A naturally occurring prfA truncation in a Listeria monocytogenes field strain contributes to reduced replication and cell-to-cell spread

Listeria (L.) monocytogenes is an environmental bacterium that may become an intracellular pathogen upon ingestion to cause gastroenteritis, septicaemia, abortions, and/or fatal infections of the central nervous system. We here describe a L. monocytogenes field strain (JF5171) isolated from a bovine placenta in the context of abortion, which exhibited attenuation in bovine brain-slice cultures. The whole genome of strain JF5171 was sequenced, and the invasion, replication, and intercellular spread of JF5171 were further analyzed by quantification of colony forming units and immunofluorescence studies. Phospholipase and hemolysis activity of JF5171 were also quantified along with transcription levels of actA, hly and prfA. The data obtained were compared to those of the widely used L. monocytogenes reference strain, EGD-e. JF5171 exhibited reduced replication and lower levels of phospholipase and hemolysis activity. Invasion and cell-to-cell spread was strongly decreased compared to EGD-e, and actin polymerization was absent. A frame shift deletion was identified in the JF5171 coding region of the major regulator for virulence, prfA. This resulted in a truncated C-terminus sequence (WEN* vs. WGKLN*). In addition, a point mutation resulted in a lysine to arginine substitution at amino acid position 197. Complementation with prfA from EGD-e and with (EGD-e) prfA-K197N increased the replication and spread efficiency of JF5171. In contrast, complementation with the truncated version of prfA had no effect. Taken together, these results suggest that the truncated C-terminus of prfA considerably contributes to the strongly attenuated phenotype observed in vitro.

Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

Role of telomere dysfunction, DNA damage response and p53 mutations in tumorigenesis and aging

The ends of eukaryotic chromosomes are protected by specialized ribonucleoprotein structures termed telomeres. Telomeres protect chromosomes from end-to-end fusions, inappropriate repair and degradation. Disruption of this complex activates an ATM/ATR DNA damage response (DDR) pathway. One component of the complex is the Protection Of Telomeres 1 (POT1) protein, an evolutionarily conserved protein which binds single-stranded 3' overhang and is required for both chromosomal end protection and telomere length regulation. The mouse contains two POT1 orthologs, Pot1a and Pot1b. Here we show that both proteins colocalize with telomeres through interaction with the adapter protein TPP1. In addition, compared to Pot1a, the OB-folds of Pot1b possess less sequence specificity for telomeres. Disruption of POT1 proteins result in telomere dysfunction and activation of an ATR-dependent DDR at telomeres, suggesting that this response is normally suppressed by POT1 binding to the single-stranded G-overhang. ^ Telomeres are maintained by telomerase, and its absence in somatic cells results in telomere progressive loss that triggers the activation of p53. Telomere dysfunction initiates genomic instability and induces both p53-dependent replicative senescence and apoptosis to suppress tumorigenesis. In the absence of functional p53, this genomic instability promotes cancer. It was previously not known which aspect of the p53 dependent DNA damage response is important to suppress tumorigenesis initiated by dysfunctional telomeres. The p53R172P knock-in mouse, which is unable to induce apoptosis but retains intact cell cycle arrest/cellular senescence pathways, allowed us to examine whether p53-dependent apoptosis is a major tumor suppression pathway initiated in the setting of telomere dysfunction. Spontaneous tumorigenesis remains potently suppressed in late generation telomerase null mice possessing the p53P/P mutation. These results suggest that suppression of spontaneous tumorigenesis initiated by dysfunctional telomeres requires activation of a p53-dependent senescence pathway. In addition, we used another knock-in mouse model with a p53R172H (p53H) point mutation to test the hypothesis that telomere dysfunction promotes chromosomal instability and accelerates the onset of tumorigenesis in vivo in the setting of this most common gain-of-function mutation in the human Li Fraumeni cancer syndrome. We unexpectedly observed that telomerase null mice possessing dysfunctional telomeres in the setting of the p53H/+ mutation develop significantly fewer tumors, die prematurely and exhibit higher level of cellular senescence, apoptosis and elevated genomic instability compared to telomerase intact p53H/+ and telomerase null p53+/+ mice. These contrasting results thus link cancer and aging to the functional status of telomeres and the integrity of the p53 pathway. ^

Development and Characterization of Novel Ras Inhibitors

Ras genes are mutated in 15% of human cancers. Ras GTPases operate as molecular switches regulating cellular processes including proliferation, differentiation, and apoptosis. The three main isoforms of Ras – H-Ras, K-Ras, and N-Ras – inhabit distinct nanodomains of the plasma membrane and intracellular compartments including the Golgi. However, the role of single endogenous Ras isoforms on these compartments remains unclear as most studies have utilized ectopically expressed and mutant forms of Ras proteins. In an effort to develop novel tools that will allow us to abrogate individual endogenous Ras isoforms, we targeted the catalytic domain of p120RasGAP to the plasma membrane with the hypervariable region (HVR) of H-Ras (GAP-CTH) or K-Ras (GAP-CTK) and to the Golgi using the HVR of H-Ras with insertion of a point mutation (GAP-CTH181S). We performed GST-RBD pull-downs on cells expressing each GAP construct and stimulated with epidermal growth factor (EGF). We found that GAP-CTH and GAP-CTK specifically inhibited H-Ras or K-Ras, respectively. However, we did not detect any effect of GAP-CTH181S on Ras activation. Additionally, we used confocal microscopy to verify the ability of GAP constructs to abrogate Ras activation in distinct sub-cellular compartments. We found that GAP-CTH inhibits H-Ras activation on the plasma membrane, while GAP-CTK inhibits K-Ras activation on the plasma membrane. On the contrary, GAP-CTH181S inhibited H-Ras activation on the Golgi. We also analyzed the effects of these GAP constructs on the activation of ERK and Akt in response to EGF stimulation. We found that EGF stimulation of the MAPK pathway was inhibited by GAP-CTK but none of the other GAP constructs, while Akt activation was not inhibited by any GAP construct. Finally, we assayed cellular proliferation and differentiation. We found that GAP-CTK and GAP-CTH were equipotent inhibitors of cellular growth, whereas GAP-CTH181S was less potent. We also found that GAP-CTK and GAP-CTH inhibited differentiation with similar potency, while GAP-CTH181S was more potent. This approach may be adapted to investigate any Ras-dependent signaling pathway. Therefore, it has the potential to become a powerful tool for studying Ras isoform-specific signaling outputs.

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform.

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^

Alterations in the ras oncogene and the p53 tumor suppressor gene in ultraviolet radiation-induced skin carcinogenesis

A rapid increase of the ultraviolet radiation (UVR)-related skin cancer incidence has attracted more and more public attention during the last few decades. Prevention and treatment of UVR-related skin cancer has become an important public health issue in the United States. Recent studies indicate that mutations in ras and/or p53 genes may be involved in UVR-induced skin tumor development but the precise molecular mechanism remains unclear. In this study, alterations of H-ras and p53 genes were investigated in different stages of carcinogenesis in a chronic UVR (solar simulator) exposure-induced Sencar mouse skin carcinogenesis model in order to clarify the role of the alterations of these genes during the skin carcinogenesis process and to further understand the mechanisms by which UVR causes skin cancer.^ Positive ras-p21 staining in cell membranes and cytosol were detected in 18/33 (55%) of squamous cell carcinomas (SCCs), but were not detected in UV-exposed skin, papillomas, or spindle cell tumors (SCTs). Positive staining of the malignant progression marker K13 was found in 17/33 (52%) of SCCs only. A significant positive correlation was observed between the K13 and the ras-p21 expression. Polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis and gene sequencing analysis revealed three point mutations, one (codon 56) in UV-exposed non-tumor bearing skin and the other two (codons 21 and 13) in SCCs. No UV-specific mutation patterns were found.^ Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. PCR-based SSCP and sequencing analysis revealed eight point mutations in exons 5 and 6 (four in SCTs, two in SCCs, and two in UV-exposed skin) including six C-T or C-A transitions. Four of the mutations occurred at a dipyrimidine (CC) sequence. The pattern of the mutations indicated that the mutagenic lesions were induced by UVR.^ These results indicate that overexpression of ras-p21 in conjunction with aberrant expression of K13 occurred frequently in UVR-induced SCCs in Sencar mouse skin. The point mutation in the H-ras gene appeared to be a rare event in UVR skin carcinogenesis and may not be responsible for overexpression of ras-p21. UVR-induced P53 gene alteration is a frequent event in UVR-induced SCCs and later stage SCT tumors in Sencar mice skin, suggesting the p53 gene mutation plays an important role in skin tumor malignant progression. ^

Signal transduction of {\it HER-2\/}/{\it neu\/} receptor tyrosine kinase

Overexpression and/or amplification of HER2/neu is frequently detected in many human cancers. Activation of p185 tyrosine kinase can be achieved by point mutation, overexpression, deletion, and heterodimerization with other class I receptors. In this study I investigated the signal transduction pathways mediating the oncogenic signal of the point mutation-activated rat p185. I demonstrated that tyrosine phosphorylation of Shc and formation of Shc/Grb2 complex correlated to the transformation of NIH3T3 cells caused by the point mutation-activated rat HER2/neu. Furthermore, I observed that association with Shc was severely impaired by deletion of most of the major autophosphorylation sites of the point-mutated p185. The truncated p185 product, however, fully retained its ability to transform NIH3T3 cells, induce Shc tyrosine phosphorylation and Shc/Grb2 complex formation. These results suggest that tyrosine phosphorylation of Shc which allows formation of Shc/Grb2 complex may play an important role in cell transformation induced by the point mutation-activated p185, and that stable binding to mutant p185 may not be necessary for Shc to mediate this signaling pathway.^ Recent studies have suggested that formation of the complex containing Sos, Grb2 and Shc is important in coupling receptor tyrosine kinases to the Ras signaling pathway. To clarify the role of this trimer in the oncogenic signaling of the activated p185, I set out to interfere with the protein-protein interactions in Shc/Grb2/Sos complex by introducing Grb2 mutants with deletions in either amino- ($\Delta$N-Grb2) or carboxyl- ($\Delta$C-Grb2) terminal SH3 domains into B104-1-1 cells derived from NIH3T3 cells that express the point mutation-activated HER-2/neu. I found that the transformed phenotypes of the B104-1-1 cells were largely reversed by expression of the $\Delta$N-Grb2. The effect of the $\Delta$C-Grb2 on phenotypic reversion was much weaker. Biochemical analysis showed that the $\Delta$N-Grb2 was able to associate Shc but not the activated p185 nor Sos, while the $\Delta$C-Grb2 bound to Shc, the activated p185, and Sos. The p185-mediated Ras activation was severely inhibited by the $\Delta$N-Grb2 but not the $\Delta$C-Grb2. Taken together, these data demonstrate that interruption of the interaction between Shc and the endogenous Grb2 by the $\Delta$N-Grb2 is able to impair the oncogenic signaling of the mutation-activated p185, indicating that (i) the $\Delta$N-Grb2 functions as a strong dominant-negative mutant, (ii) Shc/Grb2/Sos pathway plays a major role in mediating the oncogenic signal of the mutation-activated p185. Unlike the $\Delta$N-Grb2, the $\Delta$C-Grb2 appears to be a relatively weak dominant-negative mutant, probably due to its ability to largely fulfill the biological functions of the wild-type Grb2. ^

The Shc adaptor protein and its role on Her -2 /neu oncogenesis

Shc proteins are implicated in coupling receptor tyrosine kinases to the mitogen-activated protein kinase (MAPK) pathway by recruiting Grb2/SOS to the plasma membrane. To better understand the role of Shc in oncogenesis brought about by point mutation activated neu (p185*), we transfected a Shc mutant (ShcΔCH1), which lacks the Grb2 binding site Y317 by deletion of collagen-homology domain 1, into p185*-transformed NIH3T3 cells. The cellular transformation phenotypes were found to be largely suppressed by expression of ShcΔCH1. This study indicates that Shc plays a critical role in mediating the oncogenical signals of p185*. Although ShcΔCH1 still retained another Grb2 binding site (Y239/240), we did not detect its physical association with Grb2. We also found that ShcΔCH1 could associate with p185*; however, this association did not interfere with the endogenous Shc-p185* interaction or the Shc-Grb2 interaction. In addition, p185*-mediated MAPK/Elk activation, PI3-K activation and Src activation likewise was not inhibited by ShcΔCH1 expression. Taken together, our current study clearly indicates that ShcΔCH1 suppresses the p185*-induced transformation, and that this suppression is mediated through a MAPK-independent and possibly PI3-K, Src-independent pathway. These results suggest that Shc may be involved in other unidentified signal pathways which are critical for p185*-induced cellular transformation besides the three pathways that we have studied. ^

Bases moleculares de la resistencia a insecticidas en la mosca mediterránea de la fruta Ceratitis Capitata (Wiedemann)

La mosca mediterránea de la fruta Ceratitis capitata (Wiedemann, 1824) está considerada una de las plagas clave para la fruticultura. El malatión es un insecticida organofosforado que fue empleado mayoritariamente en España para el control de C. capitata hasta 2009, año en el que dejó de utilizarse por no estar incluido en el anexo I de la Directiva Europea 91/414/ECC. El incremento del uso del malatión, debido a las graves pérdidas económicas causadas por C. capitata, provocó la aparición de poblaciones de campo resistentes. El estudio de una población resistente a malatión, recogida en Castelló en 2004, permitió la identificación de dos mecanismos de resistencia: una mutación puntual (G328A) en la acetilcolinesterasa (AChE) y un mecanismo de resistencia metabólica, probablemente mediado por carboxilesterasas. Teniendo en cuenta estos antecedentes, nos propusimos estudiar los mecanismos implicados en la resistencia a malatión en C. capitata. Además, durante el desarrollo de esta Tesis, el malatión fue sustituido por otros insecticidas como el espinosad y la lambda-cialotrina para el control de la plaga. En este nuevo contexto, es extremadamente importante analizar la susceptibilidad de poblaciones de campo frente a espinosad y estudiar la posible existencia de resistencia cruzada a estos insecticidas, así como sentar las bases para el estudio de futuros mecanismos de resistencia. En primer lugar, analizamos mediante bioensayos con dosis discriminante la susceptibilidad a malatión y espinosad en doce poblaciones de C. capitata de Andalucía, Aragón, Cataluña, Comunidad Valenciana e Islas Baleares; y nuestros resultados sugirieron la presencia de individuos resistentes a malatión en la mayoría de las poblaciones analizadas. En el caso del espinosad, observamos que la susceptibilidad a este insecticida de origen biológico fue elevada en la mayoría de las poblaciones, sin embargo, la población recogida en Xàbia (Alicante) mostró un nivel de susceptibilidad unas dos veces menor al resto de poblaciones. Mediante la selección en laboratorio, obtuvimos dos líneas resistentes a malatión, W-4Km y W-10Km, con unos niveles de resistencia con respeto a la línea susceptible C de 178 y 400 veces, respectivamente. Además, se seleccionó por primera vez en C. capitata una línea altamente resistente a espinosad (Xàbia-W-100s), que actualmente es unas 500 veces más resistente que la línea de laboratorio C. Con el objetivo de escoger la estrategia más adecuada para el manejo de la plaga, estudiamos la susceptibilidad a diferentes tipos de insecticidas en la línea resistente a malatión W- 4Km. En esta línea detectamos resistencia cruzada moderada a los organofosforados fentión, diazinón, fosmet, triclorfón y metil-clorpirifos (de 7 a 16 veces) y frente al carbamato carbaril, al piretroide lambda-cialotrina y al quimioesterilizante lufenurón (de 4 a 6 veces). Por otra parte, la resistencia cruzada frente a espinosad fue baja (1,5 veces). Es importante destacar que los niveles de resistencia estimados frente a todos los insecticidas fueron de uno o dos órdenes de magnitud inferiores al observado en la línea W-4Km frente a malatión (178 veces), hecho que podría deberse, al menos, a dos posibles hipótesis: que la mutación AChE G328A confiera mayor insensibilidad al malaoxón (forma activa del malatión) que a otros insecticidas que tienen como diana la AChE y/o, en segundo lugar, que el mecanismo de resistencia mediado por carboxilesterasas hidrolice el malatión de manera más eficiente que los otros insecticidas analizados. En el estudio de nuevos mecanismos de resistencia en C. capitata, por un lado, analizamos la diversidad de enzimas citocromo P450, asociadas con resistencia metabólica en otras especies, y por otro lado, desarrollamos un sistema para la detección de nuevas mutaciones puntuales que pudiesen aparecer en los genes que codifican la AChE (Ccace2) y la aliesterasa (Ccae7). Mediante el empleo de cebadores degenerados obtuvimos 37 genes CYP, que codifican enzimas P450, pertenecientes a cinco familias. Posteriormente, en un estudio de inducción con fenobarbital, observamos que la expresión de cuatro de los seis genes analizados era susceptible de ser inducida. Por otro lado, se puso a punto un sistema que permite amplificar y secuenciar, a partir de DNA genómico, los exones de los genes Ccace2 y Ccae7 en los que se han encontrado mutaciones relacionadas con resistencia a insecticidas en otras especies. Los resultados obtenidos facilitarán el estudio de nuevos mecanismos de resistencia mediados por estas enzimas en C. capitata. Se diseñó un método PCR-RFLP para identificar los individuos portadores de la mutación AChE G328A (alelo de resistencia Ccace2R) sin la necesidad de realizar bioensayos y que, además, permite detectar resistencia cuando ésta se encuentra a baja frecuencia. Según el análisis realizado, el alelo Ccace2R se observó en 25 de las 27 localidades españolas muestreadas en el territorio español, incluyendo las Islas Baleares y Canarias. Sin embargo, este alelo no se detectó en poblaciones procedentes de once países y de cinco continentes. El análisis de la presencia del alelo Ccace2R en las líneas resistentes a malatión durante el proceso de selección en el laboratorio mostró una rápida disminución de los homocigotos, tanto para el alelo susceptible como para el alelo de resistencia, en favor de los individuos heterocigotos. Así, después de 52 generaciones de selección, se observó que la totalidad de los individuos analizados de la línea W-10Km presentaban un genotipo heterocigoto para la mutación AChE G328A. Este desequilibrio contradice la segregación mendeliana esperada para un gen con dos alelos pero podría ser explicado por la existencia de una duplicación del gen Ccace2. La demostración de la presencia de esta duplicación se realizó mediante: i) el cruzamiento de individuos heterocigotos de la línea W-10Km con homocigotos susceptibles de la línea C, que dio lugar a una descendencia en la que el 100% de los individuos eran heterocigotos; ii) la evaluación del número de copias del gen Ccace2 por PCR cuantitativa en tiempo real (qPCR), que resultó dos veces mayor en individuos de la línea W-10Km en comparación con los de la línea C; iii) el análisis del nivel de expresión de Ccace2, que fue el doble en la línea W-10Km con respecto a la línea C, y iv) el estudio de la actividad AChE, que resultó mayor en los individuos de la línea W-10Km. Según los resultados obtenidos, una duplicación del gen Ccace2 provoca la coexistencia en un mismo cromosoma del alelo silvestre y del alelo mutado y, además, las dos copias del gen Ccace2, al estar ligadas, producen una heterocigosis permanente (Ccace2RS). De esta manera se explica que el hecho de que 100% de los individuos de la línea W-10Km mostrasen un perfil de restricción correspondiente a un individuo heterocigoto ya que, en realidad, eran homocigotos estructurales para la duplicación (genotipo CCace2RS/RS). Se ha detectado un coste biológico asociado a la duplicación que consiste en un incremento en la mortalidad acumulada de los adultos a partir del séptimo día después de la emergencia. La descripción de la duplicación Ccace2RS supone la identificación de un nuevo mecanismo de resistencia a malatión en C. capitata. Finalmente, mediante el diseño de un método de doble PCR-RFLP se determinó la presencia de la duplicación Ccace2RS en la mayoría de las poblaciones españolas. La proporción de individuos portadores de la duplicación osciló entre el 5% y el 35%, observándose los mayores valores de frecuencia en las poblaciones de C. capitata recogidas en la cuenca mediterránea. Podemos por lo tanto concluir que la resistencia a malatión asociada a la mutación AChE G328A y a la duplicación Ccace2RS está ampliamente establecida en las poblaciones españolas de C. capitata. Nuestros resultados desaconsejan la utilización del malatión (si fuera de nuevo autorizado) o de otros organofosforados para el control de esta plaga. Además, una de las líneas resistentes a malatión mostró resistencia cruzada frente a insecticidas con diferentes modos de acción y que se utilizan actualmente para el control de C. capitata, tales como lambda-cialotrina y lufenurón. La alta susceptibilidad a espinosad observada en las poblaciones españolas, así como la reducida resistencia cruzada estimada para este insecticida, sugieren que su utilización es adecuada para el control de la plaga. Sin embargo, la utilización de un sólo insecticida puede entrañar riesgos por favorecer la selección de resistencia, de hecho, mediante selección en laboratorio se obtuvo una población altamente resistente a espinosad. Por tanto, es recomendable implementar programas de control integrado y de manejo de la resistencia en C. capitata utilizando distintos sistemas de control e insecticidas con diferentes mecanismos de acción que permitan su sostenibilidad en el tiempo. Los sistemas de detección de alelos de resistencia desarrollados en este trabajo permitirán la detección precoz de resistencia en campo, facilitando la decisión sobre el sistema de control más adecuado. Además, los conocimientos generados podrán contribuir al desarrollo de nuevos sistemas de detección para otros mecanismos de resistencia. Abstract. The Mediterranean fruit fly, Ceratitis capitata (Wiedemann, 1824), is considered one of the most harmful pests in fruit crops. Until 2009, when malathion use was banned due to its not inclusion in the Annex I of Directive 91/414/EEC, the application of this organophosphate (OP) insecticide in Spain increased gradually due to the large economic losses caused by C. capitata. The increase in the frequency of treatments resulted in the development of resistant field populations. The study of a malathion-resistant population, collected in 2004 in Castelló (Comunidad Valenciana), allowed the identification of two resistance mechanisms: a single point mutation (G328A) in the target acetylcholinesterase (AChE), as well as a metabolic resistance mechanism, most likely carboxylesterase-mediated. Taking all the preceding into account, we studied the malathion resistance mechanisms in C. capitata. During the development of this PhD Thesis malathion use was banned by the European Union, being replaced by other insecticides, such as spinosad and lambda-cyhalotrin. Within this new working frame, the need to analyse the possible existence of cross-resistance to these insecticides and the susceptibility to spinosad in field populations was raised. This would define the baseline for future studies on resistance mechanisms. Firstly, through discriminant dose bioassays, we analysed malathion and spinosad susceptibility in twelve C. capitata populations from Andalucia, Aragon, Cataluña, C. Valenciana and the Baleares Islands. Our results suggest the presence of malathion-resistant individuals in most of the populations analysed. Regarding spinosad, we noticed a high susceptibility to this biologically derived insecticide in most of the populations, but in the one collected in Xabia (Alicante), which had a susceptibility level two times lower than the rest of populations. Through laboratory selection, we obtained two malathion-resistant strains, W-4Km and W-10Km, with resistance levels 178- and 400-fold, respectively, compared to the control susceptible C strain. Besides, a strain highly-resistant to spinosad (Xabia-W-100s), 500-times more resistant than control C strain, was selected. In order to decide the most appropriate management strategy for the pest, we studied the susceptibility to different insecticides in the malathion-resistant W-4Km strain. We detected a moderated cross-resistance to the OPs fenthion, diazinon, phosmet, trichlorphon and methylchlorpyrifos (7- to 16-fold), and to the carbamate carbaryl, the pyretroid lambda-cyhalotrin and the chemosterilizer lufenuron (4- to 6-fold). On the other hand, cross-resistance to spinosad was low (1.5-fold). It is important to note that resistance levels to all insecticides were one or two orders of magnitude less than that observed against malathion in W-4Km strain (178-fold), a fact that might be due to, at least, two possible causes: mutation AChE G328A may provide a higher insensitivity to malaoxon (the active form of malathion) than to other insecticides having AChE as target, and/or, secondly, the carboxylesterase-mediated resistance mechanism hydrolyzes malathion more efficiently than all other analysed insecticides. To investigate new resistance mechanisms in C. capitata we analysed the diversity of the cytochrome P450 enzymes, which have been associated to metabolic resistance in insects, and we developed a new method to detect single point mutations in acetylcholinesterase (Ccace2) and aliesterase (Ccae7) genes that could appear. Using degenerate primers we obtained 37 CYP genes, coding P450 enzymes, included in five families. Afterwards, in a phenobarbital-induction study, we observed that the expression of 4 out of the 6 analysed genes could be induced. On the other hand, a system was set up to amplify and to sequence from genomic DNA the exons of genes Ccace2 and Ccae7 where mutations related to insecticide resistance have been found in other species. The results obtained could facilitate the study of new resistance mechanisms in C. capitata mediated by these enzymes. A PCR-RFLP method was designed to detect the presence of the mutation AChE G328A (resistance allele Ccace2R), with no need to perform bioassays and allowing detecting resistance at low frequency. According to the analysis, the resistance allele was found in 25 out of 27 sampled locations in Spain, including the Balearic and the Canary Islands. However, this allele was not detected in other populations collected in 11 countries from 5 continents. The follow-up of the presence of the allele Ccace2R in the malathion-resistant strains during the selection process in the laboratory showed a quick decrease in homozygous individuals, for both the susceptible and the resistant alleles, favouring heterozygous. Thus, after 52 generations of selection, all the individuals analysed from W-10Km strain showed a heterozygous genotype for mutation AChE G328A, contradicting mendelian segregation as expected for a gene with two alleles. Afterwards, we were able to demonstrate that this was caused by the presence of a duplication of the gene coding acetylcholinesterase by: i) crossing heterozygous individuals from W-10Km strain with susceptible homozygous from C strain, originating a F1 population in which 100% of individuals were heterozygous; ii) evaluating the number of copies of gen Ccace2 by quantitative PCR in real time (qPCR), that happened to be twice higher in individuals from W-10Km VII strain when compared with C strain; iii) analysing the level of expression of Ccace2, twice in W- 10Km strain when compared to C strain; iv) studying the acetylcholinesterase activity, that was higher in individuals from W-10Km strain. According to these results, duplication of gen Ccace2 originates the coexistence of the susceptible and the resistant allele in the same chromosome. The two linked copies of the gene Ccace2 provoke the existence of permanent heterozygosis (Ccace2RS). This explains why the 100% of individuals from W-10Km strain showed an heterozygous restriction pattern since, in fact, they were structural homozygotes for the duplication (genotype Ccace2RS/RS). A biological cost has been detected associated to this duplication, consisting in a rise in accumulated adult mortality from the seventh day after emergence. The Ccace2RS duplication described in this study represents a new resistance mechanism to malathion in C. capitata. Finally, by the design of a double PCR-RFLP method, the presence of Ccace2RS duplication was confirmed in most of the Spanish populations. We observed that the proportion of individuals carrying the duplication oscillated between 5 and 35%, the frequency being higher in those C. capitata populations collected in the area of the Mediterranean basin. Therefore, we can conclude that malathion resistance associated to mutation AChE G328A and to Ccace2RS duplication are widely distributed in Spanish populations of C. capitata. Our results advice against the use of malathion (if it came to be newly authorized for use) or other OPs for the control of this pest. Besides, one of the malathion-resistant strains showed cross-resistance against insecticides with diverse action modes that are currently used for pest control, such as lambdacyhalotrin and lufenuron. High susceptibility to spinosad in the Spanish populations, as well as the reduced cross-resistance estimated for this insecticide suggests its adequacy for Medfly control. However, the use of a single insecticide is a risky strategy since it favours the selection of resistance. In fact, a population highly resistant to spinosad was obtained through laboratory selection. Therefore, it is advisable to implement integrated pest management (IPM) and resistance management programs for C. capitata control. Using insecticides with different modes of action and diverse control systems would contribute to the sustainability of the pest control. The resistance allele detection systems developed through this work will allow the early detection of resistance in the field, making possible the selection of the most appropriate method for pest control. Besides, the generated knowledge may also contribute to the development of new detection systems for other resistance mechanisms.

Constitutive and regulated α-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease

Amyloid β peptide (Aβ), the principal proteinaceous component of amyloid plaques in brains of Alzheimer’s disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative α-secretase within the Aβ sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPsα into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated α-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous α-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and Aβ fragments confirm the correct α-secretase cleavage site and demonstrate a dependence on the substrate’s conformation. Our results provide evidence that ADAM 10 has α-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer’s disease.

Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.

Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of l-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an ≈2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.