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Ji Won Um Eunju Im Joongkyu Park Yohan Oh Boram Min Hyun Jung Lee Jong Bok Yoon Kwang Chul Chung 《The Journal of biological chemistry》2010,285(47):36434-36446
The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction. 相似文献
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Junko Takeuchi Masahiro Fujimuro Hideyosi Yokosawa Keiji Tanaka Akio Toh-e 《Molecular and cellular biology》1999,19(10):6575-6584
We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome. Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth. At the restrictive temperature, the Deltarpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature. We analyzed the proteasome fractions separated by glycerol density gradient centrifugation by native polyacrylamide gel electrophoresis and found that a smaller amount of the 26S proteasome was produced in the Deltarpn9 cells and that the 26S proteasome was shifted to lighter fractions than expected. The incomplete proteasome complexes were found to accumulate in the Deltarpn9 cells. Furthermore, Rpn10 was not detected in the fractions containing proteasomes of the Deltarpn9 cells. These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in the assembly and/or stability of the 26S proteasome. 相似文献
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Jianbin Yan Haiou Li Shuhua Li Ruifeng Yao Haiteng Deng Qi Xie Daoxin Xie 《The Plant cell》2013,25(2):486-498
Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCFCOI1) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCFCOI1 complexes, suggesting a function for SCFCOI1 in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCFCOI1-mediated stabilization and 26S proteasome–mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses. 相似文献
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Dikla Berko Ora Herkon Ilana Braunstein Elada Isakov Yael David Tamar Ziv Ami Navon Ariel Stanhill 《The Journal of biological chemistry》2014,289(9):5609-5618
The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps. 相似文献
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Toshinobu Tokumoto Mika Tokumoto Keiji Seto Ryo Horiguchi Yoshitaka Nagahama Shinpei Yamada Katsutoshi Ishikawa Manfred J. Lohka 《Experimental cell research》1999,247(2):313
We have prepared polyclonal antibodies againstXenopus20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development ofXenopus laevis.A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo. 相似文献
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James C. Geoghegan Michael B. Miller Aimee H. Kwak Brent T. Harris Surachai Supattapone 《PLoS pathogens》2009,5(7)
Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrPC molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrPC is pre-incubated with poly(A) RNA and PrPSc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrPSc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrPSc molecules, most likely through an epitope containing residue 172. 相似文献
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Ya-Ling Lin Shu-Chiun Sung Hwang-Long Tsai Ting-Ting Yu Ramalingam Radjacommare Raju Usharani Antony S. Fatimababy Hsia-Yin Lin Ya-Ying Wang Hongyong Fu 《The Plant cell》2011,23(7):2754-2773
Ubiquitylated substrate recognition during ubiquitin/proteasome-mediated proteolysis (UPP) is mediated directly by the proteasome subunits RPN10 and RPN13 and indirectly by ubiquitin-like (UBL) and ubiquitin-associated (UBA) domain-containing factors. To dissect the complexity and functional roles of UPP substrate recognition in Arabidopsis thaliana, potential UPP substrate receptors were characterized. RPN10 and members of the UBL-UBA–containing RAD23 and DSK2 families displayed strong affinities for Lys-48–linked ubiquitin chains (the major UPP signals), indicating that they are involved in ubiquitylated substrate recognition. Additionally, RPN10 uses distinct interfaces as primary proteasomal docking sites for RAD23s and DSK2s. Analyses of T-DNA insertion knockout or RNA interference knockdown mutants of potential UPP ubiquitin receptors, including RPN10, RPN13, RAD23a-d, DSK2a-b, DDI1, and NUB1, demonstrated that only the RPN10 mutant gave clear phenotypes. The null rpn10-2 showed decreased double-capped proteasomes, increased 20S core complexes, and pleiotropic vegetative and reproductive growth phenotypes. Surprisingly, the observed rpn10-2 phenotypes were rescued by a RPN10 variant defective in substrate recognition, indicating that the defectiveness of RPN10 in proteasome but not substrate recognition function is responsible for the null phenotypes. Our results suggest that redundant recognition pathways likely are used in Arabidopsis to target ubiquitylated substrates for proteasomal degradation and that their specific roles in vivo require further examination. 相似文献
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泛素/26S蛋白酶体途径(ubiquitin/26S proteasome pathway,UPP)是目前已知最有效的、最具特异性的蛋白质降解途径。该途径介导了真核生物80%-85%的蛋白质降解,参与了细胞多项生命活动过程,对于维持细胞正常生理功能具有重要意义。研究结果表明,植物生长发育的诸多方面以及干旱胁迫响应等过程都受到该途径的调控。概述了泛素/26S蛋白酶体途径及其在植物生长发育过程中的作用,并着重阐述了由泛素-蛋白连接酶E3介导的植物干旱胁迫响应及其作用机制的研究进展。 相似文献
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Yunuen Avalos-Padilla Abigail Betanzos Rosario Javier-Reyna Guillermina García-Rivera Bibiana Chávez-Munguía Anel Lagunes-Guillén Jaime Ortega Esther Orozco 《PLoS pathogens》2015,11(7)
Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation. 相似文献
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Harry S. Courtney Yi Li Waleed O. Twal W. Scott Argraves 《The Journal of biological chemistry》2009,284(19):12966-12971
The adhesion of bacteria to host tissues is often mediated by interactions
with extracellular matrices. Herein, we report on the interactions of the
group A streptococcus, Streptococcus pyogenes, with the extracellular
matrix protein fibulin-1. S. pyogenes bound purified fibulin-1 in a
dose-dependent manner. Genetic ablation of serum opacity factor (SOF), a
virulence determinant of S. pyogenes, reduced binding by ∼50%,
and a recombinant peptide of SOF inhibited binding of fibulin-1 to
streptococci by ∼45%. Fibulin-1 bound to purified SOF2 in a dose-dependent
manner with high affinity (Kd = 1.6 nm). The
fibulin-1-binding domain was localized to amino acid residues 457–806 of
SOF2, whereas the fibronectin-binding domain is contained within residues
807–931 of SOF2, indicating that these two domains are separate and
distinct. Fibulin-1 bound to recombinant SOF from M types 2, 4, 28, and 75 of
S. pyogenes, indicating that the fibulin-1-binding domain is likely
conserved among SOF from different serotypes. Mixed binding experiments
suggested that gelatin, fibronectin, fibulin-1, and SOF form a quaternary
molecular complex that enhanced the binding of fibulin-1. These data indicate
that S. pyogenes can interact with fibulin-1 and that SOF is a major
streptococcal receptor for fibulin-1 but not the only receptor. Such
interactions with fibulin-1 may be involved in the adhesion of S.
pyogenes to extracellular matrices of the host.Adhesion of bacteria to host surfaces is the first stage in establishing
bacterial infections in the human host, and a variety of molecular mechanisms
are utilized to initiate adhesion. A common mechanism for adhesion involves
interactions between bacterial adhesins and components of the extracellular
matrices of the host. The identification and characterization of microbial
surface components recognizing adhesive matrix molecules (MSCRAMM) has led to
important advances in vaccines and immunotherapies for preventing and treating
bacterial infections (1).The group A streptococcus, Streptococcus pyogenes, is a major
human pathogen causing diseases ranging from relative minor infections such as
pharyngitis and cellulitis to severe infections with high levels of morbidity
and mortality such as necrotizing fasciitis and toxic shock syndrome
(2). This pathogen expresses
adhesins that interact with various components of the extracellular matrix
including laminin, elastin, fibronectin, fibrinogen, and collagen
(3–7).
The interactions between fibronectin and S. pyogenes have been
intensely studied, and these investigations have revealed at least 10
different streptococcal proteins that bind fibronectin
(4).Serum opacity factor
(SOF)2 is a major
fibronectin-binding protein that is involved in adhesion to host cells
(8–11).
SOF is a virulence determinant that is expressed by approximately half of the
clinical isolates of S. pyogenes
(8). SOF opacifies serum by
binding and displacing apoA-I in high density lipoproteins
(8,
12–15).
SOF is covalently linked to the streptococcal cell wall via an LPSTG sortase
recognition site and is also released in a soluble form. SOF has two
functionally distinct domains, an N-terminal domain that opacifies serum and a
C-terminal domain that binds fibronectin. The role of SOF in adhesion involves
both its C-terminal fibronectin-binding domain and an N-terminal region (see
Fig. 1 for a schematic of
structure) (9,
11). However, the nature of
the interactions between the N-terminal region of SOF and host components is
not well characterized.Open in a separate windowFIGURE 1.A, a schematic of the structure of SOF and its functional domains
is shown. The assignment of functional domains are based on the findings of
Rakonjac et al. (33),
Kreikemeyer et al.
(34), Courtney et al.
(8,
13), and results presented in
this work. Fn, fibronectin. B, the data for the binding of
SOF peptides to fibronectin are from previous publications
(8,
13), and the data for
fibulin-1 are from the present work.Herein, we report on the interactions between a truncated form of SOF in
which its fibronectin-binding domain has been deleted and the extracellular
matrix protein fibulin-1. Fibulin-1 is a member of the fibulin family that
currently consists of seven glycoproteins. All fibulins contain epidermal
growth factor-like repeats and a unique fibulin-type module at its C terminus
that define this family (16,
17). Fibulin-1 is found within
the extracellular matrices and in human plasma at 30–50 μg/ml
(18). It interacts with many
of the components of the extracellular matrix including fibronectin, laminin,
fibrinogen, nidogen-1, endostatin, aggrecan, and versican
(16,
19). Due to its intimate
relationship with the extracellular matrix, it is not surprising that the
defects in fibulin-1 have a wide-ranging impact. Genetic evidence suggests
that fibulin-1 is involved in tissue organization, the maturation and
maintenance of blood vessels, and multiple embryonic pathways
(16,
20–22).Although it has been established that many of the other components of the
extracellular matrix can interact with bacteria, there has been no previous
report on the binding of fibulins to bacteria. Our findings indicate that
fibulin-1 does bind to streptococci and that SOF is a major streptococcal
receptor for fibulin-1. 相似文献
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Xiaofen Li Shouting Liu Hongbiao Huang Ningning Liu Chong Zhao Siyan Liao Changshan Yang Yurong Liu Canguo Zhao Shujue Li Xiaoyu Lu Chunjiao Liu Lixia Guan Kai Zhao Xiaoqing Shi Wenbin Song Ping Zhou Xiaoxian Dong Jinbao Liu 《Cell reports》2013,3(1):211-222
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Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献