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111.
112.
Bacterial diversity and activity along a salinity gradient in soda lakes of the Kulunda Steppe (Altai, Russia) 总被引:1,自引:0,他引:1
Foti MJ Sorokin DY Zacharova EE Pimenov NV Kuenen JG Muyzer G 《Extremophiles : life under extreme conditions》2008,12(1):133-145
Here we describe the diversity and activity of sulfate reducing bacteria along a salinity gradient in four different soda
lakes from the Kulunda Steppe (South East Siberia, Russia). For this purpose, a combination of culture-dependent and independent
techniques was applied. The general bacterial and SRB diversity were analyzed by denaturing gradient gel electrophoresis (DGGE)
targeting the 16S rDNA gene. DNA was used to detect the microbial populations that were present in the soda lake sediments,
whereas ribosomal RNA was used as a template to obtain information on those that were active. Individual DGGE bands were sequenced
and a phylogenetic analysis was performed. In addition, the overall activity of SRB was obtained by measuring the sulfate
reduction rates (SRR) and their abundance was estimated by serial dilution. Our results showed the presence of minor, but
highly active microbial populations, mostly represented by members of the Proteobacteria. Remarkably high SRR were measured at hypersaline conditions (200 g L−1). A relatively high viable count indicated that sulfate reducing bacteria could be highly active in hypersaline soda lakes.
Furthermore, the increase of sodium carbonate/bicarbonate seemed to affect the composition of the microbial community in soda
lakes, but not the rate of sulfate reduction. 相似文献
113.
Mirjam Froehlicher Ksenia Groh Stephan C.F. Neuhauss Rik I.L. Eggen 《Developmental biology》2009,330(1):32-129
Estrogens are known to play a role in both reproductive and non-reproductive functions in mammals. Estrogens and their receptors are involved in the development of the central nervous system (brain development, neuronal survival and differentiation) as well as in the development of the peripheral nervous system (sensory-motor behaviors). In order to decipher possible functions of estrogens in early development of the zebrafish sensory system, we investigated the role of estrogen receptor β2 (ERβ2) by using a morpholino (MO) approach blocking erβ2 RNA translation. We further investigated the development of lateral line organs by cell-specific labeling, which revealed a disrupted development of neuromasts in morphants. The supporting cells developed and migrated normally. Sensory hair cells, however, were absent in morphants' neuromasts. Microarray analysis and subsequent in situ hybridizations indicated an aberrant activation of the Notch signaling pathway in ERβ2 morphants. We conclude that signaling via ERβ2 is essential for hair cell development and may involve an interaction with the Notch signaling pathway during cell fate decision in the neuromast maturation process. 相似文献
114.
Eloisa Berman Arévalo Mirjam A. F. Ros-Tonen 《Human ecology: an interdisciplinary journal》2009,37(6):733-747
While much research on forest partnerships hitherto has been focused mainly on the drivers behind their formation, the kind
of actors and deals involved, and the factors that promote or hinder their success, much less attention has been paid to the
dynamic relationships and processes inherent in these partnerships. Based on the study of a partnership process in an indigenous
reservation in Colombian Amazonia covering a variety of projects, this paper seeks to fill part of this lacuna by analyzing
the partnership as a dynamic ‘discursive battlefield,’ in which objectives and actions are being constantly negotiated. Actors
in the Matavén partnership strategically incorporate discursive elements in order to pursue their own interests while also
endorsing those that ensure the continuation of collaboration. We conclude that discourses are embedded in partnership micro-politics.
On the one hand, discursive shifts occur as a reflection of power balances at given moments. On the other hand, discourses
constitute indispensable resources with the potential to both enhance individual actor’s negotiating power and to create opportunities
for compromise. Within an ongoing discursive tension between ‘conservation’ and ‘indigenous autonomy,’ flexible notions such
as ‘territorial ordering’ prove to be successful in allowing space for manoeuvre and granting conceptual coherence to shifts
occurring ‘on the ground.’ 相似文献
115.
Soil moisture surpasses elevated CO2 and temperature as a control on soil carbon dynamics in a multi-factor climate change experiment 总被引:1,自引:0,他引:1
Some single-factor experiments suggest that elevated CO2 concentrations can increase soil carbon, but few experiments have examined the effects of interacting environmental factors on soil carbon dynamics. We undertook studies of soil carbon and nitrogen in a multi-factor (CO2 × temperature × soil moisture) climate change experiment on a constructed old-field ecosystem. After four growing seasons, elevated CO2 had no measurable effect on carbon and nitrogen concentrations in whole soil, particulate organic matter (POM), and mineral-associated organic matter (MOM). Analysis of stable carbon isotopes, under elevated CO2, indicated between 14 and 19% new soil carbon under two different watering treatments with as much as 48% new carbon in POM. Despite significant belowground inputs of new organic matter, soil carbon concentrations and stocks in POM declined over four years under soil moisture conditions that corresponded to prevailing precipitation inputs (1,300 mm yr?1). Changes over time in soil carbon and nitrogen under a drought treatment (approximately 20% lower soil water content) were not statistically significant. Reduced soil moisture lowered soil CO2 efflux and slowed soil carbon cycling in the POM pool. In this experiment, soil moisture (produced by different watering treatments) was more important than elevated CO2 and temperature as a control on soil carbon dynamics. 相似文献
116.
Teng Ma Warren L. Grayson Mirjam Fröhlich Gordana Vunjak‐Novakovic 《Biotechnology progress》2009,25(1):32-42
Stem cells have the ability for prolonged self‐renewal and differentiation into mature cells of various lineages, which makes them important cell sources for tissue engineering applications. Their remarkable ability to replenish and differentiate in vivo is regulated by both intrinsic and extrinsic cellular mechanisms. The anatomical location where the stem cells reside, known as the “stem cell niche or microenvironment,” provides signals conducive to the maintenance of definitive stem cell properties. Physiological condition including oxygen tension is an important component of the stem cell microenvironment and has been shown to play a role in regulating both embryonic and adult stem cells. This review focuses on oxygen as a signaling molecule and the way it regulates the stem cells' development into mesenchymal tissues in vitro. The physiological relevance of low oxygen tension as an environmental parameter that uniquely benefits stem cells' expansion and maintenance is described along with recent findings on the regulatory effects of oxygen on embryonic stem cells and adult mesenchymal stem cells. The relevance to tissue engineering is discussed in the context of the need to specifically regulate the oxygen content in the cellular microenvironment in order to optimize in vitro tissue development. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
117.
Identification of Varicella-Zoster Virus-Specific CD8 T Cells in Patients after T-Cell-Depleted Allogeneic Stem Cell Transplantation
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Pim L. J. van der Heiden Renate de Boer Dirk M. van der Steen Michel G. D. Kester Menno W. A. G. van der Hoorn Wilmy M. E. Haarman Helen E. Barnby-Porritt Jeremy W. Fry C. E. Napper Erik W. A. Marijt Roel Willemze J. H. Frederik Falkenburg Mirjam H. M. Heemskerk 《Journal of virology》2009,83(14):7361-7364
To study the role of CD8 T cells in the control of varicella-zoster virus (VZV) reactivation, we developed multimeric major histocompatibility complexes to identify VZV-specific CD8 T cells. Potential HLA-A2 binding peptides from the putative immediate-early 62 protein (IE62) of VZV were tested for binding, and peptides with sufficient binding capacity were used to generate pentamers. Patients with VZV reactivation following stem cell transplantation were screened with these pentamers, leading to the identification of the first validated class I-restricted epitope of VZV. In 42% of HLA-A2 patients following VZV reactivation, these IE62-ALW-A2 T cells could be detected ex vivo.Varicella-zoster virus (VZV) infects about 95% of the population, persists throughout life, and may lead to herpes zoster when the virus reactivates. After T-cell-depleted allogeneic stem cell transplantation (TCD alloSCT), reactivation of the virus leads to considerable morbidity (10). Primary infection elicits both humoral and cellular responses, but cellular immunity is essential for preventing herpes zoster. The VZV genome comprises more than 70 unique open reading frames that encode proteins that are coordinately expressed during replication. The product of open reading frame 62, the immediate-early 62 (IE62) protein, is required for the initiation of VZV replication (9) and is expressed at high levels before viral replication has occurred (8). Previous research has demonstrated that IE62-specific T cells were detected after primary VZV infection and in immune subjects (2, 4). In addition, T cells recognizing various other IE proteins and glycoproteins of VZV, as demonstrated by gamma interferon (IFN-γ) production upon stimulation with peptides or lysate derived from these proteins, have been described (1, 6, 13). The VZV-specific memory T cells found in these studies were predominantly CD4 T cells, while no VZV-specific CD8 T cells were demonstrated without prior in vitro expansion, possibly due to the low frequency of VZV-specific CD8 T cells or to the low sensitivity of the screening methods used to detect CD8 T cells by IFN-γ production upon stimulation. Frey et al. described CD8 epitopes of IE62 detected following in vitro restimulation. However, the HLA restriction and specificity of these T cells were not confirmed (4). Due to the lack of validated VZV-derived immunodominant peptides for major histocompatibility complex (MHC) class I, the analysis of VZV-specific CD8 T-cell responses is hampered (14). To be able to analyze the role of CD8 T cells in VZV reactivation, we therefore set out to identify epitopes for VZV by using VZV-IE62-specific MHC class I peptide complexes.The predictive algorithms BIMAS (11) and SYFPEITHI (12) were used to select potential HLA-A2 binding peptides from the IE62 protein. Peptides with a score of ≥3 (BIMAS) or ≥20 (SYFPEITHI) were considered to have potentially significant binding affinity. The 81 resulting 9-mer peptides were synthesized and tested for binding affinity with the REVEAL MHC-peptide binding assay (ProImmune, Oxford, United Kingdom). HLA-A2 binding affinity was determined by the ability of the peptides to stabilize the HLA-peptide complex. Based on the binding affinity measurements, 34 high- to medium-affinity HLA-A2 binding peptides were selected and used to generate ProVE MHC pentamers (ProImmune, Oxford, United Kingdom). To enable screening of this large number of pentamers, the pentamers were divided into five pools, each containing six or seven pentamers. In the initial screening with pooled pentamers, four HLA-A2-positive patients were screened after a clinical diagnosis of VZV reactivation after TCD alloSCT. The presence of viral DNA in plasma at the time of clinical observations of VZV reactivation was confirmed by real-time PCR on plasma samples as previously described (7). After informed consent was obtained, peripheral blood mononuclear cells (PBMCs) were cryopreserved and thawed and 0.5 × 106 cells were incubated with pentamers at a concentration of 0.03 mg/ml for 10 min at room temperature in RPMI medium supplemented with 2% fetal bovine serum. After the cells were washed twice, 8 μl of FluoroTag-phycoerythrin (PE) was added for 20 min of incubation at 4°C and the cells were counterstained with CD4, CD40, and CD19-fluorescein isothiocyanate (FITC). Flow cytometric analysis was performed on a FACScalibur fluorescence-activated cell sorter (FACS; Becton-Dickinson [BD], San Jose, CA). In one of four patients, pentamer pool 6, containing pentamers 61, 62, 64, 65, 66, and 67, was positive (0.06% of CD8 T cells); no other positive signals were observed. Staining with the individual pentamers revealed that pentamer 66, containing the epitope ALWALPHAA derived from the IE62 protein of VZV (IE62-ALW-A2) was responsible for the positive signal (0.06% of CD8 T cells, Fig. Fig.1B1B).Open in a separate windowFIG. 1.Screening with pentamers containing VZV-derived immunogenic epitopes. PBMCs of a patient after VZV reactivation following TCD alloSCT were incubated with pentamers and then stained with FluoroTag-PE to detect the pentamer-positive cells (A and B) and counterstained with CD4-, CD40-, and CD19-FITC. Pentamer staining of the CD4-, CD40-, and CD19-negative cells is shown. (A) PBMCs stained with pentamer 67 containing the epitope ALPHAAAAV, showing no specific staining. (B) PBMCs stained with pentamer 66 containing the epitope ALWALPHAA, showing specific staining. IE62-ALW-A2-specific T-cell clones were sorted into a single cell per well and expanded nonspecifically. The clones were stained with an irrelevant tetramer (C) and the IE62-ALW-A2 tetramer (D) in combination with CD8-FITC. Clones 1 and 2 were stained with a Vβ kit (BD) to demonstrate that clone 1 (E) and clone 2 (F) express different T-cell receptors. The results demonstrate that we isolated different T-cell clones that specifically stain with the IE62-ALW-A2 tetramer.To confirm the specificity of the IE62-ALW-A2-specific T cells, the pentamer-positive T cells were sorted into a single cell per well with a FACSDiva (BD) and expanded as previously described (5). The expanded T-cell clones were labeled specifically with the IE62-ALW-A2 PE-conjugated tetramer that was constructed as previously described (3) (Fig. (Fig.1D),1D), and Vβ analysis with the T-cell receptor Vβ repertoire kit (BD) showed that at least two different T-cell clones were isolated, demonstrating the oligoclonal origin of IE62-ALW-A2-positive T cells (Fig. 1E and F). To assess the cytolytic capacity of IE62-ALW-A2 T cells, chromium release assays were performed as described earlier (5). 51Cr-labeled Epstein-Barr virus (EBV) lymphoblastoid cell lines (LCLs) loaded with the IE62-ALW peptide were incubated with IE62-ALW-A2 T cells for 4 h. As demonstrated in Fig. Fig.2A,2A, HLA-A2-positive EBV LCLs loaded with the IE62-ALW-A2 peptide were lysed by both T-cell clones, whereas unloaded EBV LCLs were not lysed. To determine the avidity of the T-cell clones, the IE62-ALW-A2 peptide was titrated on EBV LCLs, and after 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells by standard enzyme-linked immunosorbent assay. Half-maximum IFN-γ production of the T-cell clones was observed when the stimulator cells were loaded with 10 ng/ml peptide, indicative of high-avidity T-cell clones (Fig. (Fig.2B).2B). To determine whether the T cells recognized cells endogenously expressing the IE-62-encoding gene, COS-A2 cells were transfected with Lipofectamine (Invitrogen, Carlsbad, CA) by using pcDNA vectors coding for different VZV genes, which were kindly provided by E. Wiertz (Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands). The transfected COS-A2 cells were used 24 h after transfection as stimulator cells in this assay. After 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells. IE62-ALW-A2 T-cell clones produced IFN-γ in response to COS-A2 cells endogenously expressing the IE62 protein, as well as COS-A2 cells pulsed with the IE62-ALW-A2 peptide. No IFN-γ was produced when the COS-A2 cells were transfected with the IE63-encoding gene of VZV or pulsed with an irrelevant peptide (Fig. (Fig.2C2C).Open in a separate windowFIG. 2.IE62-ALW-A2 T cells recognize IE62-ALW-A2 peptide-loaded target cells and target cells endogenously expressing IE62. (A) The cytolytic activity of IE62-ALW-A2-positive T-cell clones 1 and 2 was analyzed with the 51Cr release assay. T cells were incubated for 4 h with IE62-ALW-A2 peptide (pep)-loaded or unloaded, HLA-A2-positive EBV LCLs at an effector-to-target ratio of 10:1. (B) IE62-ALW-A2 T-cell clone 1 was stimulated with HLA-A2-positive EBV LCLs loaded with different concentrations of the IE62-ALW-A2 peptide. Release of IFN-γ (pg/ml) after 24 h of stimulation is shown. (C) IE62-ALW-A2 T-cell clones 1 and 2 were stimulated with HLA-A2-positive COS-A2 cells, left untreated, or loaded with the IE62-ALW-A2 peptide or with the IE4-ALR-B8 peptide as an irrelevant peptide or transfected with the IE63-encoding gene (COS-A2-IE63) or the IE62-encoding gene (COS-A2-IE62). Release of IFN-γ (picograms per milliliter) after 24 h of stimulation is shown.To determine whether IE62-ALW-A2-specific T cells were present in healthy individuals, cryopreserved PBMCs from 18 healthy, VZV-seropositive, HLA-A2-positive individuals were screened with the PE-conjugated VZV tetramer. PBMCs were labeled with tetramers for 15 min at 37°C in RPMI medium without phenol supplemented with 2% fetal bovine serum, washed, and analyzed with a FACScalibur. In 3 of these 18 serologically VZV-positive individuals, IE62-ALW-A2 tetramer-positive T cells could be detected (range, 0.01 to 0.02% of CD8 T cells). These data demonstrate that IE62-ALW-A2-specific T cells can be observed and that the frequency of these T cells is low under steady-state conditions in immunocompetent persons.To assess the frequency of IE62-ALW-A2-specific T cells in a cohort of patient who suffered from VZV reactivation following TCD alloSCT, 19 HLA-A2-positive patients after VZV reactivation following TCD alloSCT were screened by using the IE62-ALW-A2 tetramer. We screened these patients at a median of 47 days after the clinical diagnosis of VZV reactivation. In 8 of these 19 patients, IE62-ALW-A2-specific T cells could be directly detected ex vivo (mean, 0.04% [range, 0.01 to 0.11%] of CD8 T cells), indicating that this epitope is recognized in 42% of the HLA-A2-positive patients during VZV reactivation (Table (Table1).1). In VZV-seronegative patients (six screened), no IE62-ALW-A2 tetramer-positive cells could be detected.
Open in a separate windowaMean percentages of IE62-ALW-A2 tetramer-positive cells of CD8 T cells of three tetramer stainings performed on different days are shown.bPBMCs were in vitro stimulated (IVS) for 7 days with IE62-ALW-A2 peptide, and the mean percentages of tetramer-positive cells of three to six stimulations are shown. A negative result was defined as <0.01% of CD8+ T cells.cND, no PBMCs were available to do the analysis.To verify the presence of the IE62-ALW-A2-specific T cells in the patient and donor cohort and to investigate whether individuals negative for IE62-ALW-A2-specific T cells were unable to mount a response against the epitope or whether the frequency of IE62-ALW-A2-specific T cells was too low to detect by FACS, the presence of these T cells was further measured after in vitro stimulation. PBMCs were cultured at a concentration of 1 × 106/ml in 24-well plates in Iscove''s modified Dulbecco''s medium supplemented with 10% human serum in the presence of IE62-ALW peptide (1 μg/ml), interleukin-2 (IL-2; 50 IU/ml), and IL-15 (10 ng/ml). After stimulation for 7 days, the presence of IE62-ALW-A2-specific T cells was reassessed by tetramer labeling. These in vitro stimulations demonstrated that IE62-ALW-A2 CD8 T cells were detectable in another four patients and confirmed the presence of IE62-ALW-A2-specific T cells in eight patients and three healthy, VZV-seropositive individuals with ex vivo-detectable IE62-ALW-A2-specific T cells (Table (Table1;1; Fig. 3A to D). Thus, in 12 (63%) of 19 patients, IE62-ALW-A2 CD8 T cells could be detected either by direct tetramer labeling or after in vitro expansion, indicating that this HLA-A2-restricted epitope is commonly used in HLA-A2-positive individuals.Open in a separate windowFIG. 3.Detection and kinetics of IE62-ALW-A2-specific T cells. PBMCs with detectable IE62-ALW-A2 T cells (A, left side), a low level of detectable tetramer-positive cells (B, left side), or no detectable tetramer-positive cells (C and D, left side) were in vitro stimulated for 7 days with IE62-ALW-A2 peptide (I μg/ml) in the presence of IL-2 and IL-15 (A to D, right side). Cells were stained with CD4-FITC, CD40-FITC, and IE62-ALW-A2 tetramer, and the percentages of CD8+ T cells that were IE62-ALW-A2 tetramer positive are indicated. CD8+ T cells are defined as CD4− CD40− lymphocytes. (E) PBMCs of a patient during the course of VZV reactivation following TCD alloSCT were stained with the IE62-ALW-A2 tetramer in combination with CD8-FITC. The percentages of IE62-ALW-A2-specific CD8 T cells before, during, and after VZV reactivation are shown. In the box, the presence of viral DNA in peripheral blood is shown as measured by real-time PCR at various time points. The bold line illustrates the use of valaciclovir to treat the VZV reactivation.To study whether the immune response against the IE62-ALW-A2 epitope correlated with clinical reactivation, the percentage of IE62-ALW-A2-positive T cells was analyzed during the course of VZV reactivation in one patient. To determine the presence of viral DNA in plasma before and during the course of VZV reactivation, real-time PCR was performed on plasma samples derived at different time points. Six days prior to clinical signs of VZV reactivation, only 0.03% of the CD8 T cells were IE62-ALW-A2 specific. At 42 days after the onset of VZV reactivation, 0.23% of the CD8 T cells were IE62-ALW-A2 specific. After the VZV infection resolved, the percentage of IE62-ALW-A2-specific CD8 T cells declined to 0.09% at day 49 and 0.03% at day 145 after reactivation (Fig. (Fig.3D).3D). The T cells present at the peak of the response were predominantly HLA-DR positive, CD45RA negative, CCR7 negative, CD28 negative, and CD27 positive, consistent with an activated effector memory phenotype.In this study, we demonstrate that CD8 T cells specific for VZV are detectable without prior in vitro stimulation in patients with VZV reactivation following TCD alloSCT. We identified the ALWALPHAA peptide derived from the IE62-encoding gene of VZV as the first validated VZV-specific HLA class I-restricted immunogenic epitope by a pentamer-based epitope discovery method. The detection of the IE62-ALW peptide as an immunogenic peptide for VZV-specific CD8 T cells demonstrates the usefulness of this procedure for discovering new immunogenic virus- or tumor-specific epitopes. We demonstrated that, despite the low frequency, it is possible to detect VZV-specific CD8 T cells, allowing ex vivo analysis of the immune response to VZV infection, reactivation, and possibly VZV vaccination. 相似文献
TABLE 1.
Presence of IE62-ALW-A2-specific T cells in HLA-A2 patients after VZV reactivation following TCD alloSCTPatient | No. of days after:
| % IE62-ALW-A2+ T cells (SD)
| ||
---|---|---|---|---|
TCD alloSCT | VZV reactivation | Before IVSa | After IVSb | |
1 | 180 | 46 | Negative | 0.22 (0.15) |
2 | 190 | 38 | 0.03 (0.01) | 0.51 (0.21) |
3 | 545 | 31 | Negative | Negative |
4 | 294 | 52 | Negative | 0.12 (0.06) |
5 | 82 | 38 | Negative | Negative |
6 | 183 | 16 | Negative | 0.01 (0.01) |
7 | 176 | 81 | 0.02 (0.01) | 0.44 (0.06) |
8 | 99 | 35 | 0.11 (0.02) | 0.22 (0.04) |
9 | 601 | 88 | Negative | 0.01 (0.01) |
10 | 95 | 63 | Negative | Negative |
11 | 90 | 83 | Negative | Negative |
12 | 179 | 48 | Negative | Negative |
13 | 1,224 | 62 | Negative | Negative |
14 | 173 | 20 | 0.03 (0.01) | 0.22 (0.12) |
15 | 514 | 21 | 0.03 (0.01) | NDc |
16 | 635 | 40 | 0.02 (0.01) | ND |
17 | 161 | 8 | Negative | Negative |
18 | 174 | 48 | 0.01 (0.00) | 0.02 (0.01) |
19 | 92 | 49 | 0.04 (0.01) | 0.06 (0.02) |
118.
Histone deacetylase inhibitors comprise a variety of natural and synthetic compounds, which have in common that they inhibit enzymes that mediate the removal of acetyl groups from a range of proteins, including nucleosomal histones. Histone deacetylase inhibitors have anti-cancer activities in vitro and in vivo and are used in the clinic for the treatment of advanced cutaneous T cell lymphoma. The molecular pathways targeted by these compounds are discussed with an emphasis on the effects of these compounds on retinoic acid signaling. 相似文献
119.
Fredrik I. Andersson Anders Tryggvesson Michal Sharon Alexander V. Diemand Mirjam Classen Christoph Best Ronny Schmidt Jenny Schelin Tara M. Stanne Bernd Bukau Carol V. Robinson Susanne Witt Axel Mogk Adrian K. Clarke 《The Journal of biological chemistry》2009,284(20):13519-13532
The Clp protease is conserved among eubacteria and most eukaryotes, and
uses ATP to drive protein substrate unfolding and translocation into a chamber
of sequestered proteolytic active sites. The main constitutive Clp protease in
photosynthetic organisms has evolved into a functionally essential and
structurally intricate enzyme. The model Clp protease from the cyanobacterium
Synechococcus consists of the HSP100 molecular chaperone ClpC and a
mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We
have purified the ClpP3/R complex, the first for a Clp proteolytic core
comprised of heterologous subunits. The ClpP3/R complex has unique functional
and structural features, consisting of twin heptameric rings each with an
identical ClpP33ClpR4 configuration. As predicted by its
lack of an obvious catalytic triad, the ClpR subunit is shown to be
proteolytically inactive. Interestingly, extensive modification to ClpR to
restore proteolytic activity to this subunit showed that its presence in the
core complex is not rate-limiting for the overall proteolytic activity of the
ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable
similarities to the 20 S core of the proteasome, revealing a far greater
degree of convergent evolution than previously thought between the development
of the Clp protease in photosynthetic organisms and that of the eukaryotic 26
S proteasome.Proteases perform numerous tasks vital for cellular homeostasis in all
organisms. Much of the selective proteolysis within living cells is performed
by multisubunit chaperone-protease complexes. These proteases all share a
common two-component architecture and mode of action, with one of the best
known examples being the proteasome in archaebacteria, certain eubacteria, and
eukaryotes (1).The 20 S proteasome is a highly conserved cylindrical structure composed of
two distinct types of subunits, α and β. These are organized in
four stacked heptameric rings, with two central β-rings sandwiched
between two outer α-rings. Although the α- and β-protein
sequences are similar, it is only the latter that is proteolytic active, with
a single Thr active site at the N terminus. The barrel-shaped complex is
traversed by a central channel that widens up into three cavities. The
catalytic sites are positioned in the central chamber formed by the
β-rings, adjacent to which are two antechambers conjointly built up by
β- and α-subunits. In general, substrate entry into the core
complex is essentially blocked by the α-rings, and thus relies on the
associating regulatory partner, PAN and 19 S complexes in archaea and
eukaryotes, respectively (1).
Typically, the archaeal core structure is assembled from only one type of
α- and β-subunit, so that the central proteolytic chamber contains
14 catalytic active sites (2).
In contrast, each ring of the eukaryotic 20 S complex has seven distinct
α- and β-subunits. Moreover, only three of the seven
β-subunits in each ring are proteolytically active
(3). Having a strictly
conserved architecture, the main difference between the 20 S proteasomes is
one of complexity. In mammalian cells, the three constitutive active subunits
can even be replaced with related subunits upon induction by
γ-interferon to generate antigenic peptides presented by the class 1
major histocompatibility complex
(4).Two chambered proteases architecturally similar to the proteasome also
exist in eubacteria, HslV and ClpP. HslV is commonly thought to be the
prokaryotic counterpart to the 20 S proteasome mainly because both are Thr
proteases. A single type of HslV protein, however, forms a proteolytic chamber
consisting of twin hexameric rather than heptameric rings
(5). Also displaying structural
similarities to the proteasome is the unrelated ClpP protease. The model Clp
protease from Escherichia coli consists of a proteolytic ClpP core
flanked on one or both sides by the ATP-dependent chaperones ClpA or ClpX
(6). The ClpP proteolytic
chamber is comprised of two opposing homo-heptameric rings with the catalytic
sites harbored within (7). ClpP
alone displays only limited peptidase activity toward short unstructured
peptides (8). Larger native
protein substrates need to be recognized by ClpA or ClpX and then translocated
in an unfolded state into the ClpP proteolytic chamber
(9,
10). Inside, the unfolded
substrate is bound in an extended manner to the catalytic triads (Ser-97,
His-122, and Asp-171) and degraded into small peptide fragments that can
readily diffuse out (11).
Several adaptor proteins broaden the array of substrates degraded by a Clp
protease by binding to the associated HSP100 partner and modifying its protein
substrate specificity (12,
13). One example is the
adaptor ClpS that interacts with ClpA (EcClpA) and targets N-end rule
substrates for degradation by the ClpAP protease
(14).Like the proteasome, the Clp protease is found in a wide variety of
organisms. Besides in all eubacteria, the Clp protease also exist in mammalian
and plant mitochondria, as well as in various plastids of algae and plants. It
also occurs in the unusual plastid in Apicomplexan protozoan
(15), a family of parasites
responsible for many important medical and veterinary diseases such as
malaria. Of all these organisms, photobionts have by far the most diverse
array of Clp proteins. This was first apparent in cyanobacteria, with the
model species Synechococcus elongatus having 10 distinct Clp
proteins, four HSP100 chaperones (ClpB1–2, ClpC, and ClpX), three ClpP
proteins (ClpP1–3), a ClpP-like protein termed ClpR, and two adaptor
proteins (ClpS1–2) (16).
Of particular interest is the ClpR variant, which has protein sequence
similarity to ClpP but appears to lack the catalytic triad of Ser-type
proteases (17). This diversity
of Clp proteins is even more extreme in photosynthetic eukaryotes, with at
least 23 different Clp proteins in the higher plant Arabidopsis
thaliana, most of which are plastid-localized
(18).We have recently shown that two distinct Clp proteases exist in
Synechococcus, both of which contain mixed proteolytic cores. The
first consists of ClpP1 and ClpP2 subunits, and associates with ClpX, whereas
the other has a proteolytic core consisting of ClpP3 and ClpR that binds to
ClpC, as do the two ClpS adaptors
(19). Of these proteases, it
is the more constitutively abundant ClpCP3/R that is essential for cell
viability and growth (20,
21). It is also the ClpP3/R
complex that is homologous to the single type in eukaryotic plastids, all of
which also have ClpC as the chaperone partner
(16). In algae and plants,
however, the complexity of the plastidic Clp proteolytic core has evolved
dramatically. In Arabidopsis, the core complex consists of five ClpP
and four ClpR paralogs, along with two unrelated Clp proteins unique to higher
plants (22). Like ClpP3/R, the
plastid Clp protease in Arabidopsis is essential for normal growth
and development, and appears to function primarily as a housekeeping protease
(23,
24).One of the most striking developments in the Clp protease in photosynthetic
organisms and Apicomplexan parasites is the inclusion of ClpR within the
central proteolytic core. Although this type of Clp protease has evolved into
a vital enzyme, little is known about its activity or the exact role of ClpR
within the core complex. To address these points we have purified the intact
Synechococcus ClpP3/R proteolytic core by co-expression in E.
coli. The recombinant ClpP3/R forms a double heptameric ring complex,
with each ring having a specific ClpP3/R stoichiometry and arrangement.
Together with ClpC, the ClpP3/R complex degrades several polypeptide
substrates, but at a rate considerably slower than that by the E.
coli ClpAP protease. Interestingly, although ClpR is shown to be
proteolytically inactive, its inclusion in the core complex is not
rate-limiting to the overall activity of the ClpCP3/R protease. In general,
the results reveal remarkable similarities between the evolutionary
development of the Clp protease in photosynthetic organisms and the eukaryotic
proteasome relative to their simpler prokaryotic counterparts. 相似文献
120.
Philip T. Liu Mirjam Schenk Valencia P. Walker Paul W. Dempsey Melissa Kanchanapoomi Matthew Wheelwright Aria Vazirnia Xiaoran Zhang Andreas Steinmeyer Ulrich Zügel Bruce W. Hollis Genhong Cheng Robert L. Modlin 《PloS one》2009,4(6)
Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen. 相似文献