共查询到20条相似文献,搜索用时 46 毫秒
1.
2.
The biochemical essence of prion replication is the molecular multiplication of the disease-associated misfolded isoform of prion protein (PrP), termed PrPSc, in a nucleic acid-free manner. PrPSc is generated by the protein misfolding process facilitated by conformational conversion of the host-encoded cellular PrP to PrPSc. Evidence suggests that an auxiliary factor may play a role in PrPSc propagation. We and others previously discovered that plasminogen interacts with PrP, while its functional role for PrPSc propagation remained undetermined. In our recent in vitro PrP conversion study, we showed that plasminogen substantially stimulates PrPSc propagation in a concentration-dependent manner by accelerating the rate of PrPSc generation while depletion of plasminogen, destabilization of its structure and interference with the PrP-plasminogen interaction hinder PrPSc propagation. Further investigation in cell culture models confirmed an increase of PrPSc formation by plasminogen. Although molecular basis of the observed activity for plasminogen remain to be addressed, our results demonstrate that plasminogen is the first cellular protein auxiliary factor proven to stimulate PrPSc propagation.Key words: prion, PrPSc, protein misfolding, auxiliary factor, plasminogen, PMCA, cell culture modelPrions are unique infectious particles that replicate in the absence of nucleic acids1 and cause fatal neurologic disorders in mammals.2 In fact, the prion particle is composed of an alternatively folded form of the cellular prion protein (PrPC) encoded by the Prnp gene. The misfolded form of PrPC, referred to as PrPSc, shares the same primary structure with PrPC,3 but exhibits distinctively different biochemical and biophysical properties.4,5 Moreover, animals lacking expression of the Prnp gene are resistant to prion infection,6 suggesting that PrPC serves as a precursor for PrPSc.The essence of prion biosynthesis is based on the protein-only hypothesis that postulates self-perpetuating replication of the prion protein.1 Although the exact replication process remains elusive, the template-assisted conversion model proposes an idea that PrPSc serves as a template to convert α-helices of PrPC into the β-sheets of PrPSc during prion replication.7 According to many lines of evidence, there exists an unidentified auxiliary factor, designated protein X, which favorably interacts with PrPC to produce a thermodynamically stable intermediate conformation called PrP*.8 By introducing PrPSc to a host, dimers will readily form between PrP* and PrPSc. This interaction induces PrP* to take on the conformation of PrPSc and results in a complex consisting of the template and the newly formed PrPSc molecules. Once the dimer disassociates protein X and the two PrPSc molecules are released and allowed to continue replicating in an exponential fashion. Recent observations that infectious material can be generated in vitro using recombinant PrP have authenticated the protein-only hypothesis.9,10 However, the infectivity of these in vitro generated PrPSc products is lower than that of brain-derived PrPSc, leading to the possibility that insufficient levels of the unidentified auxiliary factor are the limiting factor for these in vitro assays.To date, non-mammalian chaperone proteins, sulfated glycans and certain polyanionic macromolecules, such as RNA, have shown to increase the level of PrPSc in several different in vitro assays.11–14 However, defining these molecules as cellular auxiliary factors that promote the conversion of PrPC to PrPSc has been prevented for several reasons. First, overexpression of yeast heat shock protein Hsp 104 in transgenic mice does not modulate the incubation time of disease and PrPSc accumulation upon prion inoculation.15 Although mammalian Hsp 70 is upregulated in humans and animals with prion diseases,16,17 it participates in downregulation, but not upregulation, of PrPSc accumulation.18 Second, the effect of sulfated glycans on PrPSc formation has been inconsistent11–13 and certain sulfated glycans inhibit PrPSc propagation in animals and cultured cells.19–21 Lastly, although RNA is able to increase PrPSc propagation and induce the formation of PrPSc de novo from purified PrPC in the absence of a PrPSc seed,22 its specificity is in question. Thus, an auxiliary factor that positively assists PrPSc replication and is composed of a mammalian cellular protein remains to be identified.Our approach to identify an auxiliary factor relies on the idea that the auxiliary factor interacts with PrPC as proposed in the protein X hypothesis.7 Therefore, we considered PrP ligands as the best candidates for this unidentified factor. By screening a phage display cDNA expression library from ScN2a cells, we identified kringle domains of plasminogen that interact with recombinant PrP folded in an α-helical conformation (α-PrP).23 In vitro binding assays showed that interaction between plasminogen and α-PrP that represents the conformational state of PrPC was enhanced by the introduction of a dominant negative mutation and the presence of the basic N-terminal sequences in PrP.23 Interaction of PrPC and plasminogen was further confirmed by the ability of plasminogen and its kringle domains to readily interact with α-PrP.24–29 However, despite greater interaction with α-PrP, plasminogen also interacted with PrP in β-sheet conformations.23 Prior to our study, it was shown that PrPSc was immunoprecipitated with beads linked to plasminogen, its first three kringle domains [K(1+2+3)], and more recently the repeating YRG motif found within the plasminogen kringle domains.30–33 However, the ability of plasminogen to bind to PrPSc was dependent on conditions of the lipid rafts and plasminogen was actually associated with PrPC in the intact lipid rafts.34To determine the functional relevance of this interaction for PrPSc replication, we explored whether plasminogen enhances PrPSc propagation using cell culture models and the in vitro PrP conversion assay, termed protein misfolding cyclic amplification (PMCA).35 The addition of plasminogen in PMCA resulted in the generation of significantly more PrPSc in a concentration-dependent manner (Fig. 1A), suggesting that plasminogen stimulates the conversion of PrPC to PrPSc. Indeed, our kinetic studies showed that plasminogen accelerates the rate of PrPSc generation during the early stages of the in vitro PrP conversion reaction (reviewed in ref. 35). In contrast, the addition of plasminogen in PMCA lacking either PrPC or PrPSc failed to generate PrPSc (Fig. 1B and C). These results suggest that both PrPC and PrPSc are required for PrPSc replication stimulated by plasminogen and that plasminogen facilitates neither spontaneous PrP conversion nor PrPSc augmentation through aggregating pre-existing PrPSc. Incubation of plasminogen with pre-formed PrPSc followed by treatments with proteinase K failed to either increase or decrease the PrPSc level compared to controls (Fig. 1D and E), suggesting that plasminogen is not involved in stabilization of PrPSc, enhancement of PrPSc resistance to protease or promotion of PrPSc binding to the membrane for western blotting. The activity to stimulate PrP conversion was a specific property of plasminogen and was not shared with other proteins such as a known PrP ligand and proteins abundantly found in the serum or at the extracellular matrices where plasminogen is present (reviewed in ref. 35). In addition, we found that the ability of plasminogen to assist in PrPSc propagation is preserved in its kringle domains (reviewed in ref. 35). Furthermore, the activity associated with plasminogen under cell-free conditions was reproduced in cell culture models. Plasminogen and its kringle domains increased PrPSc propagation in cultured cells chronically infected with mouse-adapted scrapie or chronic wasting disease prions (Fig. 2A–D). This suggests that the activity of plasminogen in PrPSc replication has biological relevance.Open in a separate windowFigure 1The role of plasminogen in PrPSc propagation. The effect of plasminogen (Plg) was assessed by PMCA using normal brain material supplemented with or without 0.5 µM human Glu-Plg (A–D) or using Plg-deficient (Plg-/-) brain material (F and G). Pre- (−) and post- (+) PMCA samples were treated with proteinase K (PK) and analyzed by western blotting. Seeds for PMCA were diluted either as indicated or 1:900 (G) −8,100 (F). (A) Stimulation of PrPSc propagation by Plg. (B) Plg-supplemented PMCA in the absence of SBH seeds. (C) Plg-supplemented PMCA in the absence of NBH. (D) Comparison of PrPSc levels in Plg-supplemented PMCA samples (during) vs. PMCA samples only incubated with Plg prior to PK digestion (after). (E) Comparison of PrPSc levels of ScN2a cell lysate after incubation with or without Plg prior to PK digestion. (F) PMCA with brain material of Plg-/- mice and genetically unaltered littermate controls (C). (G) Restoration of PMCA using Plg-/- brain material with Plg-supplementation. NBH, normal brain homogenate; SBH, sick brain homogenate; PrPKOBH, brain homogenate of PrPC-deficient mice; CL, cell lysate. Reproduced with permission from The FASEB Journal, Mays and Ryou 2010.35Open in a separate windowFigure 2PrPSc propagation increased by plasminogen in prion-infected cells. (A) The levels of PrP in ScN2a cells incubated with 0–0.5 µM human Glu-plasminogen (Plg) for two days. (B) The levels of PrP in ScN2a cells incubated with 0, 0.1 and 1.0 µM Plg or the first three kringle domains of Plg [K(1+2+3)] for six days. (C) The levels of 3F4-tagged PrPC and nascent PrPSc formation in ScN2a cells transiently transfected (Tfx) with plasmids encoding the 3F4-tagged PrP gene (PrP-3F4) and with an empty vector (mock). The transfected cells were treated with 0 or 1 µM K (1+2+3) for three days. (D) The levels of PrP in Elk21+ cells incubated with 0 and 0.5 µM Plg for two days. PrP was detected by anti-PrP antibody D13 (A and B), 3F4 (C) or 6H4 (D) before (−) and after (+) PK treatment. Reproduced by permission of the The FASEB Journal, Mays and Ryou 2010.35Corresponding to the results that plasminogen positively assists in PrPSc replication by stimulating conversion of PrPC to PrPSc, depletion of plasminogen from the PMCA reaction by using brain material derived from plasminogen-deficient mice restricted PrPSc replication to the basal level (Fig. 1F). Supplementation with plasminogen for PMCA using plasminogen-deficient brain homogenate restored PrPSc propagation to levels equivalent to that of control PMCA in which only brain material of their non-genetically altered littermates was used (Fig. 1G). Furthermore, structural destabilization of plasminogen affected the activity of plasminogen that enhances PrPSc propagation. Because intact disulfide bonds are critical in maintaining structural integrity and the binding activity of plasminogen, we conducted PMCA supplemented with either structurally intact or modified plasminogen to investigate the functionality of plasminogen. The result showed that plasminogen pre-treated sequentially with chemical agents that disrupt disulfide bonds and modify free sulfhydryl groups failed to stimulate PrPSc propagation (reviewed in ref. 35). In addition, we showed that interference with the plasminogen-PrP interaction using L-lysine abolished the plasminogen-mediated stimulation of PrPSc propagation in PMCA (reviewed in ref. 35). Because previous observations in immunoprecipitation30 and ELISA binding assays23 described that L-lysine specifically inhibited formation of the PrP-plasminogen complex, the presence of L-lysine in PMCA is considered to saturate the lysine binding motifs of kringle domains, which competitively prevents the kringle domains of plasminogen from interacting with PrP and inhibited PrP conversion. These various inhibition studies of PrPSc propagation provides confirmatory evidence that plasminogen plays an important role in PrPSc replication.Despite our progress in understanding the role of plasminogen in PrPSc propagation, we are still unable to address mechanistic details by which plasminogen exerts its function. In fact, plasminogen shares a number of the expected characteristics of the previously proposed auxiliary factor, although there are minor but distinctive discrepancies in their properties (summarized in Fig. 3). First, plasminogen may control conformational rearrangement of PrPC to PrP*, resembling a molecular chaperone. This scenario is identical to the protein X hypothesis, in which plasminogen replaces protein X to assist the conversion process. Second, plasminogen may promote aggregation of PrPSc already converted from PrPC. This will result in efficient formation of PrPSc multimers. Third, plasminogen may stabilize the pre-existing PrPSc aggregates so that stabilized aggregates are better protected from degradation or processing by the intrinsic clearance mechanism. This will result in increased accumulation and stability of PrPSc. In the second and third scenarios, the function of plasminogen is not involved in PrPC or its conversion process, but limited to the interaction with pre-formed PrPSc. Fourth, plasminogen may play a role as a scaffolding molecule that simply brings both PrPC and PrPSc together within a proximity. This will increase the frequency of interaction between PrPC and PrPSc for conversion. This scenario is distinguished from the other three by postulating plasminogen interaction with both isoforms of PrP. In contrast, plasminogen is assumed to interact with only PrPC or PrPSc in other cases. Although accumulating data including our recent studies provide critical pieces of evidence to envision described mechanistic insights, it is still premature to conclude the mechanism involved in plasminogen-mediated stimulation of PrPSc propagation.Open in a separate windowFigure 3Plausible mechanisms for plasminogen to enhance PrPSc propagation. Plasminogen may stimulate PrPSc propagation via conformational alteration of PrPC to PrP* (i), enhancement of PrPSc aggregation (ii), stabilization of pre-exisiting PrPSc aggregates (iii) or scaffolding to gather PrPC and PrPSc together (iv).
Open in a separate windowCNS, central nervous system; CJD, Creutzfeldt-Jakob disease; α-PrP, PrP in an α-helical conformation; β-PrP, PrP in an β-sheet conformation; PMCA, protein misfolding cyclic amplification.It is essential to address a few additional issues of PrPSc propagation stimulated by plasminogen aside from the mechanistic details. First, it is necessary to confirm the authenticity of PrPSc generated in the aid of plasminogen as a bona fide infectious agent. In addition, it would be interesting to compare the structural signatures of PrPSc generated in our study and found the prion seeds. This study may provide a clue to establish a structure-infectivity relationship for PrPSc. Furthermore, the ability to repeat our PMCA results in a similar assay reconstituted with defined components would clarify whether plasminogen directly contributes to PrPSc propagation. Finally, the contribution of plasminogen to the species barrier by controlling the compatibility between the host PrPC and prion strains should be determined. Collectively, these studies will be useful to identify an intricate regulatory role for plasminogen during PrPSc replication and prion transmission.Plasminogen has shown that it stimulates PrPSc propagation in the cell-free assays and cultured cells, while its role in animal models has not been obviously clarified. In opposition to the anticipated outcomes that the course of prion disease in plasminogen-deficient animals would be delayed, intracerebral prion infection of two independent mouse lines deficient in plasminogen resulted in either unchanged or accelerated disease progression when compared to the appropriate wild-type controls.36,37 These results do not support each other and there is no good interpretation to reconcile their incongruence. However, the discrepancy between the expectation and the actual outcomes of the previous studies can be caused by the intrinsic health problems associated with the mouse lines used for the studies,38–40 which raises a question for the credibility of the model system. Some clinical phenotypes of confounding health problems in plasminogen-deficient mice overlap with the typical clinical signs of prion disease in mice. Furthermore, the drastically shortened life expectancy of plasminogen-deficient mice coincides with the incubation periods of mice inoculated with prion strains used in the previous studies. Lastly, the health problems of plasminogen-deficient mice could exacerbate the progression of the course of disease in mice inoculated with prions. Thus, either unchanged or shortened incubation periods of prion-inoculated plasminogen-deficient mice may not solely reflect the effect of plasminogen deficiency related to prion replication. Alternatively, the reason for observed outcomes from the in vivo models may be associated with the presence of functionally redundant proteins for plasminogen even under its absence. Because kringle domains of plasminogen share a well-conserved structure with those of other proteins such as tissue-type plasminogen activator, hepatocyte growth factor and apolipoprotein (a),41 it is possible to postulate that other proteins that contain kringle domains can functionally replace plasminogen. Therefore, an animal model that circumvents the drawbacks associated with the current models is needed to address the relevance of plasminogen in prion replication in vivo.Based on our study that identified plasminogen as the first cellular protein cofactor for PrPSc propagation, we anticipate an intriguing opportunity to develop future diagnosis and therapeutic intervention for prion disease. Previously, the identity of a proposed auxiliary factor was obscure so most approaches either targeted PrP isoforms or were empirical.42 Our study suggests that plasminogen is a novel target to interfere with PrPSc replication. Therefore, a variety of strategies that either deplete plasminogen or interfere with the formation of PrP-plasminogen complexes would work for development of a novel therapeutic intervention for prion disease. For instance, RNA interference of plasminogen expression, monoclonal anti-plasminogen antibody to block plasminogen binding to PrP, L-lysine that saturates the binding sites of plasminogen to PrP, and chemical agents that destabilize the structure of plasminogen are potential strategies for this purpose.In conclusion, plasminogen is a PrP ligand with the ability to stimulate PrPSc propagation. Our findings are indispensable in gaining a better understanding of the underlying mechanism for PrPSc propagation, while unveiling a new therapeutic target for prion disease. 相似文献
Table 1
Properties of plasminogen as an auxiliary factor for PrPSc propagationProtein X | Plasminogen | |
Composition | Protein; macromolecules | Protein |
Expression | Brain; neuron-specific | Brain; neuroblastoma cell line, expressed more in the non-CNS |
Subcellular localization | Plasma membrane; lipid rafts | Extracellular matrix; lipid rafts |
Association with disease | Increased protein levels in the sera of human patient with CJD | |
Interaction | Only with PrPC | PrPSc, α-PrP, β-PrP |
Binding sites on PrP |
|
|
Species specificity |
|
|
Function | An auxiliary role in conversion of PrPC to PrPSc | Enhances PrPSc propagation facilitated by PrP conversion in PMCA |
Action mechanism | Binds to PrPC and alters PrPC into PrP* that interacts with PrPSc for conversion | Unknown |
3.
4.
Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.Key words: prion diseases, prion interference, prion strainsPrion diseases are fatal neurodegenerative diseases that are caused by an abnormal isoform of the prion protein, PrPSc.1 Prion strains are hypothesized to be encoded by strain-specific conformations of PrPSc resulting in strain-specific differences in clinical signs, incubation periods and neuropathology.2–7 However, a universally agreed upon definition of prion strains does not exist. Interspecies transmission and adaptation of prions to a new host species leads to the emergence of a dominant prion strain, which can be due to selection of strains from a mixture present in the inoculum, or produced upon interspecies transmission.8,9 Prion strains, when present in the same host, can interfere with each other.Prion interference was first described in mice where a long incubation period strain 22C extended the incubation period of a short incubation period strain 22A following intracerebral inoculation.10 Interference between other prion strains has been described in mice and hamsters using rodent-adapted strains of scrapie, TME, Creutzfeldt-Jacob disease and Gerstmannn-Sträussler-Scheinker syndrome following intracerebral, intraperitoneal, intravenous and sciatic nerve routes of inoculation.10–15 We previously demonstrated the detection of PrPSc from the long incubation period DY TME agent correlated with its ability to extend the incubation period or completely block the superinfecting short incubation period HY TME agent from causing disease and results in a reduction of HY PrPSc levels following sciatic nerve inoculation.12 However, it is not known if a single long incubation period agent (e.g., DY TME) can interfere with more than one short incubation period agent or if interference can occur by a natural route of infection.To examine the question if one long incubation period agent can extend the incubation period of additional short incubation period agents, hamsters were first inoculated in the sciatic nerve with the DY TME agent 120 days prior to superinfection with the short-incubation period agents HY TME, 263K scrapie and HaCWD.16–18 The HY TME and 263K scrapie agents have been biologically cloned and have distinct PrPSc properties.19,20 The HaCWD agent used in this study is seventh hamster passage that has not been biologically cloned and therefore will be referred to as a prion isolate. Sciatic nerve inoculations were performed as previously described.11,12 Briefly, hamsters were inoculated with 103.0 i.c. LD50 of the DY TME agent or equal volume (2 µl of a 1% w/v brain homogenate) of uninfected brain homogenate 120 days prior to superinfection of the same sciatic nerve with either 104.6 i.c. LD50 of the HY TME agent, 105.2 i.c. LD50 of the HaCWD agent or 104.6 i.c. LD50/g 263K scrapie agent (Bartz J, unpublished data).16,18,21 Animals were observed three times per week for the onset of clinical signs of HY TME, 263K and HaCWD based on the presence of ataxia and hyperexcitability, while the clinical diagnosis of DY TME was based on the appearance of progressive lethargy.16–18 The incubation period was calculated as the number of days between the onset of clinical signs of the agent strain that caused disease and the inoculation of that strain. The Student''s t-test was used to compare incubation periods.12 We found that sciatic nerve inoculation of both the HaCWD agent and 263K scrapie agent caused disease with a similar incubation period to animals infected with the HY TME agent (12 In hamsters inoculated with the DY TME agent 120 days prior to superinfection with the HaCWD or 263K agents, the animals developed clinical signs of DY TME with an incubation period that was not different from the DY TME agent control group (12 The PrPSc migration properties were consistent with the clinical diagnosis and all co-infected animals had PrPSc that migrated similar to PrPSc from the DY TME agent infected control animal (Fig. 1, lanes 1–10). This data indicates that the DY TME agent can interfere with more than one isolate and that interference in the CNS may be a more generalized phenomenon of prion strains.Open in a separate windowFigure 1The strain-specific properties of PrPSc correspond to the clinical diagnosis of disease. Western blot analysis of 250 µg brain equivalents of proteinase K digested brain homogenate from prion-infected hamsters following intracerebral (i.c.), sciatic nerve (i.sc.) or per os inoculation with either the HY TME (HY), DY TME (DY), 263K scrapie (263K), hamster-adapted CWD (CWD) agents or mock-infected (UN). The unglycoyslated PrPSc glycoform of HY TME, 263K scrapie and hamster-adapted CWD migrates at 21 kDa. The unglycosylated PrPSc glycoform of DY PrPSc migrates at 19 kDa. Migration of 19 and 21 kDa PrPSc are indicated by the arrows on the left of the figure. n.a., not applicable.
Open in a separate windowaNumber affected/number inoculated;bAverage days postinfection ± standard deviation;cIncubation period similar compared to control animals inoculated with the DY TME agent alone (p > 0.05). n.a., not applicable.To examine the question if prion interference can occur following a natural route of infection, hamsters were first inoculated per os with the DY TME agent and then superinfected per os with the HY TME agent at various time points post DY TME agent infection. Hamsters were per os inoculated by drying the inoculum on a food pellet and feeding this pellet to an individual animal as described previously.22 For the per os interference experiment, 105.7 i.c. LD50 of the DY TME agent or an equal volume of uninfected brain homogenate (100 µl of a 10% w/v brain homogenate) was inoculated 60, 90 or 120 days prior to per os superinfection of hamsters with 107.3 i.c. LD50 of the HY TME agent. A 60 or 90 day interval between DY TME agent infection and HY TME agent superinfection resulted in all of the animals developing clinical signs of HY TME with incubation periods that are similar to control hamsters inoculated with the HY TME agent alone (Fig. 1, lanes 11–16). The eight-day extension in the incubation period of HY TME in the 120 day interval co-infected group is consistent with a 1 log reduction in titer.21 This is the first report of prion interference by the per os route of infection, a likely route of prion infection in natural prion disease and provides further evidence that prion strain interference could occur in natural prion disease.23–25
Open in a separate windowaNumber affected/number inoculated;bAverage days postinfection ± standard deviation;cIncubation period extended compared to control animals inoculated with the HY TME agent alone (p < 0.01); n.a., not applicable.The capacity of the DY TME agent to replicate modulates its ability to interfere with the HY TME agent. TME interference, following sciatic nerve inoculation, occurs in the lumbar spinal cord and DY PrPSc abundance in this structure correlates with the ability of the DY TME agent to interfere with the HY TME agent.12 Following extraneural routes of infection, DY TME agent replication and PrPSc deposition are not detected in spleen or lymph nodes, which is the major site of extraneural HY TME agent replication.11,21,26 The DY TME agent can interfere with the HY TME agent following intraperitoneal and per os infection, suggesting that the DY TME agent is replicating in other locations that are involved in HY TME agent neuroinvasion (11 相似文献
Table 1
Clinical signs and incubation periods of hamsters inoculated in the sciatic nerve with either the HY TME, HaCWD or 263K scrapie agents, or co-infected with the DY TME agent 120 days prior to superinfection of hamsters with the HY TME, HaCWD or 263K agentsOnset of clinical signs | |||||||
First inoculation | Interval between inoculations | Second inoculation | Clinical signs | PrP-res migration | A/Ia | After 1st inoculation | After 2nd inoculation |
Mock | 120 days | HY TME | HY TME | 21 kDa | 5/5 | n.a. | 72 ± 3b |
Mock | 120 days | HaCWD | HaCWD | 21 kDa | 5/5 | n.a. | 73 ± 3 |
Mock | 120 days | 263K | 263K | 21 kDa | 5/5 | n.a. | 72 ± 3 |
DY TME | 120 days | Mock | DY TME | 19 kDa | 4/4 | 224 ± 2 | n.a. |
DY TME | 120 days | HY TME | DY TME | 19 kDa | 5/5 | 222 ± 2c | 102 ± 2 |
DY TME | 120 days | HaCWD | DY TME | 19 kDa | 5/5 | 223 ± 3c | 103 ± 3 |
DY TME | 120 days | 263K | DY TME | 19 kDa | 5/5 | 222 ± 2c | 102 ± 2 |
Table 2
Clinical signs and incubation periods of hamsters per os inoculated with either the HY TME or DY TME agent, or per os co-infected with the DY TME agent 60, 90 or 120 days prior to superinfection of hamsters with the HY TME agentOnset of clinical signs | |||||||
First inoculation | Interval between inoculations | Second inoculation | Clinical signs | PrP-res migration | A/Ia | After 1st inoculation | After 2nd inoculation |
Mock | 120 days | HY TME | HY TME | 21 kDa | 5/5 | n.a. | 140 ± 5b |
DY TME | 60 days | HY TME | HY TME | 21 kDa | 5/5 | 195 ± 6 | 135 ± 6 |
DY TME | 90 days | HY TME | HY TME | 21 kDa | 5/5 | 230 ± 5 | 140 ± 5 |
DY TME | 120 days | HY TME | HY TME | 21 kDa | 5/5 | 269 ± 3 | 149 ± 3c |
5.
Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases. They can be sporadic, genetic, or infectious disorders involving post-translational modifications of the cellular prion protein (PrPC). Prions (PrPSc) are characterized by their infectious property and intrinsic ability to convert the physiological PrPC into the pathological form, acting as a template. The “protein-only” hypothesis, postulated by Stanley B. Prusiner, implies the possibility to generate de novo prions in vivo and in vitro. Here we describe major milestones towards proving this hypothesis, taking into account physiological environment/s, biochemical properties and interactors of the PrPC.Key words: prion protein (PrP), prions, amyloid, recombinant prion protein, transgenic mouse, protein misfolding cyclic amplification (PMCA), synthethic prionPrions are responsible for a heterogeneous group of fatal neurodegenerative diseases (1 They can be sporadic, genetic or infectious disorders involving post-translational modifications of the cellular prion protein (PrPC).2 Prions are characterized by their infectious properties and by their intrinsic ability to encipher distinct biochemical properties through their secondary, tertiary and quaternary protein structures. In particular, the transmission of the disease is due to the ability of a prion to convert the physiological PrPC into the pathological form (PrPSc), acting as a template.3 The two isoforms of PrP appear to be different in terms of protein structures, as revealed by optical spectroscopy experiments such as Fourier-transform infrared and circular dichroism.4 PrPC contains 40% α-helix and 3% β-sheet, while the pathological isoform, PrPSc, presents approximately 30% α-helix and 45% β-sheet.4,5 PrPSc differs from PrPC because of its altered physical-chemical properties such as insolubility in non-denaturing detergents and proteinases resistance.2,6,7
Open in a separate windowi, infective form; v, variant; f, familial; s, sporadic; CJD, Creutzfeldt-Jakob disease; GSS, Gerstmann-Straüssler-Sheinker disease; FFI, fatal familial insomnia; sFI, sporadic fatal insomnia; BSE, bovine spongiform encephalopathy; TME, transmissible mink encephalopathy; CWD, chronic wasting disease; FSE, feline spongiform encephalopathy.73,78The prion conversion occurring in prion diseases seems to involve only conformational changes instead of covalent modifications. However, Mehlhorn et al. demonstrated the importance of a disulfide bond between the two cysteine residues at position 179 and 214 (human (Hu) PrP numbering) to preserve PrP into its physiological form. In the presence of reducing conditions and pH higher than 7, recombinant (rec) PrP tends to assume high β-sheet content and relatively low solubility like PrPSc.8 相似文献
Table 1
The prion diseasesPrion disease | Host | Mechanism |
iCJD | humans | infection |
vCJD | humans | infection |
fCJD | humans | genetic: octarepeat insertion, D178N-129V, V180I, T183A, T188K, T188R-129V, E196K, E200K, V203I, R208H, V210I, E211Q, M232R |
sCJD | humans | ? |
GSS | humans | genetic: octarepeat insertion, P102L-129M, P105-129M, A117V-129V, G131V-129M, Y145*-129M, H197R-129V, F198S-129V, D202N-129V, Q212P, Q217R-129M, M232T |
FFI | humans | genetic: D178-129M |
Kuru | fore people | infection |
sFI | humans | ? |
Scrapie | sheep | infection |
BSE | cattle | infection |
TME | mink | infection |
CWD | mule deer, elk | contaminated soils? |
FSE | cats | infection |
Exotic ungulate encephalopathy | greater kudu, nyala, oryx | infection |
6.
7.
8.
9.
Patti J. Miller Claudio L. Afonso Erica Spackman Melissa A. Scott Janice C. Pedersen Dennis A. Senne Justin D. Brown Chad M. Fuller Marcela M. Uhart William B. Karesh Ian H. Brown Dennis J. Alexander David E. Swayne 《Journal of virology》2010,84(21):11496-11504
The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table (Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).
Open in a separate windowaRequires the addition of an exogenous protease.bProtease requirement depends on the isolate examined.cPutative.Six of these serotypes were classified in the latter half of the 1970s, when the most reliable assay available to classify paramyxoviruses was the hemagglutination inhibition (HI) assay (61). However, there are multiple problems associated with the use of serology, including the inability to classify some APMVs by comparing them to the sera of the nine defined APMVs alone (2, 8). In addition, one-way antigenicity and cross-reactivity between different serotypes have been documented for many years (4, 5, 14, 25, 29, 33, 34, 41, 51, 52, 60). The ability of APMVs, like other viruses, to show antigenic drift as it evolves over time (37, 43, 54) and the wide use and availability of precise molecular methods, such as PCR and genome sequencing, demonstrate the need for a more practical classification system.The genetic diversity of APMVs is still largely unexplored, as hundreds of avian species have never been surveyed for the presence of viruses that do not cause significant signs of disease or are not economically important. The emergence of H5N1 highly pathogenic avian influenza (HPAI) virus as the cause of the largest outbreak of a virulent virus in poultry in the past 100 years has spurred the development of surveillance programs to better understand the ecology of avian influenza (AI) viruses in aquatic birds around the globe, and in some instances it has provided opportunities for observing other viruses in wild bird populations (15, 53). In 2007, as part of a seabird health surveillance program in the Falkland Islands (Islas Malvinas), oral and cloacal swabs and serum were collected from rockhopper penguins (Eudyptes chrysocome) and environmental/fecal swab pools were collected from other seabirds.While AI virus has not yet been isolated from penguins in the sub-Antarctic and Antarctic areas, there have been two reports of serum antibodies positive to H7 and H10 from the Adélie species (11, 40). Rare isolations of APMV1, both virulent (45) and of low virulence (8), have been reported from Antarctic penguins. Sera positive for APMV1 and AMPV2 have also been reported (21, 24, 38, 40, 53). Since 1981, paramyxoviruses have been isolated from king penguins (Aptenodytes patagonicus), royal penguins (Eudyptes schlegeli), and Adélie penguins (Pygoscelis adeliae) from Antarctica and little blue penguins (Eudyptula minor) from Australia that cannot be identified as belonging to APMV1 to -9 and have not yet been classified (8, 11, 38-40). The morphology, biological and genomic characteristics, and antigenic relatedness of an APMV recently isolated from multiple penguin colonies on the Falkland Islands are reported here. Evidence that the virus belongs to a new serotype (APMV10) and a demonstration of the advantages of a whole genome system of analysis based on random sequencing followed by comparison of genetic distances are presented. Only after all APMVs are reported and classified will epidemiological information be known as to how the viruses are moving and spreading as the birds travel and interact with other avian species. 相似文献
TABLE 1.
Characteristics of prototype viruses APMV1 to APMV9 and the penguin virusStrain | Host | Disease | Distribution | Fusion cleavagec | GI accession no. |
---|---|---|---|---|---|
APMV1/Newcastle disease virus | >250 species | High mortality | Worldwide | GRRQKRF | 45511218 |
Inapparent | Worldwide | GGRQGRLa | 11545722 | ||
APMV2/Chicken/CA/Yucaipa/1956 | Turkey, chickens, psittacines, rails, passerines | Decrease in egg production and respiratory disease | Worldwide | DKPASRF | 169144527 |
APMV3/Turkey/WI/1968 | Turkey | Mild respiratory disease and moderate egg decrease | Worldwide | PRPSGRLa | 209484147 |
APMV3/Parakeet/Netherlands/449/1975 | Psittacines, passerines, flamingos | Neurological, enteric, and respiratory disease | Worldwide | ARPRGRLa | 171472314 |
APMV4/Duck/Hong Kong/D3/1975 | Duck, geese, chickens | None known | Worldwide | VDIQPRF | 210076708 |
APMV5/Budgerigar/Japan/Kunitachi/1974 | Budgerigars, lorikeets | High mortality, enteric disease | Japan, United Kingdom, Australia | GKRKKRFa | 290563909 |
APMV6/Duck/Hong Kong/199/1977 | Ducks, geese, turkeys | Mild respiratory disease and increased mortality in turkeys | Worldwide | PAPEPRLb | 15081567 |
APMV7/Dove/TN/4/1975 | Pigeons, doves, turkeys | Mild respiratory disease in turkeys | United States, England, Japan | TLPSSRF | 224979458 |
APMV8/Goose/DE/1053/1976 | Ducks, geese | None known | United States, Japan | TYPQTRLa | 226343050 |
APMV9/Duck/NY/22/1978 | Ducks | None known | Worldwide | RIREGRIa | 217068693 |
APMV10/Penguin/Falkland Islands/324/2007 | Rockhopper penguins | None Known | Falkland Islands | DKPSQRIa | 300432141 |
10.
Tremblay P Ball HL Kaneko K Groth D Hegde RS Cohen FE DeArmond SJ Prusiner SB Safar JG 《Journal of virology》2004,78(4):2088-2099
Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrPSc(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4°C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrPSc(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a β-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrPSc(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrPSc in the brains of Tg196 mice. Our results argue that MoPrPSc(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrPC(P101L) as well as in Tg196 mice expressing low levels of MoPrPC(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a β-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.The discovery that brain fractions enriched for prion infectivity contain a protein (rPrPSc) that is resistant to limited proteolytic digestion advanced prion research (8, 37). N-terminal truncation of rPrPSc produced a protease-resistant fragment, denoted PrP 27-30, that is readily measured by Western blotting, enzyme-linked immunosorbent assay, or immunohistochemistry. The measurement of PrPSc was dramatically changed with the development of the conformation-dependent immunoassay (CDI), which permitted detection of full-length rPrPSc as well as previously unrecognized protease-sensitive forms of PrPSc (39).The CDI depends on using anti-PrP antibodies that react with an epitope exposed in native PrPC but that do not bind to native PrPSc. Upon denaturation, the buried epitope in PrPSc becomes exposed and readily reacts with anti-PrP antibodies. Using the CDI, we discovered that most PrPSc is protease sensitive, which we designate sPrPSc. Whether sPrPSc is an intermediate in the formation of rPrPSc remains to be determined. In Syrian hamsters inoculated with eight different strains of prions, the ratio of rPrPSc to sPrPSc was different for each strain and the concentration of sPrPSc was proportional to the length of the incubation time (39).In earlier studies, transgenic (Tg) mice, denoted Tg2866, expressing high levels of PrP(P101L) were used to model Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L point mutation. In the brains of several lines of mice expressing high levels of PrP(P101L), no rPrPSc(P101L) was detectable (26, 27, 47). This was particularly perplexing since these Tg mice expressing high levels of PrP(P101L) developed all facets of prion-induced neurodegeneration, including multicentric PrP amyloid plaques. Moreover, brain extracts from ill Tg2866 mice transmitted disease to Tg196 mice expressing low levels of PrP(P101L) that infrequently developed spontaneous neurodegeneration (29).In humans with GSS, several different mutations of the PrP gene (PRNP) resulting in nonconservative amino acid substitutions have been identified (23). In these patients, the clinical presentation, disease course, and amounts of rPrPSc in the brain are variable. Brain extracts from humans who died of GSS were inoculated into apes and monkeys, but the transmission rates were not correlated with the levels of PrPSc in the inoculum (1, 2, 9, 32). In a limited study, GSS(P102L) was transmitted to Tg mice expressing a chimeric mouse-human (MHu2 M) PrP transgene carrying the P102L mutation but not to Tg mice expressing MHu2M PrP without the mutation (47). In another study, GSS(P102L) human prions were transmitted to Tg mice expressing MoPrP(P101L) in which the transgene was incorporated through gene replacement (31). The use of gene replacement permits all of the regulatory elements that control the wild-type (wt) MoPrP gene to modulate the expression of MoPrP(P101L). In these mice, the expression level of MoPrP(P101L) in brain is likely to be similar to that in Tg196 mice.When we synthesized a 55-mer MoPrP peptide composed of residues 89 to 143 containing the P101L mutation and folded it under conditions favoring a β-structure, it induced neurodegeneration in Tg196 mice (29). When the peptide was not folded into a β-structure, it did not produce disease in Tg196 mice. We report here that the peptide-initiated disease in Tg196 mice could be serially transmitted to other Tg196 mice using brain extracts from the peptide-inoculated Tg196 mice. Using procedures derived from the CDI, brain extracts from inoculated Tg196 mice were found to contain sPrPSc(P101L), from which a 22- to 24-kDa PrP fragment was generated by limited digestion with proteinase K (PK) at 4°C and selective precipitation with phosphotungstate (PTA) (25, 39). In the interest of clarity, we have designated digestion at 4°C as “cold PK” and simply refer to standard digestion at 37°C as “PK.” To aid in distinguishing rPrPSc(P101L) from sPrPSc(P101L), their properties based on the work reported here and in other previously published papers are listed in Table Table11 (39, 40).
Open in a separate windowa?, unknown; +, positive; −, negative.In addition to inoculating Tg196 mice with brain extracts containing sPrPSc(P101L) or with the MoPrP(89-143,P101L) peptide, we inoculated Tg196 with several strains of prions carrying wt MoPrPSc-A or MoPrPSc-B. The 301V strain carrying wt MoPrPSc-B (22) exhibited similar abbreviated incubation times in both Tg196 mice and Prnpb/b mice. In contrast, the RML and 139A strains carrying wt MoPrPSc-A showed prolonged incubation times in both Tg196 and Prnpb/b mice (12, 33). Regardless of the host mouse strain, the 301V, RML, and 139A prion strains produced large amounts of rPrPSc in the brains of inoculated mice. Thus, the discovery of sPrPSc has for the first time provided a molecular signature for GSS prions that either arise spontaneously in mice or are induced by a synthetic peptide carrying the GSS mutation. 相似文献
TABLE 1.
Characteristics of PrP(P101L) isoformsCharacteristic | Isoforma
| ||
---|---|---|---|
PrPc(P101L) | sPrPSc(P101L) | rPrPSc(P101L) | |
PrP epitopes (residues 90-125) in native state | Exposed | Buried | Buried |
Precipitatable by PTA | − | + | + |
Digestion with PK at 37°C (“PK”) | Dipeptides, tripeptides | Dipeptides, tripeptides | PrP 27-30 |
Digestion with PK at 4°C (“cold PK”) | Dipeptides, tripeptides | PrP 22-24 | PrP 27-30 |
Infectious | − | ? | + |
11.
Crystal H Johnson Brianna L Skinner Sharon M Dietz David Blaney Robyn M Engel George W Lathrop Alex R Hoffmaster Jay E Gee Mindy G Elrod Nathaniel Powell Henry Walke 《Comparative medicine》2013,63(6):528-535
Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.8, 22,28,32,42 Human-to-human, animal-to-animal, and animal-to-human spread are rare.8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.39 Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.39Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.12,37,41
B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.22 Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.30 In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.2,7,10,22,28,32 In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.7,9,21,22
Open in a separate windowaCountry reflects the location where the animal was housed at the time of diagosis.Here we describe a case of melioidosis diagnosed in a pigtail macaque (Macaca nemestrina) imported into the United States from Indonesia and the implications of the detection of a select agent identified in a laboratory research colony. We also discuss the management and care of the exposed colony, zoonotic concerns regarding the animal care staff that worked with the shipment of macaques, effects on research studies, and the procedures involved in reporting a select agent incident. 相似文献
Table 1.
Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primatesCountrya | Imported from | Date reported | Species | Reference |
Australia | Borneo | 1963 | Pongo sp. | 36 |
Brunei | Unknown | 1982 | Orangutan (Pongo pygmaeus) | 33 |
France | 1976 | Hamlyn monkey (Cercopithecus hamlyni) Patas monkey (Erythrocebus patas) | 11 | |
Great Britain | Philippines and Indonesia | 1992 | Cynomolgus monkey (Macaca fascicularis) | 10 |
38 | ||||
Malaysia | Unknown | 1966 | Macaca spp. | 30 |
Unknown | 1968 | Spider monkey (Brachytelis arachnoides) Lar gibbon (Hylobates lar) | 20 | |
Unknown | 1969 | Pig-tailed macaque (Macaca nemestrina) | 35 | |
Unknown | 1984 | Banded leaf monkey (Presbytis melalophos) | 25 | |
Singapore | Unknown | 1995 | Gorillas, gibbon, mandrill, chimpanzee | 43 |
Thailand | Unknown | 2012 | Monkey | 19 |
United States | Thailand | 1970 | Stump-tailed macaque (Macaca arctoides) | 17 |
India | Pig-tailed macaque (Macaca nemestrina) | |||
Africa | Rhesus macaque (Macaca mulatta) Chimpanzee (Pan troglodytes) | |||
Unknown | 1971 | Chimpanzee (Pan troglodytes) | 3 | |
Malaysia | 1981 | Pig-tailed macaque (Macaca nemestrina) | 2 | |
Wild-caught, unknown | 1986 | Rhesus macaque (Macaca mulatta) | 13 | |
Indonesia | 2013 | Pig-tailed macaque (Macaca nemestrina) | Current article |
12.
Interactions between endothelial cells and the surrounding extracellular matrix are continuously adapted during angiogenesis, from early sprouting through to lumen formation and vessel maturation. Regulated control of these interactions is crucial to sustain normal responses in this rapidly changing environment, and dysfunctional endothelial cell behaviour results in angiogenic disorders. The proteoglycan decorin, an extracellular matrix component, is upregulated during angiogenesis. While it was shown previously that the absence of decorin leads to dysregulated angiogenesis in vivo, the molecular mechanisms were not clear. These abnormal endothelial cell responses have been attributed to indirect effects of decorin; however, our recent data provides evidence that decorin directly regulates endothelial cell-matrix interactions. This data will be discussed in conjunction with findings from previous studies, to better understand the role of this proteoglycan in angiogenesis.Key words: decorin, angiogenesis, motility, α2β1 integrin, insulin-like growth factor I receptor, Rac GTPaseLed by appropriate cues, the vascular system undergoes postnatal remodelling (angiogenesis), to maintain tissue homeostasis. Thus while much of the mature endothelium is quiescent, locally activated endothelial cells re-enter the cell cycle, and assume a motile phenotype essential for sprouting and neo-vessel formation. Concomitantly, the surrounding extracellular matrix (ECM) is significantly altered through de novo protein expression, deposition of plasma components and protease-mediated degradation. The latter liberates cryptic binding sites and sequestered growth factors in addition to intact and degraded ECM components, which themselves possess pro- and anti-angiogenic signalling properties. For supported blood flow, endothelium quiescence and integrity is re-established, and the ECM is organized into mature, cross-linked networks. In short, endothelial cells regulate ECM synthesis, assembly and turnover while the structure and composition of ECM in turn influences cellular phenotype. The ECM therefore, plays a critical role in control of endothelial cell behaviour during angiogenesis.Decorin is a member of the small leucine-rich repeat proteoglycan (SLRP) family, which was first discovered ‘decorating’ collagen I fibrils and was subsequently shown to regulate fibrillogenesis.1,2 Both the protein core and the single, covalently attached glycosaminoglycan (GAG) moieties of decorin are involved in this function, the relevance of which is demonstrated by the phenotype of the decorin null mouse, which exhibits loose, fragile skin due to dysregulated fibrillogenesis.2 Interestingly, a role for decorin in postnatal angiogenesis was also revealed by studies in the decorin null background. Corneal neoangiogenesis was reduced.3 Conversely, neo-angiogenesis was enhanced during dermal wound healing, although surprisingly this led to delayed wound closure.4 In this case, skin fragility due to the absence of decorin may have hindered wound closure, despite an increased blood supply. It is apparent however, that decorin plays a role in inflammation-associated angiogenesis. Indeed, endothelial cells undergoing angiogenic morphogenesis in this environment express decorin, while quiescent endothelial cells do not,3–6 indicating that decorin modulates endothelial cell behaviour specifically during inflammatory-associated remodelling of the vascular system.To understand decorin effects on angiogenic morphogenesis within a minimalist environment, various in vitro models of angiogenesis have been employed (6 Similarly, decorin expression enhanced tube formation on matrigel,8 but in other studies utilising this substrate was found to either have no influence9 or to inhibit tubulogenesis induced by growth factors.10 In yet another study, decorin inhibited tube formation when presented as a substrate prior to addition of collagen I.7 These contrasting observations may reflect the importance of the micro-environment within which decorin is presented. Alternatively, controversial results could result from different sources of decorin since cell types differ in their post-translational modifications of the GAG moiety. Hence, varying length or sulfation patterns of GAG chains may account for different biological activities of decorin. Discrepancies can also be explained as artefacts due to different purification protocols, such as when denaturing conditions are used to extract decorin from tissue. Taken together however, these observations suggest that decorin is neither a pro- nor an anti-angiogenic factor per se, but rather a regulator of angiogenesis, dependent on local cues for different activities. Further, that decorin is capable of both enhancing and inhibiting tubulogenesis may suggest a role in balancing vessel regression versus persistence. Immature vessels have a period of plasticity prior to maturation, during which they can be remodelled, and either regress, or given the appropriate signals, proceed to maturity.11 As a modulator of tube formation, it is tempting to speculate that decorin could influence the switch from immature to mature vessels, favouring one or the other in conjunction with signals from the local environment.
Open in a separate windowDecorin has been demonstrated to influence cell adhesion and motility, in particular, its influence on endothelial cell adhesion, migration and tube formation is controversial, and is the main focus of this table. Some additional key effects of decorin on fibroblast and platelet adhesion and motility are also summarised. In each case, the extracellular matrix environment in which the assay was conducted is shown, and where known, the proposed mechanism is stated.What are the molecular mechanisms by which decorin influences tubulogenesis? Since endothelial cell-matrix interactions control all aspects of angiogenesis, from motility, sprouting and lumen formation, to survival and proliferation, the role of decorin should be considered in this regard. Indirectly, decorin could quite feasibly modulate cell-matrix interactions through regulation of matrix structure and organisation2,12 and growth factor activity.13 However in vitro studies have begun to unravel rather more direct mechanisms. Studies on fibroblasts indicate that decorin can inhibit cell-matrix interactions by binding to and masking integrin attachment sites in matrix substrates. For instance, decorin inhibits fibroblast adhesion by competing with cell-surface GAG-containing CD44 for GAG binding sites on collagen XIV;14 similarly, decorin inhibits fibroblast adhesion to thrombospondin by interacting with the cell-binding domain of this substrate15 and may compete with fibroblast cell-surface heparin sulphate proteoglycans for binding to fibronectin.16 While such studies are rather lacking in endothelial cell systems, any one of these interactions could be relevant to endothelial cells. However, that decorin slightly enhanced endothelial cell attachment to fibronectin and collagen I in our system points to the existence of alternative mechanisms.17Indeed, a recent study demonstrated that decorin is an important signalling molecule in endothelial cells, where it both signals through the insulin-like growth factor I receptor (IGF-IR) and competes with the natural ligand for interaction.18 Further, decorin appears to be biologically available and relevant for interaction with this receptor in vivo. Increased receptor expression was observed in both native and neo-vessels in decorin knockout mouse cornea in conjunction with reduced neoangiogenesis. In accordance with this, decorin downregulates the IGF-IR in vitro,18 indicating that signalling through, and control of IGF-IR levels by decorin could be an important factor in regulating angiogenesis. Additionally, immobilised decorin supports platelet adhesion through interactions with the collagen I-binding integrin, α2β1.19 We have shown that decorin—α2β1 integrin interaction may play a part in modulating endothelial cell—collagen I interactions, and further, have demonstrated that decorin promotes motility in this context through activation of IGF-IR and the small Rho GTPase, Rac.17 Similarly, decorin stimulates fibroblast motility through activation of small Rho GTPases,20 supporting a direct mechanism by which decorin influences cell-matrix interactions and motility, via activation of key regulators of cytoskeleton and focal adhesion dynamics. It should also be noted that signalling by decorin directly through ErbB receptors has also been extensively demonstrated in cancer cell systems where these receptors are frequently overexpressed.21 This interaction was not relevant to human umbilical vein endothelial cells18 although a recent study found that decorin activated the epidermal growth factor receptor in mouse cerebral endothelial cells.8 These differences presumably depend on cell-specific factors such as receptor availability as well as relative receptor affinities. In a complex system such as angiogenesis, multiple mechanisms doubtlessly are involved. However, it is clear that modulation of cell-matrix interactions by decorin could certainly be expected to play a key role in contributing to regulation of postnatal angiogenesis.Signals from the extracellular matrix via integrins and from growth factors to their receptors are co-ordinately integrated into the complex angiogenic cascade. Evidence exists to suggest that decorin could regulate cell-matrix interactions during early tube formation, i.e., endothelial cell sprouting and cell alignment, through both influencing integrin activity and signalling through IGF-IR.17 Later stages of angiogenesis, such as lumen formation and maturation are also potentially regulated by decorin through activation of Rac and α2β1 integrin,17 since activity of both these molecules is integral to this phase of angiogenesis.22 Additionally, Rac activity is implicated in regulating endothelium permeability and integrity,23 providing further possibilities in control of endothelium function by decorin. Further investigations would be required however, to establish whether decorin exerts its effects on tubulogenesis through these molecular mechanisms.Of relevance to α2β1 integrin-dependent endothelial cell interaction with collagen I, sprouting endothelial cells would encounter interstitial ECM, of which collagen I is a major component. Further, a ‘provisional’ matrix containing collagen I is secreted by sprouting endothelial cells and may be required for motility,24 and tube formation.25 Theoretically, various interactions could exist between decorin, collagen type I and α2β1 integrin in this context, which may be differentially supported through various stages of angiogenesis. Up to eleven interaction sites of α2β1 integrin have been postulated to exist within collagen I, albeit with different affinities towards this receptor. Some of these binding sites may only be recognized by the integrin in its highly active conformation.26 By influencing the collagen I binding activity of α2β117 decorin could thus alter the number of endothelial cell—collagen I contacts, thereby modulating adhesion and motility. Additionally, some decorin and α2β1 integrin binding sites may overlap, or are in close proximity.27 By virtue of this location, decorin would be ideally placed to locally modulate collagen I—binding activity of the integrin. Interestingly, modulation of activity of both α2β1 integrin and the small Rho GTPase Rac by decorin also could have implications for collagen I fibrillogenesis, which in turn, would indirectly influence cell-matrix interactions. Both the related Rho GTPase RhoA, and α2β1 integrin are involved in cellular control of pericellular collagen I fibrillogenesis.28 Thus in addition to regulating cell independent fibrillogenesis1 decorin could potentially influence cell-mediated aspects of this process. Pertinent questions remain therefore, as to under which biological situations is the interaction between α2β1 integrin and decorin relevant, and does decorin influence α2β1 integrin activity on the cell-surface through direct interactions, and/or by inside-out signalling through the IGF-I receptor (or alternative receptors)? Further, how do differential decorin/α2β1 integrin/collagen I interactions mediate fibrillogenesis and cell-matrix interactions?Interaction of decorin with multiple binding partners makes it challenging to fully understand the role of decorin in angiogenesis (Fig. 1). A consideration of the relative accessibility and affinity of binding sites on both decorin and its'' binding partners would facilitate further understanding. It is still an open question whether collagen I—bound decorin can simultaneously interact with other ligands. In the case of the IGF-IR, the binding site on the concave surface of decorin overlaps with that of collagen I, thus mutually exclusive interactions seem more likely. That decorin clearly influences both collagen I matrix integrity and IGF-IR activity in vivo, would suggest that decorin is not exclusively associated with collagen I. Perhaps decorin occurs in a more ‘soluble’ form when locally secreted by endothelial cells undergoing angiogenic morphogenesis. Does collagen-bound decorin interact simultaneously with α2β1 integrin? This could be a possibility, since decorin core protein interacts with collagen I, allowing the possibility of GAG—integrin interaction. In this scenario however, interaction of α2β1 integrin with the GAG moiety of decorin in preference to collagen I might sound improbable. Nevertheless, during remodelling, interactions such as these could occur in a transient manner, and be crucial in controlling cell-matrix interactions in a rapidly changing environment. Interestingly, decorin interacts with IGF-IR via the core protein,18 and with α2β1 integrin via the GAG moiety17 raising yet another possibility of simultaneous decorin interaction with multiple binding partners. Additionally, while it is a matter of some debate whether decorin exists predominantly as a monomer or as a dimer in a physiologically relevant environment, it has been proposed that collagen-bound decorin could support simultaneous interactions of decorin with additional binding partners, and that dimer-monomer transitions also could facilitate differential interactions.29 Perhaps supporting multiple simultaneous interactions of decorin, the phenotype of patients with a progeroid variant of Ehlers-Danlos Syndrome indicates an essential role for properly glycosylated decorin (and the related SLRP biglycan). These patients exhibit skeletal and craniofacial abnormalities, loose skin and deficiencies in wound healing as a direct result of abnormal decorin and biglycan glycosylation, such that approximately half the population of decorin is secreted as the core protein only.30 Notably, the defect in loose skin and in wound healing is similar to the phenotype of the decorin knockout mouse.2,4 Evidently, the core protein alone cannot maintain normal function in vivo, despite being responsible for several important interactions of decorin, in particular, binding to collagen I and the IGF-IR. These studies may therefore support a requirement for simultaneous interactions of the core protein and GAG moieties for proper function of decorin.Open in a separate windowFigure 1Decorin influences cell-matrix interactions through multiple mechanisms. Decorin signals through the IGF-IR via the core protein moiety (grey diamond), and may simultaneously interact with the α2 subunit (cross-hatched subunit) of α2β1 integrin via the GAG moiety (wavy black line) (A). Activation of Rac through IGF-IR enhances motility by modulating cytoskeleton dynamics and may influence α2β1 integrin activity for collagen I through inside-out signalling (B). Decorin induces large, peripheral vinculin (grey oval)-positive focal adhesions by signalling through IGF-IR and/or α2β1 integrin (C and D). Decorin could also directly influence α2β1 integrin activity through binding to the α2 subunit and/or simultaneous interactions with collagen I (thick wavy black line) through the core protein. Collagen I interacts with the A-domain (white circle) of the α2 subunit at a site distinct to that of decorin (D). In summary, activation of IGF-IR, Rac and modulation of α2β1 integrin affinity for collagen I by decorin modulates cell-matrix interactions and contributes to enhanced motility and tubulogenesis in a collagen I environment.Modulation of cell-matrix interactions by decorin plays a key role in modulating endothelial cell motility and angiogenesis in vivo, and some of the mechanisms responsible have been elucidated in conjunction with in vitro studies. The large number of potential interactions of decorin with multiple matrix components and cell-surface receptors makes a clear understanding difficult. However, direct activation of signalling pathways by decorin has been highlighted recently as likely to play an important role. In conclusion, a better understanding of the mechanisms by which decorin regulates vessel formation and persistence would contribute to understanding how angiogenesis is dysregulated in a clinical setting, and how rational therapeutic strategies can be developed to restore tissue function and homeostasis. 相似文献
Table 1
Summary of the key functions of decorin in controlling cell behaviourCell type | Function | Decorin addition | Environment/Mechanism | References |
Endothelial (HUVEC derived) | Enhanced tubulogenesis | Overexpression | Collagen I lattices, enhanced survival potentially IGF-IR mediated | 6, 18 |
Mouse cerebral endothelial cells | Enhanced tubulogenesis | Overexpression | Matrigel substrate, EGFR activation leads to VEGF upregulation | 8 |
HUVEC | No effect on tubulogenesis | Exogenous | Matrigel substrate | 9 |
HUVEC | Inhibited tubulogenesis | Exogenous | Matrigel substrate, growth factor induced | 10 |
HUVEC, HDMEC | Inhibited tubulogenesis | Substrate | Collagen I lattice overlay | 7 |
HUVEC | Minimal adhesion | Substrate | Decorin substrate | 7 |
HUVEC | Inhibited adhesion | Exogenous | Collagen I and fibronectin | 10 |
HUVEC | Inhibited migration | Exogenous | VEGF-mediated chemotaxis through gelatin | 10 |
Endothelial (HUVEC derived) | Enhanced adhesion | Exogenous | Collagen I, fibronectin | 17 |
BAE | Inhibited migration | Overexpression | Collagen I, enhanced fibronectin fibrilllogenesis by decorin | 12 |
Endothelial (HUVEC derived) | Enhanced motility | Exogenous | Collagen I, Decorin activates IGF-IR/Rac-1 and α2β1 integrin activity | 17 |
Human lung fibroblast | Enhanced motility | Exogenous | Decorin activates Rho GTPases, mediators of motility | 20 |
Human foreskin fibroblast | Inhibited adhesion | Exogenous | Decorin GAG moiety competes with CD44 for binding to collagen XIV | 14 |
Mouse Fibroblast (3T3) | Inhibited adhesion | Exogenous | Decorin competes with cells for interaction with thrombospondin at the cell-binding domain | 15 |
Human fibroblast | Inhibits adhesion | Exogenous | Decorin GAG competes with cell-surface heparin-sulphate for interaction with fibronectin | 16 |
Platelets | Supported adhesion | Substrate | Decorin interacts with, and signals through α2β1 integrin on platelets | 19 |
13.
Nicholas Holton Kate Harrison Takao Yokota Gerard J Bishop 《Plant signaling & behavior》2008,3(1):54-55
Brassinosteroids (BRs) are perceived by Brassinosteroid Insensitive 1 (BRI1), that encodes a leucine-rich repeat receptor kinase. Tomato BRI1 has previously been implicated in both systemin and BR signalling. The role of tomato BRI1 in BR signalling was confirmed, however it was found not to be essential for systemin/wound signalling. Tomato roots were shown to respond to systemin but this response varied according to the species and growth conditions. Overall the data indicates that mutants defective in tomato BRI1 are not defective in systemin-induced wound signalling and that systemin perception can occur via a non-BRI1 mechanism.Key words: tomato BRI1, brassinosteroids, systemin, wound signallingBrassinosteroids (BRs) are steroid hormones that are essential for normal plant growth. The most important BR receptor in Arabidopsis is BRASSINOSTERIOD INSENSITIVE 1 (BRI1), a serine/threonine kinase with a predicted extracellular domain of ∼24 leucine-rich repeats (LRRs).1,2 BRs bind to BRI1 via a steroid-binding domain that includes LRR 21 and a so-called “island” domain.2,3 In tomato a BRI1 orthologue has been identified that when mutated, as in the curl3 (cu3) mutation, results in BR-insensitive dwarf plants.4 Tomato BRI1 has also been purified as a systemin-binding protein.5 Systemin is an eighteen amino acid peptide, which is produced by post-translational cleavage of prosystemin. Systemin has been implicated in wound signalling and is able to induce the production of jasmonate, protease inhibitors (PIN) and rapid alkalinization of cell suspensions (reviewed in ref. 6).To clarify whether tomato BRI1 was indeed a dual receptor it was important to first confirm its role in BR signalling. Initially this was carried out by genetic complementation of the cu3 mutant phenotype.7 Overexpression of tomato BRI1 restored the dwarf phenotype and BR sensitivity and normalized BR levels (35S:TomatoBRI1 complemented line Wt* cu3* 6-deoxocathasterone 566 964 676 6-deoxoteasterone nd 47 48 3-dehydro-6-deoxoteasterone 87 62 69 6-deoxotyphasterol nd 588 422 6-deoxocastasterone 1,755 6,247 26,210 castasterone 255 637 17,428 brassinolide nd nd nd