which proteins of the complex interact directly with Fhit, and 2) the biological role of these interactions in vivo. Using cancer cells expressing exogenous WT and mutant Fhit proteins, we were able to examine the structural features of Fhit that affect the direct interaction with its effectors, participate in ROS production, and are necessary for tumor suppression activity.