共查询到20条相似文献,搜索用时 109 毫秒
1.
B. C. Maddison C. A. Baker H. C. Rees L. A. Terry L. Thorne S. J. Bellworthy G. C. Whitelam K. C. Gough 《Journal of virology》2009,83(16):8293-8296
The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrPSc within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.PrPSc, a disease-specific marker for prion diseases and the likely infectious agent, is widely distributed within the central nervous system (CNS) and lymphoreticular tissues (LRS) in ovine scrapie, human variant Creutzfeldt-Jakob disease (vCJD), and cervine chronic wasting disease (CWD) during both clinical and preclinical stages (4, 11, 25). Furthermore, while the LRS distribution of PrPSc is much more restricted in bovine spongiform encephalopathy (BSE), sheep experimentally infected with BSE display a PrPSc tissue distribution more akin to that of ovine scrapie (11).For rodent-adapted scrapie and cervine CWD, the disease agent is detected in excreta when animals are in the clinical stages of disease, a process likely to contribute to environmental reservoirs of infectivity and lateral disease transmission (5, 13, 21). Within an experimental rodent model, it has also been demonstrated that the shedding of PrPSc and concomitant infectivity in feces occurs during preclinical scrapie (21).Evidence now also demonstrates that milk provides a vehicle for the transmission for prion diseases. Scrapie-free lambs fed milk from clinical scrapie-affected ewes propagate PrPSc within their LRS (8). Additionally, a recent study using a transgenic mouse bioassay demonstrated the secretion of infectivity in milk from preclinical animals where scrapie infectivity was found in milk months before the onset of clinical signs in animals with an ARQ/VRQ PrP genotype (10). The presence of scrapie infectivity within milk was irrespective of mammary gland pathology or PrPSc accumulation, and these animals were estimated to have considerable accumulation of immunohistochemically (IHC) detectable PrPSc within the LRS at the time of sampling.Here, we applied serial protein misfolding cyclic amplification (sPMCA) to the in vitro detection of PrPSc within sheep milk (Fig. (Fig.1)1) (Table (Table11).Open in a separate windowFIG. 1.sPMCA analysis of ovine milk samples. Milk was clarified and seeded into brain homogenate from sheep unexposed to the scrapie agent. Samples underwent sPMCA, and products were digested with proteinase K before analysis of 10 μl of each sample. PrP was detected with monoclonal antibodies SHA31 and P4; molecular mass markers are indicated (kDa). Milk was sampled from animals not exposed to the scrapie agent (U), those displaying clinical signs of scrapie (C), or those exposed to a scrapie-positive farm environment but not displaying clinical disease (S). NS, non-seeded PMCA brain substrate subjected to identical sPMCA conditions at the same time as positive samples were analyzed. Samples from the four nonexposed animals were analyzed 18 to 20 times each by sPMCA. Samples from clinically affected or clinically normal scrapie-exposed animals were analyzed in triplicate. For this triplicate analysis of each sample, the sPMCA round at which samples became positive is indicated under the appropriate lane. n, negative at round 12. Each sample was PrPSc negative until the stated round and thereafter was positive.
Open in a separate windowaAll animal procedures were performed under Home Office (United Kingdom) and local ethical review committee approval and compliance with the Animal (Scientific Procedures) Act of 1986.bAmino acid residues at positions 136, 154, and 171 of the PRNP gene.cIntroduction into a scrapie-affected flock.dDays postlactation to postmortem or as of 12 December 2008 for animals that were still alive at the time this paper was written.eClinical disease usually included head tremors and pruritus with associated wool loss and nervousness. The indicated clinical status was applicable throughout lactation to either postmortem or as of 12 December 2008 for animals that were still alive at the time this paper was written.fPrPSc was analyzed by IHC, Western blot analysis, or enzyme-linked immunosorbent assay. All animals with a positive result contained PrPSc within both brain and lymphatic tissues.gsPMCA was used for PrPSc detection and the results are tallied within this column. Replica analysis of a single milk sample from each animal was carried out. For animals 0695/07, 0334/07, 0335/07, 0350/07, 0333/07, 0142/07, and 0326/07, multiple milk samples were collected during the lactation period indicated and multiple samples from an individual animal were pooled before analysis.hNA, not applicable.iNonexposed animals were 750 to 1,110 days old at lactation and where applicable were 1,200 to 1,650 days old at postmortem.PMCA was first described by Saborio and colleagues (20) and allows the amplification of minute quantities of PrPSc (18). In rodent scrapie models, this methodology has detected PrPSc in both blood (2, 18) and brain (22) material in the clinical and preclinical stages of disease as well as in urine excreted during clinical disease (14). This technique has recently been applied to the high-level amplification of PrPSc from natural hosts of prion diseases, including vCJD (7), CWD (9), and scrapie (23). Fresh ovine milk was obtained from individual sheep at least 7 days postpartum. Milk was collected from individual animals into sterile containers and stored on ice for shipping. Within 48 h of collection, milk samples were stored at −80°C. Colostrum was not analyzed. After thawing milk samples, samples from the same individual animal were pooled and EDTA, Nonidet P-40, and sodium deoxycholate were added to final concentrations of 50 mM, 0.5% (vol/vol), and 0.5% wt/vol, respectively. Samples (1 ml) were centrifuged for 10 min at 16,000 × g. After cooling on ice for 5 min, clarified milk supernatant was withdrawn from under the solidified fat layer.sPMCA was carried out as described by Thorne and Terry (23), who demonstrated that samples from a range of animals containing at least one VRQ PrP allele could be amplified by this technique. Clarified milk supernatant was diluted 1 in 10 into PMCA brain homogenate substrate (10% [wt/vol] brain homogenate from a VRQ/VRQ PrP genotype animal within 150 mM NaCl, 4 mM EDTA, pH 8.0, 1.0% [wt/vol] Triton X-100, and miniprotease inhibitor; Roche) to a final volume of 100 μl. Samples contained within sealed 0.2-ml PCR tubes were placed in a rack within an ultrasonicating water bath (model 3000; Misonix) that held the bottom of the tubes 0.4 cm above the sonicator horn. Water was added to the water bath up to the rack surface, immersing the sonicator horn. The water bath was held at 37°C, and sonications were performed for 40 s at 200 W, equivalent to 80% of the maximum power output of the machine. Sonications were repeated once every 30 min for 24 h (one PMCA round), after which the amplified samples were diluted 1 in 3 with PMCA substrate in a final volume of 100 μl and the sample was subjected to further rounds of PMCA. Twelve PMCA rounds were performed for each sample, a total of 576 sonications over 12 days. PMCA samples were digested with 50 μg/ml proteinase K for 1 h at 37°C before analysis of 10 μl of each sample by Western blotting using 12% (wt/vol) acrylamide NuPAGE precast Bis-Tris gels (as described in reference 15). All clinical scrapie-affected animals or those exposed to the scrapie agent were challenged by introduction into the Ripley flock (Veterinary Laboratories Agency, United Kingdom), where natural scrapie is endemic with a high incidence since 1996. Ryder and coworkers (17) reported that all animals with PrP genotypes VRQ/VRQ and ARQ/VRQ that were born into this flock developed scrapie, with incubation periods of 21 to 28 months and 28 to 39 months, respectively. When ARQ/VRQ animals were introduced into the flock at 6 to 26 months of age, 77% of the animals had subclinical scrapie 24 to 30 months later, as detected by IHC analysis of the LRS. Here, PrPSc was detected in the milk from clinically affected animals at a rate of 92% (24 analyses; triplicate analyses of samples from 8 animals) and from scrapie-exposed, clinically normal sheep at a rate of 78% (27 analyses; triplicate analyses of samples from 9 animals) (Fig. (Fig.1)1) (Table (Table1).1). All scrapie-exposed sheep, both clinically affected and clinically normal, tested positive for PrPSc in at least one sPMCA reaction. PrPSc was amplified from the milk of sheep with VRQ/VRQ, ARR/VRQ, ARQ/VRQ, and AHQ/VRQ PrP genotypes (Table (Table1).1). It required at least four to eight rounds of sPMCA to produce detectable PrPSc within a milk sample from each of the scrapie-exposed sheep (Fig. (Fig.1).1). Replica analysis of a pooled milk sample from each individual sheep occasionally demonstrated high variability in the round that samples became positive for PrPSc (Fig. (Fig.1);1); this result may indicate the presence of very low levels of PrPSc (19) and/or heterogeneity within milk samples. Analyses of ovine milk from a New Zealand-derived scrapie-free flock kept under strict biosecurity conditions (ADAS, United Kingdom) did not amplify PrPSc within 12 rounds of sPMCA (78 analyses; up to 20 replica analyses of samples from 4 animals). For each of the sPMCA analyses, both positive and negative samples were analyzed concurrently within the same run on the same sonicator. These data demonstrate that PrPSc amplified from samples from scrapie-exposed animals is not due to spontaneous PrPSc formation or cross-contamination between samples within the sPMCA procedure. It is of note that prions were shed within milk from clinically normal, scrapie-exposed animals with multiple PrP genotypes. The ARQ/VRQ genotype is indicative of a high level of disease penetrance and widespread preclinical PrPSc accumulation within the LRS system, whereas AHQ/VRQ and ARR/VRQ genotype animals typically have much lower disease penetrance (24) and LRS involvement (11). This indicates the secretion of prions within milk regardless of high-level PrPSc accumulation within the LRS and also the very likely detection of subclinical as well as preclinical disease in some of these animals.No clinical scrapie-affected animals displayed clinical mastitis, and PrPSc was not detected within mammary gland tissue from five sheep with clinical scrapie (Fig. (Fig.22 and data not shown). This is in agreement with the study by Lacroux et al. (10), indicating that while the accumulation of PrPSc within mammary gland tissue can occur, it is not a prerequisite for its deposition within milk. Here, postmortem detection of PrPSc was carried out by routine diagnosis using IHC and Western blot analysis of the obex. Exceptions were animals 1349/08 and 1348/08, where obex tissue was analyzed by Bio-Rad TeSeE enzyme-linked immunosorbent assay (Table (Table1).1). Postmortem IHC examination of palatine tonsil, ileal Peyer''s patches, medial retropharyngeal lymph node, and mesenteric lymph node tissue was also carried out. Scrapie-exposed animals were shown to secrete PrPSc within their milk irrespective of whether they could be confirmed as scrapie positive by postmortem immunoassay detection of PrPSc within the CNS and LRS (Table (Table1).1). This discrepancy in PrPSc detection may well reflect the greater sensitivity of sPMCA compared to immunoassay detection of PrPSc; these results also indicate that PrPSc is secreted during the early stages of disease progression. Scrapie-exposed animals had PrPSc detected within their milk at least 20 months prior to possible clinical onset of disease, and this was not apparently influenced by the PrP genotype.Open in a separate windowFIG. 2.Detection of protease-resistant PrPSc within CNS and mammary gland tissues of animals displaying clinical scrapie. Tissues were prepared as 10% or 40% (wt/vol) homogenates for spinal cord and mammary gland tissue, respectively, as described previously elsewhere (15). Native or proteinase K-digested homogenates (25 μg/ml; 1 h at 37°C) were analyzed as indicated. Protease-resistant PrPSc was readily detectable within spinal cord tissue (SC; lanes 1 to 2) but was not detectable within mammary gland samples (MG; lanes 3 to 6). Either 0.33 mg (0350/07) or 0.165 mg (0326/07 and 0344/07) of spinal cord tissue and 1.32 mg (lanes 3 and 4) and 6 mg (lane 5) of mammary gland tissue was analyzed per lane. Protease-resistant PrPSc was still undetectable from 20 mg of mammary gland tissue following precipitation with sodium phosphotungstic acid (25) prior to analysis (lane 6). PrPSc within scrapie-positive brain tissue (63 μg) was readily detected by this method after spiking into 20 mg of mammary gland homogenate (B, lane 7). Full-length and fragmented protease-sensitive PrPC was readily detected within mammary gland tissue (lane 3). Animal numbers are indicated. PrP was detected with monoclonal antibody SHA31; molecular mass markers are indicated (kDa).These data clearly demonstrate that the secretion of PrPSc within milk occurs in natural scrapie. There are several routes through which the prion protein could be secreted into milk. Evidence suggests that within ovine mammary gland tissue, PrPC is actively produced within epithelial cells, and its secretion is most likely by exocytosis and the apocrine secretion of fat globules (3). It is unknown whether PrPSc is produced within epithelial cells and secreted into milk through similar mechanisms. Alternative mechanisms are through vesicular transcytosis or paracellular transport of PrPSc from the blood. It is established that prion-infected animals harbor infectivity and PrPSc within the blood during preclinical disease (6, 18) and that blood components are secreted within milk, including cell types known to colocalize with PrPSc within ovine mammary glands (12).Results indicate the potential transmission of scrapie in the milk of infected sheep for a prolonged period prior to clinical onset. As well as ewe-to-lamb disease transmission, this process is also likely to contribute to lateral transmission, as lambs fed milk from clinically infected ewes were the source for the transmission of scrapie between lambs within the first few months after birth (8). It is unknown whether other prion diseases result in the secretion of prions within milk. CWD, vCJD, and experimental ovine BSE share similarities with scrapie in the tissue distribution of infectivity, and it seems plausible that an analogous secretion mechanism may occur. Given the extended preclinical stages and the purported importance of subclinical states for these diseases (16), such an outcome would have significant implications for the transmission of prion diseases from apparently healthy animals and humans.With regard to ovine milk and milk products, scrapie is not transmissible to humans, and to date there is no evidence of the natural occurrence of ovine BSE. As such, the reported findings do not indicate the likely introduction of zoonotic prions from sheep into the human food chain. Nevertheless, the presented data do indicate caution in the risk assessment associated with such foods. Also, it is unknown if analogous shedding of prions into milk occurs with bovine BSE; evidence from previous epidemiological and bioassay studies would suggest that such a scenario seems unlikely to cause clinical disease (1, 26). However, the present report demonstrates that prions are secreted within the milk of sheep with PrP genotypes not typically associated with LRS accumulation of PrPSc and that prions were secreted from animals devoid of IHC-detectable PrPSc within their LRS. Such PrPSc tissue distribution is similar to bovine BSE, and given the importance of bovine milk in the human diet, the potential presence of low levels of prions within bovine milk warrants further investigation.Finally, analyzing milk samples by sPMCA offers a methodology with a clear potential for the identification of clinically sick animals and those with preclinical/subclinical scrapie. Such a noninvasive live-animal assay has the potential to contribute to the epidemiological study, management, and control of prion diseases within farmed animals. 相似文献
TABLE 1.
Timeline of exposure of animals to a scrapie-positive farm environment, sample collection, and scrapie statusAnimala (PrP genotypeb) | Age at exposurec | Days postexposure at lactation | Days postlactation to clinical scrapied | Clinical statuse | PrPSc detection at postmortemf | PrPSc detection in milk (positive tests/total tests)g |
---|---|---|---|---|---|---|
1349/08 (VRQ/VRQ) | Not exposed | NAh,i | NA | Negative | Negative | 0/20 |
K489 (VRQ/VRQ) | Not exposed | NAi | NA | Negative | NA (still alive) | 0/18 |
0618/06 (VRQ/VRQ) | Not exposed | NAi | NA | Negative | Negative | 0/20 |
1348/08 (VRQ/VRQ) | Not exposed | NAi | NA | Negative | Negative | 0/20 |
0695/07 (VRQ/VRQ) | Birth | 666-680 | 0 | Positive | Positive | 3/3 |
0334/07 (VRQ/VRQ) | Birth | 661-666 | 0 | Positive | Positive | 3/3 |
0335/07 (VRQ/VRQ) | Birth | 666-674 | 0 | Positive | Positive | 3/3 |
0350/07 (VRQ/VRQ) | Birth | 663-676 | 0 | Positive | Positive | 3/3 |
0333/07 (VRQ/VRQ) | Birth | 667-675 | 0 | Positive | Positive | 3/3 |
0142/07 (VRQ/VRQ) | Birth | 665-673 | 0 | Positive | Positive | 3/3 |
0326/07 (VRQ/VRQ) | Birth | 670-676 | 0 | Positive | Positive | 2/3 |
0199/07 (VRQ/VRQ) | Birth | 664 | 0 | Positive | Positive | 2/3 |
0692/07 (ARQ/VRQ) | ∼480 days | 1,003 | >450 | Negative | Positive | 2/3 |
0480/07 (ARQ/VRQ) | ∼480 days | 1,003 | >355 | Negative | Positive | 3/3 |
0349/07 (ARQ/VRQ) | ∼480 days | 1,003 | >348 | Negative | Positive | 3/3 |
0822/07 (ARQ/VRQ) | Birth | 760 | >564 | Negative | Negative | 2/3 |
2295 (AHQ/VRQ) | ∼120 days | 1,376 | >621 | Negative | Negative | 3/3 |
3148 (ARR/VRQ) | Birth | 1,288 | >621 | Negative | NA (still alive) | 2/3 |
1514 (ARR/VRQ) | Unknown | 597 | >621 | Negative | NA (still alive) | 2/3 |
1518 (ARR/VRQ) | Unknown | 597 | >621 | Negative | NA (still alive) | 1/3 |
1244 (ARR/VRQ) | Birth | 1,130 | >621 | Negative | NA (still alive) | 3/3 |
2.
C. P. A. de Haan R. Kivist? M. L. H?nninen 《Applied and environmental microbiology》2010,76(20):6942-6943
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.
Open in a separate windowaIn 0.3% of the strains, neither isoform was found. NF, not found.bNA, not associated.A total of 28 isolates (representing 6 CCs and 13 STs) were sequenced for fspA1 and compared to reference strains NCTC 11168 and 81-176. All isolates in the ST-22 CC showed the same one-nucleotide (nt) difference with both NCTC 11168 and 81-176 strains, resulting in a Thr→Ala substitution in the predicted protein sequence (represented by isolate FB7437, GenBank accession number ; Fig. HQ104931Fig.1).1). Eight other isolates in different CCs showed a 2-nt difference (isolate 1970, GenBank accession number ; Fig. HQ104932Fig.1)1) compared to strains NCTC 11168 and 81-176, although this did not result in amino acid substitutions. All 28 isolates were predicted to encode a full-length FspA1 protein.Open in a separate windowFIG. 1.Comparison of FspA1 and FspA2 isoforms. FspA1 is represented by 81-176, FB7437, and 1970. FspA2 is represented by C. jejuni strains 76763 to 1960 (GenBank accession numbers ). Scale bar represents amino acid divergence.In total, 62 isolates (representing 7 CCs and 35 STs) were subjected to fspA2 sequence analysis. Although a 100% sequence similarity between different STs was found for isolates in the ST-21, ST-45, ST-48, ST-61, and ST-206 CCs, fspA2 was generally more heterogeneous than fspA1 and we found 13 predicted FspA2 amino acid sequence variants in total (Fig. HQ104933 to HQ104946(Fig.1).1). In several isolates with uncommon and often unassigned (UA) STs, the proteins were truncated (Fig. (Fig.1),1), with most mutations being ST specific. For example, all ST-58 isolates showed a 13-bp deletion (isolate 3074_2; Fig. Fig.1),1), resulting in a premature stop codon. Also, all ST-1332 CC isolates were predicted to have a premature stop codon by the addition of a nucleotide between nt 112 and nt 113 (isolate 1960; Fig. Fig.1),1), a feature shared with two isolates typed as ST-4002 (UA). A T68A substitution in ST-1960 (isolate T-73494) also resulted in a premature stop codon. Interestingly, ST-1959 and ST-4003 (represented by isolate 4129) both lacked one triplet (nt 235 to 237), resulting in a shorter FspA2 protein. SignalP analysis showed the probability of a signal peptide between nt 22 and 23 (ACA-AA [between the underlined nucleotides]). An A24C substitution in two other strains, represented by isolate 76580, of ST-693 and ST-993 could possibly result in a truncated FspA2 protein as well.In conclusion, our results showed that FspA1 and FspA2 showed host and MLST associations. The immunogenic FspA1 seems to be conserved among C. jejuni strains, in contrast to the heterogeneous apoptosis-inducing FspA2, of which many isoforms were truncated. FspA proteins could serve as virulence factors for C. jejuni, although their roles herein are not clear at this time. 相似文献
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 CCsHost or ST complex/ST (no. of isolates) | % of strains witha: | P valueb | |
---|---|---|---|
fspA1 | fspA2 | ||
Host | |||
All (669) | 64.3 | 35.4 | |
Human (367) | 69.5 | 30.0 | <0.001 |
Poultry (183) | 79.2 | 20.8 | <0.001 |
Bovine (119) | 25.2 | 74.8 | <0.0001 |
ST complex and STs | |||
ST-21 complex (151) | 2.6 | 97.4 | <0.0001 |
ST-50 (76) | NF | 100 | <0.0001 |
ST-53 (19) | NF | 100 | <0.0001 |
ST-451 (9) | NF | 100 | <0.0001 |
ST-883 (11) | NF | 100 | <0.0001 |
ST-22 complex (22) | 100 | NF | <0.0001 |
ST-22 (11) | 100 | NF | <0.01 |
ST-1947 (9) | 100 | NF | 0.03 |
ST-45 complex (268) | 99.3 | 0.7 | <0.0001 |
ST-11 (7) | 100 | NF | NA |
ST-45 (173) | 99.4 | 0.6 | <0.0001 |
ST-137 (22) | 95.5 | 4.5 | 0.001 |
ST-230 (14) | 100 | NF | <0.0001 |
ST-48 complex (18) | 44.4 | 55.6 | NA |
ST-48 (7) | 100 | NF | NA |
ST-475 (8) | NF | 100 | <0.001 |
ST-52 complex (5) | NF | 100 | <0.01 |
ST-52 (4) | NF | 100 | 0.02 |
ST-61 complex (21) | NF | 100 | <0.0001 |
ST-61 (11) | NF | 100 | <0.0001 |
ST-618 (3) | NF | 100 | 0.04 |
ST-206 complex (5) | NF | 100 | <0.01 |
ST-283 complex (24) | 100 | NF | <0.0001 |
ST-267 (23) | 100 | NF | <0.0001 |
ST-677 complex (59) | 100 | NF | <0.0001 |
ST-677 (48) | 100 | NF | <0.0001 |
ST-794 (11) | 100 | NF | <0.001 |
ST-692 complex (3) | NF | 100 | 0.04 |
ST-1034 complex (5) | NF | 80 | NA |
ST-4001 (3) | NF | 100 | 0.04 |
ST-1287 complex/ST-945 (8) | 100 | NF | NA |
ST-1332 complex/ST-1332 (4) | NF | 100 | 0.02 |
Unassigned STs | |||
ST-58 (6) | NF | 100 | <0.01 |
ST-586 (6) | 100 | NF | NA |
3.
In Vivo Fitness Cost of the M184V Mutation in Multidrug-Resistant Human Immunodeficiency Virus Type 1 in the Absence of Lamivudine 总被引:1,自引:0,他引:1
Roger Paredes Manish Sagar Vincent C. Marconi Rebecca Hoh Jeffrey N. Martin Neil T. Parkin Christos J. Petropoulos Steven G. Deeks Daniel R. Kuritzkes 《Journal of virology》2009,83(4):2038-2043
4.
Flavia Pichiorri Hiroshi Okumura Tatsuya Nakamura Preston N. Garrison Pierluigi Gasparini Sung-Suk Suh Teresa Druck Kelly A. McCorkell Larry D. Barnes Carlo M. Croce Kay Huebner 《The Journal of biological chemistry》2009,284(2):1040-1049
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 kcat and
increases as large as 30-fold in Km. Mutants with
2–7-fold increases in Km had significantly reduced
apoptotic indices and the mutant with a 30-fold increase in
Km 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 Km and
kcat values ∼2- and ∼7-fold lower, respectively,
than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic
kinetics; one site had Km and kcat
values ∼2- and ∼140-fold lower, respectively, than for
unphosphorylated Fhit; the second site had a Km
∼60-fold higher and a kcat ∼6-fold lower than for
unphosphorylated Fhit (13).
Thus, it was possible that the alterations in Km and
kcat 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
Km (mm) kcat (s–1) A549 MKN74 Hsp60 Fdxr Hsp60 Fdxr
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