Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

Theileria induces oxidative stress and HIF1α activation that are essential for host leukocyte transformation.

Complex links between infection and cancer suggest that we still can learn much about tumorigenesis by studying how infectious agents hijack the host cell machinery. We studied the effects of an intracellular parasite called Theileria that infects bovine leukocytes and turns them into invasive cancer-like cells. We investigated the host cells pathways that are deregulated in infected leukocytes and might link infection and lymphoproliferative disease. We show that intracellular Theileria parasites drive a Warburg-like phenotype in infected host leukocytes, characterized by increased expression of metabolic regulators, increased glucose uptake and elevated lactate production, which were lost when the parasite was eliminated. The cohabitation of the parasites within the host cells leads to disruption of the redox balance (as measured by reduced/oxidized glutathione ratio) and elevated ROS (reactive oxygen species) levels, associated with chronic stabilization of the hypoxia-inducible factor 1 alpha (HIF1α). Inhibition of HIF1α (pharmacologically or genetically), or treatment with antioxidants, led to a marked reduction in expression of aerobic glycolytic genes and inhibited the transformed phenotype. These data show that stabilization of HIF1α, following increased ROS production, modulates host glucose metabolism and is critical for parasite-induced transformation. Our study expands knowledge about the molecular strategy used by the parasite Theileria to induce the transformed phenotypes of infected cells via reprogramming of glucose metabolism and redox signaling.

Identification of proteins undergoing expression level modifications in WI-38 SV40 fibroblasts overexpressing methionine sulfoxide reductase A.

Methionine sulfoxide reductase A overexpressing WI-38 SV40 human fibroblasts have been previously shown to exhibit higher resistance to oxidative stress by decreasing intracellular reactive oxygen species content and oxidative damage to proteins [C.R. Picot, I. Petropoulos, M. Perichon, M. Moreau, C. Nizard, B. Friguet, Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H(2)O(2)-mediated oxidative stress, Free Radic Biol Med 39 (2005) 1332-1341]. In order to get further insight into the molecular mechanisms underlying this resistance to oxidative stress, proteins that are differentially expressed in methionine sulfoxide reductase A overexpressing cells were identified by 2D gel and Western blot quantitative analyses. Five proteins were shown to be differentially expressed and were identified by mass spectrometry, some of them were related to either cellular protection against oxidative stress, apoptosis or premature ageing.

Rat peptide methionine sulphoxide reductase: cloning of the cDNA, and down-regulation of gene expression and enzyme activity during aging.

Peptide methionine sulphoxide reductase (PMSR, EC 1.8.4.6), the msrA or pmsR gene product, is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins. Decreased expression and/or activity of the PMSR with age could explain, at least in part, the accumulation of oxidized protein observed upon aging. To test this hypothesis, the rat pmsR cDNA was cloned and sequenced. The recombinant protein was expressed, its catalytic activity checked with a synthetic substrate and polyclonal antibodies were raised against recombinant PMSR. The expression of the pmsR gene and protein as well as its catalytic activity were then analysed as a function of age in the rat brain and in two organs that express the most PMSR, liver and kidney. It appears that pmsR gene expression decreases with age in liver and kidney as early as 18 months, whereas protein level and protein activity are reduced in the three organs at the very end of the life of the rat (26 months). These results suggest that the down-regulation of PMSR can contribute to the accumulation of oxidized protein that has been associated with the aging process.

Inhibition of nitric oxide synthase activity by early and advanced glycation end products in cultured rabbit proximal tubular epithelial cells.

Nitric oxide (NO) is important in the regulation of renal tubular function. We have investigated whether glycated proteins could impair the NO production by examining the effects of Amadori products (AP-BSA) and advanced glycation end products (AGE-BSA) on primary cultures of rabbit proximal tubular epithelial (PTE) cells. Nitric oxide synthase activity was assessed by measurement of the conversion of L-arginine to L-citrulline and by production of NO, after short-term (30 min) or long-term (1 or 3 days) incubation. Short incubations of PTE cells with either 200 microg/ml AP-BSA or 40 microg/ml AGE-BSA significantly decreased NO production. AP-BSA (3000 microg/ml) inhibited the Ca(2+)-dependent NOS activity even though above 50 microg/ml it increased Ca(2+)-independent NOS activity. In contrast, 40 microg/ml AGE-BSA inhibited both isoforms of NOS. Longer incubations with 200 microg/ml AP-BSA or 250 microg/ml AGE-BSA decreased NO release and inhibited Ca(2+)-dependent and -independent NOS activities. APs did not affect NO release by S-nitroso-N-acetyl-penicillamine (SNAP), while 250 microg/ml AGEs decreased it. After 3 days incubation, glycation products had no effect on the NOS cell content. Cell viability and proliferation were not modified under these experimental conditions, suggesting that the fall in NO production was not due to there being fewer cells. These data indicate that APs and AGEs directly inhibit NOS activity, and additionally that AGEs quench released NO. Thus, both types of glycated proteins alter the production of NO by PTE cells and could participate in the renal tubule dysfunction associated with aging and diabetes.

Accumulation of advanced glycation endproducts in the rat nephron: link with circulating AGEs during aging.

The accumulation of advanced glycosylation end products (AGEs) is believed to be a factor in the development of aging nephropathy. We have attempted to establish a link between the formation of AGEs and the onset of renal impairment with aging, indicated by albuminuria, using a fluorescence assay and immunohistochemical detection of AGEs in the renal extracellular matrix in rats. The fluorescence of collagenase-digested Type IV collagen from GBM increased with age, from 1.65 +/- 0.05 AU/mM OHPro (3 months) and 1.58 +/- 0.04 (10 months) to 2.16 +/- 0.06 (26 months) (p < 0.001) and 2.53 +/- 0.18 (30 months) (p < 0.001). In contrast, the extent of early glycation products significantly decreased from 5.35 +/- 0.25 nmol HCHO/nmol OHPro at 3 months to 3.14 +/- 0.19 at 10 months (p < 0.001), 3.42 +/- 0.38 at 26 months, and 0.74 +/- 0.08 at 30 months (p < 0.001). The urinary fluorescence of circulating AGE rose from 2.42 +/- 0.15 AU/mg protein (3 months), 1.69 +/- 0.07 (10 months), to 4.63 +/- 0.35 (26 months) (p < 0.01) and 4.73 +/- 0.72 (30 months), while the serum fluorescence increased from 0.39 +/- 0.02 AU/mg protein at 3 months and 0.43 +/- 0.02 at 10 months to 0.59 +/- 0.04 at 26 months (p < 0.001) and 0.54 +/- 0.03 at 30 months (p < 0.04). Polyclonal antibodies raised against AGE RNase showed faint areas of AGE immunoreactivity in mesangial areas in the nephrons of young rats. The immunolabeling of Bowman's capsule, the mesangial matrices, and the peripheral loops of glomerular and tubule basement membranes increased with rat age. The increase in circulating AGE peptides parallels the accumulation of AGEs in the nephron, and this parallels the pattern of extracellular matrix deposition, suggesting a close link between AGE accumulation and renal impairment in aging rats.

Characterization and distribution of albumin binding protein in normal rat kidney.

The mechanism by which proteins that pass through the glomerular basal lamina are taken up by proximal tubule cells is incompletely characterized. Past work has identified the kinetics of albumin binding to renal brush-border membrane. We have now purified and characterized albumin binding protein (ABP) and shown its distribution in renal proximal tubular cells. ABP was purified from rat renal proximal tubular cell brush-border membrane by affinity chromatography with rat serum albumin-Sepharose. The resulting ABP had two apparent molecular masses (55 and 31 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies to ABP were raised in rabbits and checked by immunoassay and immunoblotting. Light-microscopic immunohistochemistry showed ABP all along the proximal tubule in the pars convoluta and pars recta. Electron-microscopic immunohistochemistry showed labeling on microvilli and in apical endocytic vacuoles, dense apical tubules, and lysosomes. These results indicate that ABP is involved in proximal tubule endocytosis.

Effect of glycation of albumin on its binding to renal brush-border membrane vesicles: influence of aging in rats.

Aging is associated with the loss of preferential urinary excretion of Amadori-product glycated albumin. We have measured the binding of 125I-labeled glycated albumin to the renal brush-border membrane vesicles from young and old rats to determine whether a specific receptor-mediated endocytosis system may be involved. 125I-Glycated albumin was specifically bound by renal brush-border membrane vesicles in a time- and temperature-dependent manner; the binding was concentration-dependent, saturable and reversible. Scatchard plots gave an apparent dissociation constant Km of 488 +/- 17 nM, and a number of binding sites N of 33.5 +/- 3.4 pmol/mg protein/min in membrane vesicles from young (3 months old) rats; the binding of native [125I]albumin, gave a Km of 1194 +/- 200 nM (P < 2%) and N of 82.4 +/- 16.3 pmol/mg protein/min (P < 3%). Vesicles from 10-month-old rats had a similar Km (619.6 +/- 135.3 nM) and N (21.91 +/- 2.98 pmol/mg protein/min), while those from older (30 months old) rats had significantly increased Km (1344 +/- 237 nM, P < 3%) and N (81.3 +/- 10.9 pmol/mg protein/min, P < 1%) for 125I-glycated albumin binding. 125I-Glycated HSA was not displaced by unlabeled native HSA in less than 100-fold excess and native [125I]HSA was only displaced by a 10-fold excess of unlabeled glycated HSA. The binding of native [125I]HSA was partly inhibited (85%) by unlabeled glycated HSA. Thus, there appear to be two different binding sites, one for glycated and the other for native albumin, lying close together; and the glycation site on albumin is the discriminatory recognition factor.

Glycation of albumin with aging and diabetes in rats: changes in its renal handling.

Albumin glycation was investigated in old rats to elucidate the link between the preferential excretion of glycated albumin and age-related microalbuminuria. Postprandial blood glucose and the glycated albumin in the serum and urine of 3-, 10- and 30-month-old Wistar rats and in streptozotocin diabetic rats were determined. Blood glucose increased from 1.46 +/- 0.046 g l-1 in 3-month-old rats to 2.08 +/- 0.06 (10 months) and 1.75 +/- 0.23 (30 months) (P < 0.05). Albumin glycation level in the serum increased from 0.79 +/- 0.07 nmol HCHO/nmol albumin (3 months) to 1.41 +/- 0.14 (10 months) and 1.73 +/- 0.21 (30 months) (P < 0.05); urinary level increased from 1.63 +/- 0.39 nmol HCHO/nmol albumin (3 months) to 2.92 +/- 0.57 (10 months) and 2.39 +/- 0.36 (30 months) (P < 0.01). The percent glycated albumin in serum rose from 3.33 +/- 0.64 to 6.81 +/- 0.63 and 6.99 +/- 1.79% of total albumin (P < 0.05), whereas the urine percentage decreased from 12.81 +/- 3.97 to 12.64 +/- 2.87 and 2.63 +/- 0.97% (P < 0.05) in 3-, 10- and 30-month-old rats, respectively. Editing decreased with aging from 4.28 +/- 0.83 (3 months) to 1.84 +/- 0.32 (10 months) and 0.52 +/- 0.14 (30 months) (P < 0.01). Editing in microproteinuric diabetic rats was lower (0.95 +/- 0.08) than in 3-month-old control rats (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Anonymous marker loci within 400 kb of HLA-A generate haplotypes in linkage disequilibrium with the hemochromatosis gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3 and is less than 1 cM from the HLA class I genes; however, the precise physical location of the gene has remained elusive and controversial. The unambiguous identification of a crossover event within hemochromatosis families is very difficult; it is particularly hampered by the variability of the phenotypic expression as well as by the sex- and age-related penetrance of the disease. For these practical considerations, traditional linkage analysis could prove of limited value in further refining the extrapolated physical position of HFE. We therefore embarked upon a linkage-disequilibrium analysis of HFE and normal chromosomes from the Brittany population. In the present report, 66 hemochromatosis families yielding 151 hemochromatosis chromosomes and 182 normal chromosomes were RFLP-typed with a battery of probes, including two newly derived polymorphic markers from the 6.7 and HLA-F loci located 150 and 250 kb telomeric to HLA-A, respectively. The results suggest a strong peak of existing linkage disequilibrium focused within the i82-to-6.7 interval (approximately 250 kb). The zone of linkage disequilibrium is flanked by the i97 locus, positioned 30 kb proximal to i82, and the HLA-F gene, found 250 kb distal to HLA-A, markers of which display no significant association with HFE. These data support the possibility that HFE resides within the 400-kb expanse of DNA between i97 and HLA-F. Alternatively, the very tight association of HLA-A3 and allele 1 of the 6.7 locus, both of which are comprised by the major ancestral or founder HFE haplotype in Brittany, supports the possibility that the disease gene may reside immediately telomeric to the 6.7 locus within the linkage-disequilibrium zone. Additionally, hemochromatosis haplotypes possessing HLA-A11 and the low-frequency HLA-F polymorphism (allele 2) are supportive of a separate founder chromosome containing a second, independently arising mutant allele. Overall, the establishment of a likely "hemochromatosis critical region" centromeric boundary and the identification of a linkage-disequilibrium zone both significantly contribute to a reduction in the amount of DNA required to be searched for novel coding sequences constituting the HFE defect.

[Molecular genetics of hemochromatosis]. / Génétique moléculaire de l'hémochromatose.

Haemochromatosis is an inherited disorder of iron metabolism characterized by a general iron over loading. Without diagnosis and early treatment, it is a serous and potentially fatal disease by cardiac failure or hepatocellular carcinoma in particular. Gene prevalence was estimated at 0.06 in Brittany, so that haemochromatosis may be the most common genetic disease in this area. The biochemical defect of the disease is unknown; only one fact is well established: the iron absorption through duodenal mucosa is excessive. However, we don't know if it is a primary event. The gene is also unknown but in 1975 it was located on the short arm of chromosome 6, closely linked to the HLA class I region, less than 1 cM from HLA-A. None of the genes coding for the known iron proteins could be the haemochromatosis gene because of their chromosomal localization. In order to locate this gene with precision, we have used a reverse genetic approach now called positional cloning. Characterization of new polymorphic markers and linkage disequilibrium analysis have led us to locate the gene within a 350 kb region around HLA-A. We have then searched for all the structural genes in this region. Seven new genes have been so identified and located with precision. A structural analysis of these genes was undertaken to find an eventual abnormality in patients.

Age-related changes in albumin binding by renal brush-border membrane vesicles.

A selective proteinuria occurs with normal aging. We investigated the contribution of a defect in the receptor-mediated endocytosis to the age-related albuminuria by measuring albumin binding by renal brush-border membrane vesicles from young and old female Wistar rats using a filtration method. Old (24 months) rats had a significantly higher proteinuria (13.29 +/- 5.25 mg prot/24 h/100 g bw) than did young (3 months) rats (1.23 +/- 0.55 mg prot/24 h/100 g bw). Scatchard analysis of the kinetic parameters of 125I-albumin binding revealed a decrease in the binding capacity of brush-border membrane vesicles from old rats. The number of binding sites, N (pmol/mg protein/min) was 236.84 +/- 97.50 in old rat preparations and 380.27 +/- 178.36 in young rat vesicles (P < 0.05). By contrast, Km did not change significantly with age (478.86 +/- 259.29 nM in old rat vesicles and 498.00 +/- 220.36 nM in young rat preparations). Consequently the index of adsorptive endocytosis efficiency (the N/Km ratio) decreased drastically with age from 0.782 +/- 0.238 at 3 months to 0.547 +/- 0.199 at 24 months (P < 0.05). These data indicate that defective receptor-mediated endocytosis could, at least partly, explain the age-dependent rise in urinary albumin excretion.

[Molecular genetics of hemochromatosis]. / Génétique moléculaire de l'hémochromatose.

Haemochromatosis is an inherited disorder of iron metabolism characterized by a general iron over loading. Without diagnosis and early treatment, it is a serious and potentially fatal disease by cardiac failure or hepatocellular carcinoma in particular. Gene prevalence was estimated at 0.06 in Brittany, so that haemochromatosis may be the most common genetic disease in this area. The biochemical defect of the disease is unknown; only one fact is well established: the iron absorption through duodenal mucosa is excessive. However we don't know if it is a primary event. The gene is also unknown but in 1975 it was located on the short arm of chromosome 6, closely linked to the HLA class I region, less than 1 cM from HLA-A. None of the genes coding for the known iron proteins could be the haemochromatosis gene because of their chromosomal localization. In order to locate this gene with precision, we have used a reverse genetic approach now called positional cloning. Characterization of new polymorphic markers and linkage disequilibrium analysis, have led us to locate the gene within a 350 kb region around HLA-A. We have then searched for all the structural genes in this region. Seven new genes have been so identified and located with precision. A structural analysis of these genes was undertaken to find an eventual abnormality in patients.

Familial screening for genetic haemochromatosis by means of DNA markers.

Genetic haemochromatosis (HFE) is a frequent and potentially fatal disease. Early phlebotomies may prevent complications. The recessive gene for HFE is unknown but closely linked to the HLA-A locus. No direct test for homozygosity for HFE is currently available, apart from HLA typing within the family of a patient with confirmed HFE. During a reverse genetic approach to identify the gene, we found three anonymous genomic probes (P3, P5, and I.82) derived from previously cloned YACs and physically mapped in the HLA class I region. P3 and P5 probes recognise 3 loci (P3A, P3B, P5) and I.82 one locus about 100 kb from HLA-A. Using five biallelic polymorphisms (I.82/BglII, P3B/EcoRV, P3B/PstI, P5/HindIII, P3A/PstI), we tested 198 HLA typed subjects from the families of 22 haemochromatosis patients. The information from the five polymorphisms was sufficient to identify unequivocally extended restriction haplotypes in all families. The restriction haplotypes cosegregate with the HFE allele and enable identification of genotypically identical sibs in all families studied. The linked DNA markers described in this article avoid the disadvantages of HLA serological typing and can be used in genetic counselling of HFE families.

Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis.

Two yeast artificial chromosomes of the HLA class I region were subcloned. Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series. Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis. Only one marker presents an unequivocal association with the haemochromatosis gene and is 100 kb centromeric to HLA-A. This association however is not as strong as with HLA-A3. The results suggest two possible locations for the haemochromatosis gene: less than 100 kb centromeric to the HLA-A locus, or on the telomeric side.

Age-related changes in the plasma membrane proteoglycans of rat kidney glomerular cells.

In a previous in vivo study, we showed that the glomerular cells of rat kidney synthesize both peripheral and integral plasma membrane proteoglycans. The present work focuses on the age-related changes in these cell membrane proteoglycans. The peripheral proteoglycans in "adult control" rats aged 3 months were found to be heparan sulfate, dermatan sulfate, and chondroitin sulfate, with heparan sulfate being the main glycosaminoglycan. The integral membrane proteoglycans contained mainly dermatan sulfate plus less amounts of heparan sulfate. The relative proportions of the glycosaminoglycans in the integral membrane proteoglycans changed between 1 and 3 months. In addition, the degree of sulfation increased in both families of proteoglycans, and this was associated with an increase in glycosaminoglycan synthesis in the peripheral proteoglycans. The nature and relative proportions of the glycosaminoglycans forming the proteoglycans, did not change with age, after 10 months, and neither did the amount of glycosaminoglycans. But, the degree of sulfation of both peripheral and integral membrane proteoglycans decreased. De novo synthesized proteoglycans from 24-month-old rats had a higher overall charge than did those at other ages, owing to the presence of sulfate and carboxylic groups. We conclude that, as for glomerular basement membrane proteoglycans, biochemical alterations affect the glomerular cell membrane proteoglycans with aging.

HLA class I gene polymorphism in genetic hemochromatosis.

The authors have studied HLA class I polymorphism in an attempt to identify a genomic marker of the hemochromatosis gene. Five enzymes were used (HindIII, EcoRI, EcoRV, PvuII, and HincII) in 47-106 unrelated patients and 71-91 controls. Both populations were HLA-typed. The relationship between the restriction fragments and HLA-A antigens was considered. Comparison of the two groups revealed a significantly high frequency of one 14 kb EcoRI fragment among the HLA-A3 hemochromatosis patients. The fragment hybridizes to an HLA-A specific probe but not to an HLA-B specific probe, and is associated with the hemochromatosis gene, which is itself associated with HLA-A3.

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process.

1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the filtration method. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant Kd of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.