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1.
Ventzislava A. Hristova Steven A. Beasley R. Jane Rylett Gary S. Shaw 《The Journal of biological chemistry》2009,284(22):14978-14986
Missense mutations in park2, encoding the parkin protein, account
for ∼50% of autosomal recessive juvenile Parkinson disease (ARJP) cases.
Parkin belongs to the family of RBR
(RING-between-RING) E3 ligases involved in
the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R
and synphillin-1. The proposed architecture of parkin, based largely on
sequence similarity studies, consists of N-terminal ubiquitin-like and
C-terminal RBR domains. These domains are separated by a ∼160-residue
unique parkin sequence having no recognizable domain structure. We used
limited proteolysis experiments on bacterially expressed and purified parkin
to identify a new domain (RING0) within the unique parkin domain sequence.
RING0 comprises two distinct, conserved cysteine-rich clusters between
Cys150–Cys169 and
Cys196–His215 consisting of
CX2-3CX11CX2C
and
CX4–6CX10–16-CX2(H/C)
motifs. The positions of the cysteine/histidine residues in this region bear
similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING
domains. However, in parkin a 26-residue linker region separates the motifs,
which is not typical of other RING domain structures. Further, the RING0
domain includes all but one of the known ARJP mutation sites between the
ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass
spectrometry and inductively coupled plasma-atomic emission spectrometry
analysis, we determined that the RING0, RING1, IBR, and RING2 domains each
bind two Zn2+ ions, the first observation of an E3 ligase with the
ability to bind eight metal ions. Removal of the zinc from parkin causes near
complete unfolding of the protein, an observation that rationalizes
cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y)
that form cellular inclusions and/or are defective for ubiquitination likely
because of poor zinc binding and misfolding. The identification of the RING0
domain in parkin provides a new overall domain structure for the protein that
will be important in assessing the roles of ARJP mutations and designing
experiments aimed at understanding the disease.Autosomal recessive juvenile Parkinson disease
(ARJP)2 is a
neurodegenerative disorder arising from the loss of dopaminergic neurons in
the substantia nigra of the midbrain. ARJP is characterized by the onset of
Parkinsonian symptoms such as tremors, rigidity, and bradykinesia. It is
distinguished from the idiopathic form of Parkinson disease by the onset of
symptoms, prior to the age of forty. The hereditary nature of ARJP implicates
a number of mutations in the genes encoding the proteins parkin, PINK1, LRRK2,
and DJ-1 as the cause of dopaminergic neurodegeneration
(1–4).
A variety of deletion, truncation, and point mutations distributed throughout
the park2 gene, which encodes the protein parkin, have been reported
in ARJP patients (1,
5–18).Parkin functions as a ubiquitin ligase (E3) and belongs to a family of RBR
(RING-between-RING) ubiquitin ligase enzymes
involved in proteosome-mediated protein degradation
(19–21).
The currently accepted domain architecture of parkin, deduced from multiple
sequence alignment, shows that the C terminus of the protein is characterized
by two ∼50-residue RING (really interesting
new gene) domains separated by a 51-residue IBR
(In-Between-RING) domain (22,
23). The RING domains of
parkin are proposed to interact with the ubiquitin-conjugating enzymes UbcH7,
UbcH8, Ubc7, and Ubc13 and control parkin-mediated ubiquitination of a variety
of substrates such as Pael-R, synphilin-1, Sept5, and PICK1 among others
(24–31).
Other members of the RBR family include the human homolog of
Drosophila Ariadne (HHARI), DORFIN, and HOIL-1, which share close
domain architecture
(32–35).
Traditionally, RING domains coordinate two Zn2+ ions through a
C3HC4 metal-binding consensus sequence. However, the
RING2 domain of HHARI binds a single Zn2+
(36), and because this is the
only RING2 structure available for an RBR protein, it suggests that there may
be variability in the number of Zn2+ ions coordinated by different
RING domains. The recent three-dimensional structure of the parkin IBR domain
(23) revealed a two-site
zinc-binding motif with a novel fold compared with other zinc-binding motifs
(37). However, despite the
potential importance of zinc binding to the RING domains (or other portions)
of parkin, the ability and capacity for zinc coordination or its impact on
structure has not been identified for parkin.The N terminus of parkin comprises a ubiquitin-like domain (UblD) proposed
to facilitate the delivery and degradation of ubiquitinated substrates by the
26 S proteosome via interactions with the S5a subunit
(38,
39). The central ∼150
residues of parkin separating the UblD from the RBR region are referred to as
the unique parkin domain (UPD). This segment of parkin is essential for
function, and ARJP associated mutations within this region have been shown to
lead to dysfunction of parkin E3 ligase activity
(40,
41). However, the absence of
any sequence similarity to other proteins or the identification of a distinct
domain within the UPD has made these experiments difficult to interpret. Other
than the isolated UblD and IBR domains of parkin, there has been limited
success with the purification and characterization of parkin, especially when
lacking affinity tags in the final purified form. Bacterially expressed parkin
typically shows a heterogeneous mixture of full-length and degraded protein
species, making characterization of the protein difficult
(42). In this work we have
used purified parkin to identify a novel zinc-binding
C4C3(C/H) domain upstream of the RBR region and within
the UPD. We have used limited proteolysis and electrospray ionization mass
spectrometry (ESI-MS) to show that this domain coordinates two Zn2+
ions in addition to six other Zn2+ ions in the RBR C terminus. The
presence of a new parkin-specific zinc-binding domain provides insight into
the structure of parkin and opens the door to establish the importance of this
domain in ARJP for this new subclass of RBR E3 ligases. 相似文献
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Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.Dictyostelium discoideum is a widely used model organism for studying the organization and function of the endocytic pathway. In Dictyostelium, the organization of the endocytic pathway is similar to that in higher eukaryotes. The pathway in Dictyostelium can be divided into four steps (see Fig. S1 in the supplemental material): uptake at the plasma membrane of particles and medium, transfer through early acidic endocytic compartments (lysosomes), passage into less acidic postlysosomes (PLs), and finally, exocytosis of undigested materials (17, 20). Thus, Dictyostelium recapitulates many of the functions of the endocytic pathway in mammalian cells, including some features observed in most cell types (lysosome biogenesis) and some observed only in specialized cells (phagocytosis, macropinocytosis, and lysosome secretion).Dictyostelium amoebae live in the soil, where they feed by ingesting and digesting other microorganisms. In addition, axenic laboratory strains can macropinocytose medium to ensure their growth. Accordingly, both in natural situations and in laboratory settings, the endocytic pathway plays a key role in the acquisition of nutrients by Dictyostelium cells. In agreement with this notion, several observations suggest that the physiology of the endocytic pathway is sensitive to nutrient availability. In particular, starvation induces secretion of lysosomal enzymes by an unknown mechanism (11). The morphology of the endocytic pathway is also sensitive to nutritional cues, as shown for example by the observation that formation of multilamellar endosomes is enhanced in cells fed with bacteria (18).Here, we analyzed the effect of starvation on the organization as well as the dynamics of the endocytic pathway. We found that, while the overall organization was not extensively modified in starved cells, the dynamics of endocytic compartments were altered. Moreover, analysis of two specific knockout mutants, the apm3 (6) and lvsB (8) strains, revealed that their phenotype was profoundly altered upon starvation, providing further insight about the role of Apm3 and LvsB in the endocytic pathway. 相似文献
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Barrie J. Anthony Kylie R. James Geoffrey N. Gobert Grant A. Ramm Donald P. McManus 《PloS one》2013,8(6)
Hepatic fibrosis induced by egg deposition is the most serious pathology associated with chronic schistosomiasis, in which the hepatic stellate cell (HSC) plays a central role. While the effect of Schistosoma mansoni eggs on the fibrogenic phenotype of HSCs has been investigated, studies determining the effect of eggs of
S
. japonicum
on HSCs are lacking. Disease caused by
S
. japonicum
is much more severe than that resulting from S. mansoni infection so it is important to compare the pathologies caused by these two parasites, to determine whether this phenotype is due to the species interacting differently with the mammalian host. Accordingly, we investigated the effect of
S
. japonicum
eggs on the human HSC cell line, LX-2, with and without TGF-β (Transforming Growth Factor beta) co-treatment, so as to determine the impact on genes associated with fibrogenesis, inflammation and matrix re-organisation. Activation status of HSCs was assessed by αSMA (Alpha Smooth Muscle Actin) immunofluorescence, accumulation of Oil Red O-stained lipid droplets and the relative expression of selected genes associated with activation. The fibrogenic phenotype of HSCs was inhibited by the presence of eggs both with or without TGF-β treatment, as evidenced by a lack of αSMA staining and reduced gene expression of αSMA and Col1A1 (Collagen 1A1). Unlike S. mansoni-treated cells, however, expression of the quiescent HSC marker PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) was not increased, nor was there accumulation of lipid droplets. In contrast,
S
. japonicum
eggs induced the mRNA expression of MMP-9 (Matrix Metalloproteinase 9), CCL2 (Chemokine (C-C motif) Ligand 2) and IL-6 (Interleukin 6) in HSCs indicating that rather than inducing complete HSC quiescence, the eggs induced a proinflammatory phenotype. These results suggest HSCs in close proximity to
S
. japonicum
eggs in the liver may play a role in the proinflammatory regulation of hepatic granuloma formation. 相似文献
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The purpose of this paper is to show the interactions of Cd and Zn in the freshwater crab Sinopotamon henanense through metallothionein (MT) and malondialdehyde (MDA) level measurements. Laboratory acclimated S.henanense were exposed to Cd (50 µg/L, 100 µg/L, 500 µg/L ), and Zn (100 µg/L, 1000 µg/L) alone and in combined treatments (100 µg/L Zn+50 µg/L Cd, 100 µg/L Zn+100 µg/L Cd, 100 µg/L Zn+500 µg/L Cd, 1000 µg/L Zn+50 µg/L Cd, 1000 µg/L Zn+100 µg/L Cd, 1000 µg/L Zn+500 µg/L Cd) for 7, 14, 21, 28, 35 days. The results demonstrated that the MDA contents increased with exposure time and dose and showed time- and dose-dependence in both gills and hepatopancreas of S.henanense after single Cd exposure, while the changes of MDA levels were not significant with single Zn exposure. The MDA levels decreased when the crabs were exposed to metal mixtures compared to Cd exposure alone, indicating that Zn mediated the cellular toxicity of Cd. MT contents increased after single Cd exposure and also showed a time- and dose-dependence, in a tissue-specific way. Zn showed a limited ability of MT induction both in gills and hepatopancreas of S.henanense. The MT contents represented not a simple addition of single metal exposures but were enhanced at a higher concentration of Zn combined with different Cd concentrations compared to single metal exposure. Whether MT can be used as a biomarker for complex field conditions need to be considered cautiously since different induction patterns of MT were found among single Zn, Cd and combined groups. It is suggested that several biomarkers together as a suite should be used in the monitoring of heavy metal pollution in the aquatic environment. 相似文献
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Yixin H. Ye Megan Woolfit Edwige Rancès Scott L. O'Neill Elizabeth A. McGraw 《PLoS neglected tropical diseases》2013,7(8)
Background
Wolbachia infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural Wolbachia infections in Drosophila melanogaster with the mosquito vector Aedes aegypti artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-Wolbachia associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect.Methodology/Findings
Here we assess whether the level of innate immune priming induced by different Wolbachia strains in A. aegypti is correlated with the degree of protection conferred against bacterial pathogens. We show that Wolbachia strains wMel and wMelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The wMelPop strain provides stronger, more broad-based protection than wMel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that Wolbachia densities themselves decline during pathogen infection, likely as a result of the immune induction.Conclusions/Significance
This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether Wolbachia may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness. 相似文献13.
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Adults of Romanomermis culicivorax obtained from mass cultures were examined by scanning and transmission electron microscopy to determine the organization of their anterior sense organs. The normal pattern apparently consists of two lateral amphids plus six cephalic papillae. Lateral cephalic papillae contain two sense organs, each with a cuticular pore, while subdorsal and subventral papillae have three sense organs, each with a cuticular pore. About 30% of females and 80% of males examined showed aberrant developments in these sense organs. Both cuticle and underlying cells (hypodermis and neurons) are affected; some sense organs are absent while others are incompletely formed. Few aberrant worms were found in a smaller collection of worms reared in lower population densities. Perhaps aberrant forms are examples of teratological development resulting from, or promoted by, conditions used for mass rearing of biological control agents. 相似文献
19.
Three Drought-Responsive Members of the Nonspecific
Lipid-Transfer Protein Gene Family in Lycopersicon
pennellii
Show Different Developmental Patterns of
Expression 总被引:6,自引:0,他引:6 下载免费PDF全文
Genomic clones of two nonspecific lipid-transfer protein genes from a drought-tolerant wild species of tomato (Lycopersicon pennellii Corr.) were isolated using as a probe a drought- and abscisic acid (ABA)-induced cDNA clone (pLE16) from cultivated tomato (Lycopersicon esculentum Mill.). Both genes (LpLtp1 and LpLtp2) were sequenced and their corresponding mRNAs were characterized; they are both interrupted by a single intron at identical positions and predict basic proteins of 114 amino acid residues. Genomic Southern data indicated that these genes are members of a small gene family in Lycopersicon spp. The 3′-untranslated regions from LpLtp1 and LpLtp2, as well as a polymerase chain reaction-amplified 3′-untranslated region from pLE16 (cross-hybridizing to a third gene in L. pennellii, namely LpLtp3), were used as gene-specific probes to describe expression in L. pennellii through northern-blot analyses. All LpLtp genes were exclusively expressed in the aerial tissues of the plant and all were drought and ABA inducible. Each gene had a different pattern of expression in fruit, and LpLtp1 and LpLtp2, unlike LpLtp3, were both primarily developmentally regulated in leaf tissue. Putative ABA-responsive elements were found in the proximal promoter regions of LpLtp1 and LpLtp2. 相似文献