共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
3.
4.
S. N. Moralev E. V. Rozengart 《Journal of Evolutionary Biochemistry and Physiology》2001,37(5):469-491
An analytical review is performed of the literature data on the hydrolysis rate, affinity of substrate to active center, and constants of the substrate inhibition (K
ss) at hydrolysis of the choline (acetyl-, propyonyl-, butyrylcholine, acetyl--methylcholine) and/or of corresponding thiocholine substrates by 59 cholinesterases from 49 different animals (chordate, insects, molluscs, nematodes). The characteristic peculiarities of enzymes from different groups of animals are revealed. The absence of regular changes of parameters of the cholinesterase substrate specificity in the course of evolutionary development is shown. 相似文献
5.
6.
7.
Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids
《Biophysical journal》2020,118(4):957-966
Diacylglycerol kinase ε (DGKε) is a membrane-bound enzyme that catalyzes the ATP-dependent phosphorylation of diacylglycerol to form phosphatidic acid (PA) in the phosphatidylinositol cycle. DGKε lacks a putative regulatory domain and has recently been reported to be regulated by highly curved membranes. To further study the effect of other membrane properties as a regulatory mechanism of DGKε, our work reports the effect of negatively charged phospholipids on DGKε activity and substrate acyl chain specificity. These studies were conducted using purified DGKε and detergent-free phospholipid aggregates, which present a more suitable model system to access the impact of membrane physical properties on membrane-active enzymes. The structural properties of the different model membranes were studied by means of differential scanning calorimetry and 31P-NMR. It is shown that the enzyme is inhibited by a variety of negatively charged phospholipids. However, PA, which is a negatively charged phospholipid and the product of DGKε catalyzed reaction, showed a varied regulatory effect on the enzyme from being an activator to an inhibitor. The type of feedback regulation of DGKε by PA depends on the particular PA molecular species as well as the physical properties of the membrane that the enzyme binds to. In the presence of highly packed PA-rich domains, the enzyme is activated. However, its acyl chain specificity is only observed in liposomes containing 1,2-dioleoyl PA in the presence of Ca2+. It is proposed that to endow the enzyme with its substrate acyl chain specificity, a highly dehydrated (hydrophobic) membrane interface is needed. The presence of an overlap of mechanisms to regulate DGKε ensures proper phosphatidylinositol cycle function regardless of the trigged stimulus and represents a sophisticated and specialized manner of membrane-enzyme regulation. 相似文献
8.
Elena Feraru Tomasz Paciorek Mugurel I. Feraru Marta Zwiewka Ruth De Groodt Riet De Rycke Jürgen Kleine-Vehn Ji?í Friml 《The Plant cell》2010,22(8):2812-2824
Plant vacuoles are essential multifunctional organelles largely distinct from similar organelles in other eukaryotes. Embryo protein storage vacuoles and the lytic vacuoles that perform a general degradation function are the best characterized, but little is known about the biogenesis and transition between these vacuolar types. Here, we designed a fluorescent marker–based forward genetic screen in Arabidopsis thaliana and identified a protein affected trafficking2 (pat2) mutant, whose lytic vacuoles display altered morphology and accumulation of proteins. Unlike other mutants affecting the vacuole, pat2 is specifically defective in the biogenesis, identity, and function of lytic vacuoles but shows normal sorting of proteins to storage vacuoles. PAT2 encodes a putative β-subunit of adaptor protein complex 3 (AP-3) that can partially complement the corresponding yeast mutant. Manipulations of the putative AP-3 β adaptin functions suggest a plant-specific role for the evolutionarily conserved AP-3 β in mediating lytic vacuole performance and transition of storage into the lytic vacuoles independently of the main prevacuolar compartment-based trafficking route. 相似文献
9.
The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability. 相似文献
10.
Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression. 相似文献
11.
Wei-Li Yu Yong-Liang Jiang Andreas Pikis Wang Cheng Xiao-Hui Bai Yan-Min Ren John Thompson Cong-Zhao Zhou Yuxing Chen 《The Journal of biological chemistry》2013,288(21):14949-14958
The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6′P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6′P (a non-metabolizable analog of cellobiose-6′P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)8 TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr126, Tyr303, and Trp338, at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser424, Lys430, and Tyr432 of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite −1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family. 相似文献
12.
The action of the environmental toxic Pb2+ on photosynthetic electron transport was studied in thylakoid membranes isolated from spinach leaves. Fluorescence and thermoluminescence techniques were performed in order to determine the mode of Pb2+ action in photosystem II (PSII). The invariance of fluorescence characteristics of chlorophyll a (Chl a) and magnesium tetraphenylporphyrin (MgTPP), a molecule structurally analogous to Chl a, in the presence of Pb2+ confirms that Pb cation does not interact directly with chlorophyll molecules in PSII. The results show that Pb interacts with the water oxidation complex thus perturbing charge recombination between the quinone acceptors of PSII and the S2 state of the Mn4Ca cluster. Electron transfer between the quinone acceptors QA and QB is also greatly retarded in the presence of Pb2+. This is proposed to be owing to a transmembrane modification of the acceptor side of the photosystem. 相似文献
13.
《Bioscience, biotechnology, and biochemistry》2013,77(12):3174-3179
Δ12 and ω3 fatty acid desaturases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs), which are important constituents of membrane glycerolipids and also precursors to signaling molecules in many organisms. In this study, we determined the substrate specificity and regioselectivity of the Δ12 and ω3 fatty acid desaturases from Saccharomyces kluyveri (Sk-FAD2 and Sk-FAD3). Based on heterologous expression in Saccharomyces cerevisiae, it was found that Sk-FAD2 converted C16–20 monounsaturated fatty acids to diunsaturated fatty acids by the introduction of a second double bond at the ν+3 position, while Sk-FAD3 recognized the ω3 position of C18 and C20. Furthermore, fatty acid analysis of major phospholipids suggested that Sk-FAD2 and Sk-FAD3 have no strong substrate specificity toward the lipid polar head group or the sn-positions of fatty acyl groups in phospholipids. 相似文献
14.
Saija Kiljunen Neeta Datta Svetlana V. Dentovskaya Andrey P. Anisimov Yuriy A. Knirel Jos�� A. Bengoechea Otto Holst Mikael Skurnik 《Journal of bacteriology》2011,193(18):4963-4972
φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis. 相似文献
15.
Wei Chen Jizhong Lou Jen Hsin Klaus Schulten Stephen C. Harvey Cheng Zhu 《PLoS computational biology》2011,7(2)
Integrins may undergo large conformational changes during activation, but the dynamic processes and pathways remain poorly understood. We used molecular dynamics to simulate forced unbending of a complete integrin αVβ3 ectodomain in both unliganded and liganded forms. Pulling the head of the integrin readily induced changes in the integrin from a bent to an extended conformation. Pulling at a cyclic RGD ligand bound to the integrin head also extended the integrin, suggesting that force can activate integrins. Interactions at the interfaces between the hybrid and β tail domains and between the hybrid and epidermal growth factor 4 domains formed the major energy barrier along the unbending pathway, which could be overcome spontaneously in ∼1 µs to yield a partially-extended conformation that tended to rebend. By comparison, a fully-extended conformation was stable. A newly-formed coordination between the αV Asp457 and the α-genu metal ion might contribute to the stability of the fully-extended conformation. These results reveal the dynamic processes and pathways of integrin conformational changes with atomic details and provide new insights into the structural mechanisms of integrin activation. 相似文献
16.
17.
Pedro A. Pérez-Mancera Camino Bermejo-Rodríguez Manuel Sánchez-Martín Fernando Abollo-Jiménez Belén Pintado Isidro Sánchez-García 《PloS one》2008,3(7)
Background
FUS-DDIT3 is a chimeric protein generated by the most common chromosomal translocation t(12;16)(q13;p11) linked to liposarcomas, which are characterized by the accumulation of early adipocytic precursors. Current studies indicate that FUS-DDIT3- liposarcoma develops from uncommitted progenitors. However, the precise mechanism whereby FUS-DDIT3 contributes to the differentiation arrest remains to be elucidated.Methodology/Principal Findings
Here we have characterized the adipocyte regulatory protein network in liposarcomas of FUS-DITT3 transgenic mice and showed that PPARγ2 and C/EBPα expression was altered. Consistent with in vivo data, FUS-DDIT3 MEFs and human liposarcoma cell lines showed a similar downregulation of both PPARγ2 and C/EBPα expression. Complementation studies with PPARγ but not C/EBPα rescued the differentiation block in committed adipocytic precursors expressing FUS-DDIT3. Our results further show that FUS-DDIT3 interferes with the control of initiation of translation by upregulation of the eukaryotic translation initiation factors eIF2 and eIF4E both in FUS-DDIT3 mice and human liposarcomas cell lines, explaining the shift towards the truncated p30 isoform of C/EBPα in liposarcomas. Suppression of the FUS-DDIT3 transgene did rescue this adipocyte differentiation block. Moreover, eIF4E was also strongly upregulated in normal adipose tissue of FUS-DDIT3 transgenic mice, suggesting that overexpression of eIF4E may be a primary event in the initiation of liposarcomas. Reporter assays showed FUS-DDIT3 is involved in the upregulation of eIF4E in liposarcomas and that both domains of the fusion protein are required for affecting eIF4E expression.Conclusions/Significance
Taken together, this study provides evidence of the molecular mechanisms involve in the disruption of normal adipocyte differentiation program in liposarcoma harbouring the chimeric gene FUS-DDIT3. 相似文献18.
Hong Cao Jing Chen Eugene W. Krueger Mark A. McNiven 《Molecular and cellular biology》2010,30(3):781-792
The mechanisms by which epithelial cells regulate clathrin-mediated endocytosis (CME) of transferrin are poorly defined and generally viewed as a constitutive process that occurs continuously without regulatory constraints. In this study, we demonstrate for the first time that endocytosis of the transferrin receptor is a regulated process that requires activated Src kinase and, subsequently, phosphorylation of two important components of the endocytic machinery, namely, the large GTPase dynamin 2 (Dyn2) and its associated actin-binding protein, cortactin (Cort). To our knowledge these findings are among the first to implicate an Src-mediated endocytic cascade in what was previously presumed to be a nonregulated internalization process.Iron is an essential element for all mammalian organisms that plays essential roles in hemoglobin and myoglobin production (23). Altered iron transport can lead to disease states such as hemochromatosis (23), anemia (5, 23), and neuronal disorders (23). The transferrin receptor (TfR) is an important component of iron regulation in cells. There are two distinct TfRs in humans sharing 45% identity that are homodimeric and bind iron-associated transferrin (Tf) at markedly different affinities (26). While significant attention has been paid toward understanding the basic endocytic machinery that supports the efficient internalization and recycling of the TfR1 and its associated iron-bound ligand, it has been assumed that this transport process is constitutive in nature. This is in direct contrast to the highly regulated internalization pathway used by members of the receptor tyrosine kinase family (RTKs) and the family of G-coupled protein receptors (GPCRs) that utilize phosphorylation and/or ubiquination as signaling modules to regulate internalization.To test if TfR1 internalization might be regulated in a similar fashion, we focused on two essential components of the endocytic machinery: the large GTPase Dyn2 that mediates endocytic vesicle scission (35) and Cort that binds to Dyn2 via an SH3-PRD interaction and has been postulated to regulate actin dynamics to facilitate vesicle invagination and release (36, 40). Both Dyn2 and Cort have shown to be phosphorylated in vivo and in vitro by a variety of kinases (51, 58). Dyn1 interacts with (17) and is phosphorylated by Src in neuronal cells and in other excitable cells in response to activation of GPCRs and epidermal growth factor (EGF) (1, 2). While the Src phosphorylation motifs of dynamin are conserved in the epithelial expressed form of Dyn2, it is unclear if Dyn2 is phosphorylated in response to ligands that induce clathrin-based endocytosis.Cort possesses a series of C-terminal tyrosines that are heavily Src-phosphorylated and implicated in regulating actin remodeling during cell motility (20). In this study, we demonstrate that addition of Tf to cultured epithelial cells results in an internalization of the TfR1 mediated by a Src kinase-dependent phosphoactivation of the Dyn2-Cort-based endocytic machinery. In support of these findings, dominant negative forms of c-Src kinase, when expressed in a hepatocyte-derived cell line (Clone 9), attenuate Tf internalization. Remarkably, cells exposed to Tf showed a 3- to 4-fold increase in Dyn2 and Cort phosphorylation compared to that shown by untreated cells, an increase exceeding that observed in cells treated with EGF. These findings provide new insights into the regulation of what was thought to be a constitutive endocytic process. 相似文献
19.
Markus P. Kummer Hiroko Maruyama Claudia Huelsmann Sandra Baches Sascha Weggen Edward H. Koo 《The Journal of biological chemistry》2009,284(4):2296-2306
The formation of insoluble cross β-sheet amyloid is pathologically
associated with disorders such as Alzheimer, Parkinson, and Huntington
diseases. One exception is the nonpathological amyloid derived from the
protein Pmel17 within melanosomes to generate melanin pigment. Here we show
that the formation of insoluble MαC intracellular fragments of Pmel17,
which are the direct precursors to Pmel17 amyloid, depends on a novel
juxtamembrane cleavage at amino acid position 583 between the furin-like
proprotein convertase cleavage site and the transmembrane domain. The
resulting Pmel17 C-terminal fragment is then processed by the
γ-secretase complex to release a short-lived intracellular domain
fragment. Thus, by analogy to the Notch receptor, we designate this cleavage
the S2 cleavage site, whereas γ-secretase mediates proteolysis at the
intramembrane S3 site. Substitutions or deletions at this S2 cleavage site,
the use of the metalloproteinase inhibitor TAPI-2, as well as small
interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17
reduced the formation of insoluble Pmel17 fragments. These results demonstrate
that the release of the Pmel17 ectodomain, which is critical for melanin
amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position.Folding of proteins is a highly regulated process ensuring their correct
three-dimensional structure. Under pathological circumstances, a soluble
protein can be folded into highly stable cross β-sheet amyloid
structures, which are believed to play pathological roles in disorders such as
Alzheimer, Parkinson, and Huntington diseases. An exception to this general
concept is the physiological amyloid structure of the melanosomal matrix
formed by the protein Pmel17. Melanosomes are lysosome-related organelles that
contain pigment granules (melanin) in melanocytes and retinal epithelial cells
(reviewed in Ref. 1).
Melanogenesis is believed to proceed through several sequential maturation
steps, classified by melanosomes from stage I to stage IV. Maturation of stage
II melanosomes requires the formation of Pmel17 intralumenal fibers
(2,
3).Pmel17 (also called gp100, ME20, RPE1, or silver) is a type I transmembrane
glycoprotein of up to 668 amino acids in humans (reviewed in Ref.
4). The requirement of Pmel17
for the generation of functional melanin has been shown in a number of
different organisms, because, for example, certain point mutations in the
Pmel17/silver gene result in hypopigmentation phenotypes
(5–7).
The most characteristic domain within Pmel17 is a specific lumenal
proline/serine/threonine rich repeat domain (see
Fig. 1A), that is
imperfectly repeated 13 times in the Mα fragment. Importantly, deletion
of the rich repeat domain results in a complete loss of fibril formation,
pointing to the requirement of Pmel17, and especially the rich repeat domain,
in melanin formation (8).
Pmel17 exists in different isoforms generated by alternative splicing.
Pmel17-i2 is the most
abundant isoform, whereas the Pmel17-l isoform contains a 7-amino acid
insertion close to the transmembrane domain
(9,
10).Open in a separate windowFIGURE 1.Effect of the γ-secretase inhibitor DAPT on Pmel17 processing.
A, schematic diagram of Pmel17 and epitopes of antibodies. Pmel17
contains five potential N-glycosylation sites indicated by branched
structures. The long form of Pmel17, Pmel17-l, is characterized by a seven
amino acid insertion (VPGILLT) within the lumenal domain close to the
transmembrane domain (TM), which is absent in Pmel17-i. NVS marks a
potential N-glycosylation site near this insertion. The epitopes of
antibodies αPep13h and HMB45 are indicated. Cleavage by a furin-like PC
results in the formation of the Mα and the membrane-bound 26-kDa Mβ
fragment, which are connected via disulfide bonds. Release and further
processing of the Mα fragment into MαN and MαC fragments
results in the formation of fibrils and marks the transition of stage I to
stage II melanosomes (dashed line). B, human MNT-1 cells
were incubated with increasing amounts of DAPT for 18 h, and then the lysates
were separated by SDS-PAGE and analyzed by immunoblotting with αPep13h
antibody. DAPT treatment resulted in the accumulation of a C-terminal fragment
of Pmel17 (CTF), whereas Pmel17 P1 and Mβ fragment were unchanged.
C, probing the Triton-soluble fraction with HMB45 revealed increased
amounts of the highly glycosylated P2 form of Pmel17 after DAPT incubation.
D, detection of Pmel17 amyloidogenic fragments (MαC) in the
SDS-extracted insoluble pellet using antibody HMB45. E, murine B16-FO
cells treated with increasing concentrations of DAPT. Immunoblotting using
antibodyαPep13h revealed the formation of CTF of similar size as in
MNT-1 cells. F, time course analysis of Pmel17, Mβ, and
Pmel17-CTF after DAPT treatment. The cell lysates were immunoblotted using
αPep13h. Pmel17-CTF was detectable after 10 min of incubation with 1
μm DAPT. G, the size of the Pmel17-CTF was determined
using an unstained low molecular range peptide standard. The marker peptides
were detected by Ponceau S staining and Pmel17-CTF were detected by immunoblot
using αPep13h.Pmel17 traffics through the secretory pathway as a 100-kDa protein (called
P1). In the late Golgi compartment it undergoes further glycosylation,
resulting in a short lived 120-kDa protein (called P2). P2 is rapidly cleaved
within the post-Golgi by a furin-like proprotein convertase (PC) to generate
two fragments that remain tethered to each other by disulfide bonds: a
C-terminal polypeptide containing the transmembrane domain (Mβ) and a
large N-terminal ectodomain (Mα)
(2)
(Fig. 1A).
Consequently, inhibition of this furin-like activity not only prevents the
generation of Mα and Mβ fragments but also inhibits the formation
of melanosomal striation in HeLa cells
(3). These findings suggest
that Mα must first be dissociated from the Mβ for melanogenesis to
proceed. It is unclear how Mα is released from the membrane. Reduction
of disulfide bonds would release Mα from Mβ; alternatively,
proteolytic digestion of Mβ should also free Mα from the membrane
tether. It has been speculated that, given the presence of lysosomal
hydrolases in melanosomes and proteolytic maturation of Pmel17, proteolysis is
the more likely mechanism (4).
Recently, it was shown that recombinant Mα is able to form amyloid
structures in vitro in an unprecedented rapidity, and furthermore,
Pmel17 amyloid also accelerated melanin formation
(11). These findings
demonstrate that mammalian amyloid formed by Pmel17 is functional and
physiological.The insoluble pool of Pmel17 in cells consists mostly of truncated Mα
C-terminal fragments (MαC) of heterogeneous sizes, indicating that
further processing of Mα occurs after its release from the membrane
(8,
12). MαC fragments are
found in the insoluble fraction of melanocytes as well as in nonmelanotic
cells, the latter after overexpression of Pmel17
(8), and are reduced or absent
in amelanotic cells (8,
13,
14). Meanwhile, the C-terminal
fragment derived from the Mβ fragment and recognized by a C-terminal
specific epitope antibody is less stable, indicating rapid turnover
(2).The presenilin (PS) family of proteins consists of two homologous integral
transmembrane proteins, PS1 and PS2, which are part of the γ-secretase
complex. The latter consists of presenilin 1 or 2, nicastrin, APH-1, and PEN-2
(15) and catalyzes the
cleavage of the hydrophobic transmembrane domain of a burgeoning list of
proteins, also called regulated intramembrane cleavage. Other substrates for
the γ-secretase-mediated intramembrane cleavage include Notch, amyloid
precursor protein (APP), cadherin (E-cadherin), nectin-1, the low density
lipoprotein-related receptor, CD44, ErbB-4, the voltage-gated sodium channel
β2-subunit, and the Notch ligands Delta and Jagged. Importantly, in
Alzheimer disease, the presenilin-mediated γ-secretase cleavage of APP
releases the amyloid β-protein fragment, a peptide believed to play a key
role in Alzheimer disease pathogenesis. Interestingly, a recent report
described the absence of melanin pigment in presenilin-deficient animals, an
observation confirmed by the lack of melanin formation in cells treated with
γ-secretase inhibitors
(16). The mechanism
responsible for this finding is unclear, leading us to ask whether Pmel17
processing is a presenilin-dependent process and, if so, whether this cleavage
is involved in melanogenesis.In this study, we show the presence of an endoproteolytic activity that
cleaves the extracellular domain of Pmel17-i at a juxtamembrane position
between the known PC cleavage site and the transmembrane domain, which we term
the S2 cleavage site, by a TAPI-sensitive ADAM (a disintegrin
and metalloproteinase protein) protease. This
intracellular shedding of Pmel17 after S2 cleavage results in the liberation
of the Mα N-terminal ectodomain, the precursor to Pmel17 amyloid, which
is able to form insoluble Pmel17 aggregates. The C-terminal transmembrane
fragment generated by S2 cleavage is further processed by γ-secretase
(S3 cleavage) to release the Pmel17 intracellular domain, which is then
rapidly degraded. 相似文献
20.
Ishfaq Ahmed Sheikh Amit Kumar Singh Nagendra Singh Mau Sinha S. Baskar Singh Asha Bhushan Punit Kaur Alagiri Srinivasan Sujata Sharma Tej P. Singh 《The Journal of biological chemistry》2009,284(22):14849-14856
The crystal structure of the complex of lactoperoxidase (LPO) with its
physiological substrate thiocyanate (SCN–) has been
determined at 2.4Å resolution. It revealed that the
SCN– ion is bound to LPO in the distal heme cavity. The
observed orientation of the SCN– ion shows that the sulfur
atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of
SCN– forms a hydrogen bond with a water (Wat) molecule at
position 6′. This water molecule is stabilized by two hydrogen bonds
with Gln423 Nε2 and Phe422 oxygen. In
contrast, the placement of the SCN– ion in the structure of
myeloperoxidase (MPO) occurs with an opposite orientation, in which the
nitrogen atom is closer to the heme iron than the sulfur atom. The site
corresponding to the positions of Gln423, Phe422 oxygen,
and Wat6′ in LPO is occupied primarily by the side chain of
Phe407 in MPO due to an entirely different conformation of the loop
corresponding to the segment Arg418–Phe431 of LPO.
This arrangement in MPO does not favor a similar orientation of the
SCN– ion. The orientation of the catalytic product
OSCN– as reported in the structure of
LPO·OSCN– is similar to the orientation of
SCN– in the structure of LPO·SCN–.
Similarly, in the structure of
LPO·SCN–·CN–, in which
CN– binds at Wat1, the position and orientation of
the SCN– ion are also identical to that observed in the
structure of LPO·SCN.Lactoperoxidase
(LPO4; EC 1.11.1.7) is
a Fe3+ heme enzyme that belongs to the mammalian peroxidase family
(1). The family of mammalian
peroxidases comprises lactoperoxidase
(2), eosinophil peroxidase
(3), thyroid peroxidase
(4), and myeloperoxidase (MPO)
(5). LPO, eosinophil
peroxidase, and MPO are responsible for antimicrobial function and innate
immune responses
(6–8),
whereas thyroid peroxidase plays a key role in thyroid hormone biosynthesis
(9). These peroxidases are
different from plant and fungal peroxidases because unlike plant and fungal
enzymes, the prosthetic heme group in mammalian peroxidases is covalently
linked to the protein (10).
There are also several striking structural and functional differences among
the mammalian peroxidases
(11). The heme group in MPO is
attached to the protein via three covalent linkages
(12), whereas LPO
(12,
13), eosinophil peroxidase
(12), and thyroid peroxidase
(12) contain only two ester
linkages. These covalent and various non-covalent linkages contribute
differentially to the high stability of the heme core as well as for the
peculiar values of their redox potentials
(2,
14). Furthermore, MPO consists
of two disulfide-linked protein chains, whereas LPO, eosinophil peroxidase,
and thyroid peroxidase are single chain proteins, although their chain lengths
differ greatly. In addition, their sequences contain several critical amino
acid differences that may also contribute to the variations in the
stereochemical environments of the substrate-binding sites. As a consequence
of these differences, the mammalian enzymes oxidize various inorganic ions
such as SCN–, Br–, Cl–, and
I– with differing specificities and potencies. Biochemical
studies have shown that LPO catalyzes preferentially the conversion of
SCN– to OSCN–
(15,
16), whereas MPO uses halides
(17,
18) with a preference for
chloride ion as the substrate. The preferences of eosinophil peroxidase and
thyroid peroxidase are bromide and iodide, respectively. However, the
stereochemical basis of the reported preferences for the substrates by
mammalian heme peroxidases is still unclear. So far, the structures of only
two mammalian enzymes, MPO and LPO, have been determined
(12,
13). It is of considerable
importance to identify the structural parameters that are responsible for the
subtle specificities. In the present work, we have attempted to address this
question through the new crystal structures of LPO complexes with
SCN– ions using goat, bovine, and buffalo lactoperoxidases.
Because the overall structures of complexes of SCN– with LPO
from all three species were found to be identical, the structure of the
complex of buffalo LPO with SCN– and the ternary complex with
SCN– and CN– will be discussed here, and
buffalo LPO will be termed hereafter as LPO. To highlight the factors
pertaining to binding specificity of SCN–, a comparison of
the structures of LPO·SCN– and
MPO·SCN– has also been made, revealing many valuable
differences pertaining to the observed orientations of the common substrate,
SCN– ion, when bound at the substrate-binding site in the
distal heme cavity of the two structures. The structures of
LPO·SCN– and MPO·SCN– clearly
show that the bound SCN– ions are present in the distal heme
cavity of two enzymes with opposite orientations. In the structure of
LPO·SCN–, the sulfur atom is closer to the heme iron
than the nitrogen atom, whereas in that of MPO·SCN–,
the nitrogen atom is closer to the heme iron than the sulfur atom. As a result
of this, the interactions of the SCN– ion in the distal site
of two proteins differ drastically. Gln423, a conserved water (Wat)
molecule at position 6′, and a well aligned carbonyl oxygen of
Phe422 in the proximity of the substrate-binding site in LPO
against a protruding Phe407 in MPO seem to play the key roles in
inducing the observed orientations of SCN– ions in LPO and
MPO. The structure of LPO·SCN– has also been compared
with the structure of its ternary complex with SCN– and
CN– ions. 相似文献