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1.
Xiaojun Li C. T. Ranjith-Kumar Monica T. Brooks S. Dharmaiah Andrew B. Herr Cheng Kao Pingwei Li 《The Journal of biological chemistry》2009,284(20):13881-13891
The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded
RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate
innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that
lacks signaling capability, regulates the signaling of the RLRs. To establish
the structural basis of dsRNA recognition by the RLRs, we have determined the
2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound
to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of
dsRNA with minimal contacts between the protein molecules. Gel filtration
chromatography and analytical ultracentrifugation demonstrated that LGP2 binds
blunt-ended dsRNA of different lengths, forming complexes with 2:1
stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do
not stimulate the activation of RIG-I efficiently in cells. Surprisingly,
full-length LGP2 containing mutations that abolish dsRNA binding retained the
ability to inhibit RIG-I signaling.The innate immune response is the first line of defense against invading
pathogens; it is the ubiquitous system of defense against microbial infections
(1). Toll-like receptors
(TLRs)3 and RIG-I
(retinoic acid-inducible gene
1)-like receptors (RLRs) play key roles in innate immune response
toward viral infection
(2-5).
Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the
endosome following phagocytosis of the pathogens
(6). RIG-I-like receptors RIG-I
and MDA5 detect viral RNA from replicating viruses in infected cells
(3,
7,
8). Stimulation of these
receptors leads to the induction of type I interferons (IFNs) and other
proinflammatory cytokines, conferring antiviral activity to the host cells and
activating the acquired immune responses
(4,
9).RIG-I discriminates between viral and host RNA through specific recognition
of the uncapped 5′-triphosphate of single-stranded RNA (5′ ppp
ssRNA) generated by viral RNA polymerases
(10,
11). In addition, RIG-I also
recognizes double-stranded RNA generated during RNA virus replication
(7,
12). Transfection of cells
with synthetic double-stranded RNA stimulates the activation of RIG-I
(13,
14). Synthetic dsRNA mimics,
such as polyinosinic-polycytidylic acid (poly(I·C)), can activate MDA5
when introduced into the cytoplasm of cells. Digestion of poly(I·C)
with RNase III transforms poly(I·C) from a ligand for MDA5 into a
ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I
recognizes short dsRNA (15).
Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of
these receptors in antiviral immune responses and demonstrated that they sense
different sets of RNA viruses
(12,
16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N
termini, a DEX(D/H) box RNA helicase domain, and a C-terminal
regulatory or repressor domain (CTD). The helicase domain and the CTD are
responsible for viral RNA binding, whereas the CARDs are required for
signaling (3,
8). The current model of RIG-I
activation suggests that under resting conditions RIG-I is in a suppressed
conformation, and viral RNA binding triggers a conformation change that leads
to the exposure of the CARDs for the recruitment of the downstream protein
IPS-1 (also known as MAVS, Cardif, or VISA)
(14,
17). Limited proteolysis of
the RIG-I·dsRNA complex showed that RIG-I residues 792-925 of the CTD
are involved in dsRNA and 5′ ppp ssRNA binding
(14). The CTD of RIG-I
overlaps with the C terminus of the previously identified repressor domain
(18). The structures of RIG-I
and LGP2 (laboratory of genetics and
physiology 2) CTD in isolation have been determined by
x-ray crystallography and NMR spectroscopy
(14,
19,
20). A large, positively
charged surface on RIG-I recognizes the 5′ triphosphate group of viral
ssRNA (14,
19). RNA binding studies by
titrating RIG-I CTD with dsRNA and 5′ ppp ssRNA suggested that
overlapping sets of residues on this charged surface are involved in RNA
binding (14). Mutagenesis of
several positively charged residues on this surface either reduces or disrupts
RNA binding by RIG-I, and these mutations also affect the induction of
IFN-β in vivo
(14,
19). However, the exact nature
of how the RLRs recognize viral RNA and how RNA binding activates these
receptors remains to be established.LGP2 is a homolog of RIG-I and MDA5 that lacks the CARDs and thus has no
signaling capability (21,
22). The expression of LGP2 is
inducible by dsRNA or IFN treatment as well as virus infection
(21). Overexpression of LGP2
inhibits Sendai virus and Newcastle disease virus signaling
(21). When coexpressed with
RIG-I, LGP2 can inhibit RIG-I signaling through the interaction of its CTD
with the CARD and the helicase domain of RIG-I
(18). LGP2 could suppress
RIG-I signaling by three possible ways
(23): 1) binding RNA with high
affinity, thereby sequestering RNA ligands from RIG-I; 2) interacting directly
with RIG-I to block the assembly of the signaling complex; and 3) competing
with IKKi (IκB kinase ε) in the NF-κB signaling pathway for a
common binding site on IPS-1. To elucidate the structural basis of dsRNA
recognition by the RLRs, we have crystallized human LGP2 CTD (residues
541-678) bound to an 8-bp double-stranded RNA and determined the structure of
the complex at 2.0 Å resolution. The structure revealed that LGP2 CTD
binds to the termini of dsRNA. Mutagenesis and functional studies showed that
dsRNA binding is likely not required for the inhibition of RIG-I signaling by
LGP2. 相似文献
2.
3.
4.
Taeko Shigemoto Maiko Kageyama Reiko Hirai JiPing Zheng Mitsutoshi Yoneyama Takashi Fujita 《The Journal of biological chemistry》2009,284(20):13348-13354
Retinoic acid-inducible gene I (RIG-I) and melanoma
differentiation-associated gene 5 (MDA5) are essential for detecting viral RNA
and triggering antiviral responses, including production of type I interferon.
We analyzed the phenotype of non-synonymous mutants of human RIG-I and MDA5
reported in databases by functional complementation in cell cultures. Of seven
missense mutations of RIG-I, S183I, which occurs within the second caspase
recruitment domain repeat, inactivated this domain and conferred a dominant
inhibitory function. Of 10 mutants of MDA5, two exhibited loss of function. A
nonsense mutation, E627*, resulted in deletion of the C-terminal region and
double-stranded RNA (dsRNA) binding activity. Another loss of function
mutation, I923V, which occurs within the C-terminal domain, did not affect
dsRNA binding activity, suggesting a novel and essential role for this residue
in the signaling. Remarkably, these mutations are implicated in resistance to
type I diabetes. However, the A946T mutation of MDA5, which has been
implicated in type I diabetes by previous genetic analyses, affected neither
dsRNA binding nor IFN gene activation. These results provide new
insights into the structure-function relationship of RIG-I-like receptors as
well as into human RIG-I-like receptor polymorphisms, antiviral innate
immunity, and autoimmune diseases.Innate and adaptive immune systems constitute the defense against
infections by pathogens. Immediately after an infection occurs, various cells
in the body sense the virus and initiate antiviral responses in which type I
IFN2 plays a critical
role, both in viral inhibition and in the subsequent adaptive immune response
(1). The production of IFN is
initiated when sensor molecules such as Toll-like receptors (TLRs) and RLRs
detect virus-associated molecules. TLRs detect pathogen-associated molecular
patterns (PAMPs) at the cell surface or in the endosome in immune cells such
as dendritic cells and macrophages
(2). RLRs sense viral RNA in
the cytoplasm of most cell types and induce antiviral responses, including the
activation of IFN genes
(3). RLRs include RIG-I, MDA5,
and laboratory of genetics and physiology 2 (LGP2).It is proposed that RLRs sense and activate antiviral signals through the
coordination of their functional domains
(4). The N-terminal region of
RIG-I and MDA5 is characterized by two repeats of CARD and functions as an
activation domain (3). This
domain is responsible for the transduction of signals downstream to IFN-β
promoter stimulator 1 (IPS-1) (also known as MAVS, VISA, and Cardif). The
primary sequence of the CTD, consisting of ∼140 amino acids, is conserved
among RLRs. The CTD of RIG-I functions as a viral RNA-sensing domain as
revealed by biochemical and structural analyses
(5,
6). Both dsRNA and
5′-ppp-ssRNA, which are generated in the cytoplasm of virus-infected
cells, are recognized by a basic cleft structure of RIG-I CTD. In addition to
its RNA recognition function, the CTD of RIG-I and LGP2 functions as a
repression domain through interaction with the activation domain. The
repression domain is responsible for keeping RIG-I inactive in non-stimulated
cells (3,
7). The helicase domain, with
DEXD/H box-containing RNA helicase motifs, is the largest domain
found in RLRs. Once dsRNA or 5′-ppp-ssRNA is recognized by the CTD, the
helicase domain causes structural changes to release the activation domain.
ATP binding and/or its hydrolysis is essential for the conformational change
because Walker''s ATP-binding site within the helicase domain is essential for
signaling by RIG-I and MDA5.Analyses of knock-out mice have revealed that RIG-I and MDA5 recognize
distinct RNA viruses (8,
9). Picornaviruses are detected
by MDA5, but many other viruses such as influenza A, Sendai, vesicular
stomatitis, and Japanese encephalitis are detected by RIG-I. The difference is
based on the distinct non-self RNA patterns generated by viruses, as
demonstrated by the finding that RIG-I is selectively activated by dsRNA or
5′-ppp ssRNA, whereas MDA5 is activated by long dsRNA
(10–12).Single nucleotide polymorphisms (SNPs) of the human RIG-I and
MDA5 genes including several non-synonymous SNPs (nsSNPs), which
potentially alter the function of the proteins encoded, are reported in
databases. In this report, we investigated the functions of nsSNPs of RIG-I
and MDA5 by functional complementation using respective knock-out cells. We
identified loss of function mutations of RIG-I and MDA5. Notably, two MDA5
mutations, E627* and I923V, recently reported to have a strong association
with resistance to T1D (13),
were severely inactive. The results suggest a novel molecular mechanism for
the activation of RLRs and will contribute to our understanding of the
functional effects of RLR polymorphisms and the critical relationship between
RLR nsSNPs and diseases. 相似文献
5.
Dennis Ding-Hwa Wang Zhanyong Shu Scot A. Lieser Phang-Lang Chen Wen-Hwa Lee 《The Journal of biological chemistry》2009,284(31):20812-20821
6.
7.
8.
9.
Elucidating the temporal order of silencing 总被引:1,自引:0,他引:1
Izaurralde E 《EMBO reports》2012,13(8):662-663
10.
11.
12.
13.
Jason E. Pike Peter M. J. Burgers Judith L. Campbell Robert A. Bambara 《The Journal of biological chemistry》2009,284(37):25170-25180
We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.Eukaryotic cellular DNA is replicated semi-conservatively in the 5′ to 3′ direction. A leading strand is synthesized by DNA polymerase ϵ in a continuous manner in the direction of opening of the replication fork (1, 2). A lagging strand is synthesized by DNA polymerase δ (pol δ)3 in the opposite direction in a discontinuous manner, producing segments called Okazaki fragments (3). These stretches of ∼150 nucleotides (nt) must be joined together to create the continuous daughter strand. DNA polymerase α/primase (pol α) initiates each fragment by synthesizing an RNA/DNA primer consisting of ∼1-nt of RNA and ∼10–20 nt of DNA (4). The sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the DNA by replication factor C (RFC). pol δ then complexes with PCNA and extends the primer. When pol δ reaches the 5′-end of the downstream Okazaki fragment, it displaces the end into a flap while continuing synthesis, a process known as strand displacement (5, 6). These flap intermediates are cleaved by nucleases to produce a nick for DNA ligase I (LigI) to seal, completing the DNA strand.In one proposed mechanism for flap processing, the only required nuclease is flap endonuclease 1 (FEN1). pol δ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7–9). FEN1 binds at the 5′-end of the flap and tracks down the flap cleaving only at the base (5, 10, 11). Because pol δ favors the displacement of RNA-DNA hybrids over DNA-DNA hybrids, strand displacement generally is limited to that of the initiator RNA of an Okazaki fragment (12). In addition, the tightly coordinated action of pol δ and FEN1 also tends to keep flaps short. However, biochemical reconstitution studies demonstrate that some flaps can become long (13, 14). Once these flaps reach ∼30 nt, they can be bound by the eukaryotic single strand binding protein replication protein A (RPA) (15). Binding by RPA to a flap substrate inhibits cleavage by FEN1 (16). The RPA-bound flap would then require another mechanism for proper processing.This second mechanism is proposed to utilize Dna2 (16) in addition to FEN1. Dna2 is both a 5′-3′ helicase and an endonuclease (17, 18). Like FEN1, Dna2 recognizes 5′-flap structures, binding at the 5′-end of the flap and tracking downward toward the base (19, 20). Unlike FEN1, Dna2 cleaves the flap multiple times but not all the way to the base, such that a short flap remains (20). RPA binding to a flap has been shown to stimulate Dna2 cleavage (16). Therefore, if a flap becomes long enough to bind RPA, Dna2 binds and cleaves it to a length of 5–10 nucleotides from which RPA dissociates (21). FEN1 can then enter the flap, displace the Dna2, and then cleave at the base to make the nick for ligation (16, 18, 22). The need for this mechanism may be one reason why DNA2 is an essential gene in Saccharomyces cerevisiae (23, 24). It has been proposed that, in the absence of Dna2, flaps that become long enough to bind RPA cannot be properly processed, leading to genomic instability and cell death (23).In reconstitution of Okazaki fragment processing with purified proteins, even though some flaps became long enough to bind RPA, FEN1 was very effective at cleaving essentially all of the generated flaps (13, 14). Evidently, FEN1 could engage the flaps before binding of RPA. However, these reconstitution assays did not include the 5′-3′ helicase Pif1 (25, 26). Pif1 is involved in telomeric and mitochondrial DNA maintenance (26) and was first implicated in Okazaki fragment processing from genetic studies in S. cerevisiae. Deletion of PIF1 rescued the lethality of dna2Δ, although the double mutant was still temperature-sensitive (27). The authors of this report proposed that Pif1 creates a need for Dna2 by promoting longer flaps. Further supporting this conclusion, deletion of POL32, which encodes the subunit of pol δ that interacts with PCNA, rescued the temperature sensitivity of the dna2Δpif1Δ double mutant (12, 27). Importantly, pol δ exhibited reduced strand displacement activity when POL32 was deleted (12, 28, 29). The combination of pif1Δ and pol32Δ is believed to create a situation in which virtually no long flaps are formed, eliminating the requirement for Dna2 flap cleavage (27).We recently performed reconstitution assays showing that Pif1 can assist in the creation of long flaps. Inclusion of Pif1, in the absence of RPA, increased the proportion of flaps that lengthened to ∼28–32 nt before FEN1 cleavage (14). With the addition of RPA, the appearance of these long flap cleavage products was suppressed. Evidently, Pif1 promoted such rapid flap lengthening that RPA bound some flaps before FEN1 and inhibited cleavage. The RPA-bound flaps would presumably require cleavage by Dna2 for proper processing.Only a small fraction of flaps became long with Pif1. However, there are hundreds of thousands of Okazaki fragments processed per replication cycle (30). Therefore, thousands of flaps are expected to be lengthened by Pif1 in vivo, a number significant enough that improper processing of such flaps could lead to cell death.Our goal here was to determine whether Pif1 can influence the flow of Okazaki fragments through the two proposed pathways. We first questioned whether Pif1 stimulates strand displacement synthesis by pol δ. Next, we asked whether Pif1 lengthens short flaps so that Dna2 can bind and cleave. Finally, we used a complete reconstitution system to determine whether Pif1 promotes creation of RPA-bound flaps that require cleavage by both Dna2 and FEN1 before they can be ligated. Our results suggest that Pif1 promotes the two-nuclease pathway, and reveal the mechanisms involved. 相似文献
14.
15.
16.
Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg2+ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.Viruses with single-strand positive-sense RNA genomes represent the largest class of viruses, which includes numerous pathogens of humans, plants, and animals. In these viruses, RNA replication occurs through negative-strand RNA intermediate, which may also act as the template for synthesis of subgenomic RNAs in some viruses. During replication, various nonstructural proteins remain associated with the viral polymerase in a small compartmentalized replisome. Most of the other accessory proteins are obtained from the cellular machinery.Helicase seems to be essential for RNA replication by many positive-sense RNA viruses (19). Many positive-strand RNA viruses encode their own RNA helicases and besides RNA-dependent RNA polymerase, helicase is the most conserved viral sequence in these viruses. It has been shown by direct mutagenesis studies in poliovirus (26, 39), alphaviruses (31), brome mosaic virus (2, 41), nidoviruses (40), and flaviviruses (15) that helicase functions are essential for viral replication. In addition, it may be involved in RNA translocation, genome packaging, protection of RNA at the replication center, modulating RNA-protein interactions, etc.Helicases are classified into six superfamilies, SF-1 to SF-6 (11, 35), and can be classified further into subfamilies, A (3′→5′) or B (5′→3′) depending on their unwinding directionality. Classic helicases (exhibiting both NTPase and unwinding activities) are referred to as subtype α, while translocases (with no unwinding activity) are referred to as subtype β (35). SF-1 and SF-2 constitute largest of these superfamilies with seven signature motifs (I, Ia, II, III, IV, V, and VI), which form core of the enzyme. Although these motifs are not comparable between SF-1 and SF-2, universal features of core domains include (i) conserved residues involved in binding and hydrolysis of the NTP and (ii) an arginine finger that plays a key role in energy coupling.Hepatitis E virus (HEV) is a nonenveloped virus in the genus Hepevirus of the family Hepeviridae. Hepatitis E is an important public health disease in many developing countries and is also endemic in some industrialized countries (8). Infection by HEV has a known association with increased mortality during pregnancy (22, 23). HEV has a positive-sense RNA genome of ∼7.2 kb, consisting of a 5′ noncoding region (5′NCR) of 27 to 35 nucleotides (nt), followed by three open reading frames (ORFs)—ORF1, ORF2, and ORF3—and a 3′NCR of 65 to 74 nt, ending with a poly(A) tail of variable length (37). The 5′ end has m7G cap (18). ORF1 is known to encode for the viral nonstructural polyprotein with a proposed molecular mass of ∼186 kDa (3). Based on protein sequence homology, the ORF1 polyprotein is proposed to contain four putative domains indicative of methyltransferase, papain-like cysteine protease, RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) (24). ORF2 encodes the major structural protein (capsid protein), which has N-terminal signal peptide and three glycosylation sites and is translocated across the endoplasmic reticulum (ER). ORF2 protein associates with the 5′ end of the viral RNA, suggesting its regulatory role in the virus replication (36, 37, 44, 45). ORF3 encodes a protein which gets phosphorylated by the cellular mitogen activated protein kinase and is associated with cellular membranes and cytoskeleton fractions (43).HEV belongs to an “alpha-like” supergroup of positive-sense single-stranded RNA (+ssRNA) viruses with conserved motifs of replication-related proteins in the ORF1, with typical signature sequences homologous with the other members of the family (11, 12, 13). ORF1 of HEV encodes additional domains such as the Y domain, papainlike protease, “proline-rich hinge,” and the X domain. Methyltransferase (25), RdRp (1), and X domain (binding to poly-ADP-ribose) (9) in ORF1 have been characterized, whereas the functions of the other domains are yet to be identified. Intracellularly expressed RdRp localizes itself in the ER membranes (30), suggesting that HEV replicates probably in ER in the cytosolic compartment of the cells. It is still unknown whether ORF1 polyprotein undergoes cleavages to form separate functional units of the replication machinery or functions as a single protein with multiple functional domains.The putative RNA helicase of HEV contains all of the seven conserved segments typical of the SF-1 helicase (12, 13). Putative SF-1 helicases are extremely widespread among +ssRNA viruses. Based on sequence comparisons, such helicases have been identified in a variety of plant virus families, as well as in animal viruses such as alphavirus, rubivirus, hepatitis E virus, and coronavirus (11). When compared to other +ssRNA viral helicases belonging to SF-1, HEV helicase showed the highest overall similarity with the helicase of beet necrotic yellow vein virus, a plant furovirus. HEV helicase was speculated to have N-terminal NTPase and C-terminal RNA-binding domains (24). A major obstacle in studying HEV replication has been lack of cell culture system. We report here experimental verification of the helicase activity of the recombinant helicase domain protein of HEV. 相似文献
17.
Michele L. Forster James J. Mahn Billy Tsai 《The Journal of biological chemistry》2009,284(19):13045-13056
Protein-disulfide isomerase (PDI), an endoplasmic reticulum (ER)-resident
protein, is primarily known as a catalyst of oxidative protein folding but
also has a protein unfolding activity. We showed previously that PDI unfolds
the cholera toxin A1 (CTA1) polypeptide to facilitate the ER-to-cytosol
retrotranslocation of the toxin during intoxication. We now provide insight
into the mechanism of this unfoldase activity. PDI includes two redox-active
(a and a′) and two redox-inactive (b and
b′) thioredoxin-like domains, a linker (x), and a
C-terminal domain (c) arranged as
abb′xa′c. Using recombinant PDI
fragments, we show that binding of CTA1 by the continuous
PDIbb′xa′ fragment is necessary and sufficient
to trigger unfolding. The specific linear arrangement of
bb′xa′ and the type a domain
(a′ versus a) C-terminal to
bb′x are additional determinants of activity. These
data suggest a general mechanism for the unfoldase activity of PDI: the
concurrent and specific binding of bb′xa′ to
particular regions along the CTA1 molecule triggers its unfolding.
Furthermore, we show the bb′ domains of PDI are indispensable
to the unfolding reaction, whereas the function of its a′
domain can be substituted partially by the a′ domain from ERp57
(abb′xa′c) or ERp72
(ca°abb′xa′), PDI-like proteins
that do not unfold CTA1 normally. However, the bb′ domains of
PDI were insufficient to convert full-length ERp57 into an unfoldase because
the a domain of ERp57 inhibited toxin binding. Thus, we propose that
generating an unfoldase from thioredoxin-like domains requires the
bb′(x) domains of PDI followed by an a′
domain but not preceded by an inhibitory a domain.Protein-disulfide isomerase
(PDI)2 is a
multifunctional protein that resides in the endoplasmic reticulum (ER) lumen
of all eukaryotic cells (reviewed in Ref.
1). Mammalian PDI was first
identified as a catalyst of oxidative protein folding
(2), but it is now also known
to mediate viral infection (3,
4), antigen processing
(5), collagen assembly
(6), and ER-associated
degradation
(7–9).
To participate in this variety of cellular processes, PDI performs multiple
activities. For example, during oxidative protein folding, PDI catalyzes the
oxidation and isomerization of disulfide bonds and induces conformational
changes in non-native polypeptides
(10). Independently of redox
chemistry, PDI is a molecular chaperone, binding polypeptides to prevent their
aggregation
(11–13).
PDI also acts as a structural subunit of the prolyl 4-hydroxylase (P4H) and
microsomal triglyceride transfer protein complexes; however, this function is
similar to its chaperone activity
(14–19).
In contrast to its protein folding activities, PDI unfolds the catalytic A1
polypeptide of cholera toxin (CTA1) in preparation for the retrotranslocation
of the toxin from the ER lumen into the cytosol
(8,
20).Cholera toxin (CT) is a pathogenic factor that causes secretory diarrhea in
animals (reviewed in Ref. 21).
The holotoxin includes a single catalytic A subunit (CTA) and a homopentameric
B subunit (CTB) joined noncovalently
(22). Upon secretion from the
bacterium Vibrio cholerae, CTA is cleaved into the A1 and A2
polypeptides, which are joined by a disulfide bond and noncovalent
interactions (22,
23). To intoxicate a cell, CTB
binds the ganglioside GM1 on the surface of the cell, and the holotoxin is
transported in a retrograde manner to the ER lumen
(24). In the ER, CTA is
reduced to generate CTA1, and PDI unfolds and dissociates CTA1 from the
holotoxin (20). The unfolded
toxin is subsequently transported across the ER membrane
(25,
26). Upon reaching the
cytosol, CTA1 refolds and induces toxicity
(27,
28).We showed previously that PDI acts as a redox-dependent chaperone to unfold
CTA1 (20). In the reduced
state of PDI, it binds and unfolds the toxin. Subsequent oxidation of PDI by
ER oxidase 1 causes PDI to release unfolded CTA1
(25). Aside from this
information, nothing is known about the mechanism of the unfolding activity of
PDI.PDI is a modular protein comprising two a-type thioredoxin-like
domains (a and a′), two b-type
thioredoxin-like domains (b and b′), a flexible linker
(x), and an extended C-terminal domain (c) arranged as
abb′xa′c
(29–31).
The a-type domains are characterized by the presence of the catalytic
sequence CXXC and are therefore redox-active, whereas the
b-type domains lack this sequence and are redox-inactive
(32). The thioredoxin-like
domains of PDI differ from each other in primary structure despite having a
common fold. The crystal structure of yeast PDI shows the bb′
domains form a rigid base from which the a-type domains extend like
flexible arms (33,
34). This base is thought to
be the core of a substrate-binding groove formed by all four thioredoxin-like
domains (30,
33,
35).To understand the mechanism of the unfoldase activity of PDI, we analyzed
the contribution of each domain to the ability of PDI to bind and unfold CTA1
using recombinant PDI fragments. Unfolded CTA1 was detected by an established
in vitro trypsin sensitivity assay that relies on tryptic cleavage
sites hidden in the folded toxin to be exposed in the unfolded toxin
(20). Because CTA1 likely
mimics a misfolded host cell protein for its recognition and unfolding by PDI
(22,
36,
37), this study has
implications for how PDI unfolds endogenous misfolded proteins in preparation
for their retrotranslocation and subsequent ER-associated degradation.There are nearly 20 mammalian PDI-like proteins, characterized by the
presence of one or more thioredoxin-like domains and ER localization (reviewed
in Refs. 38,
39). We previously
demonstrated that two PDI-like proteins, ERp72 and ERp57, do not facilitate
CTA1 retrotranslocation (8). In
contrast to PDI, ERp72 retains CTA1 in the ER and either stabilizes its native
conformation or renders it more compact
(8). To understand how these
structurally homologous proteins are functionally unique, we tested whether
the various thioredoxin-like domains of ERp57 and ERp72 could functionally
replace the corresponding PDI domains to unfold CTA1. Thus, in addition to
suggesting a general mechanism for the unfoldase activity of PDI, our data
indicate functional similarities and differences among thioredoxin-like
domains of PDI family proteins. 相似文献
18.
19.
20.
Zhaohui Wang Krzysztof Treder W. Allen Miller 《The Journal of biological chemistry》2009,284(21):14189-14202
RNAs of many positive strand RNA viruses lack a 5′ cap structure and
instead rely on cap-independent translation elements (CITEs) to facilitate
efficient translation initiation. The mechanisms by which these RNAs recruit
ribosomes are poorly understood, and for many viruses the CITE is unknown.
Here we identify the first CITE of an umbravirus in the 3′-untranslated
region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of
the ∼100-nucleotide PEMV RNA 2 CITE (PTE), and
mutagenesis revealed that it forms a long, bulged helix that branches into two
short stem-loops, with a possible pseudoknot interaction between a C-rich
bulge at the branch point and a G-rich bulge in the main helix. The PTE
inhibited translation in trans, and addition of eIF4F, but not
eIFiso4F, restored translation. Filter binding assays revealed that the PTE
binds eIF4F and its eIF4E subunit with high affinity. Tight binding required
an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a
strong correlation between PTE-eIF4E binding affinity and ability to stimulate
cap-independent translation. We conclude that the PTE recruits eIF4F by
binding eIF4E. The PTE represents a different class of translation enhancer
element, as defined by its structure and ability to bind eIF4E in the absence
of an m7G cap.Regulation of translation occurs primarily at the initiation step. This
involves recognition of the 5′ m7G(5′)ppp(5′)N
cap structure on the mRNA by initiation factors, which recruit the ribosome to
the 5′-end of the mRNA
(1–5).
The 5′ cap structure and the poly(A) tail are necessary for efficient
recruitment of initiation factors on eukaryotic mRNAs
(3,
6–8).
The cap is recognized by the eIF4E subunit of eukaryotic translation
initiation factor complex eIF4F (or the eIFiso4E subunit of eIFiso4F in higher
plants). The poly(A) tail is recognized by poly(A)-binding protein. In plants,
eIF4F is a heterodimer consisting of eIF4E and eIF4G, the core scaffolding
protein to which the other factors bind. eIF4A, an ATPase/RNA helicase,
interacts with eIF4F but is not part of the eIF4F heterodimer
(9,
10). For translation
initiation, the purpose of eIF4E is to bring eIF4G to the capped mRNA. eIF4G
then recruits the 43 S ternary ribosomal complex via interaction with
eIF3.The RNAs of many positive sense RNA viruses contain a cap-independent
translation element
(CITE)3 that allows
efficient translation in the absence of a 5′ cap structure
(11–13).
In animal viruses and some plant viruses, the CITE is an internal ribosome
entry site (IRES) located upstream of the initiation codon. Most viral IRESes
neither interact with nor require eIF4E, because they lack the
m7GpppN structure, which, until this report, was thought to be
necessary for mRNA to bind eIF4E with high affinity
(3,
14). Translation initiation
efficiency of mRNA is also influenced by the length of, and the degree of
secondary structure in the 5′ leader
(15–17).Many uncapped plant viral RNAs harbor a CITE in the 3′-UTR that
confers highly efficient translation initiation at the 5′-end of the
mRNA
(18–22).
These 3′ CITEs facilitate ribosome entry and apparently conventional
scanning at the 5′-end of the mRNA
(17,
23,
24). A variety of unrelated
structures has been found to function as 3′ CITEs, suggesting that they
recruit the ribosome by different interactions with initiation factors
(13).The factors with which a plant CITE interacts to recruit the ribosome have
been identified for only a potyvirus, a luteovirus, and a satellite RNA. The
143-nt 5′-UTR CITE of the potyvirus, tobacco etch virus is an IRES that
functions by binding of its AU-rich pseudoknot structure with eIF4G
(25). It binds eIF4G with up
to 30-fold greater affinity than eIFiso4G and does not require eIF4E for IRES
activity. In addition to RNA elements, the genome-linked viral protein (VPg)
of potyviruses may participate in cap-independent translation initiation by
interacting with the eIF4E and eIFiso4E subunits of eIF4F and eIFiso4F,
respectively
(26–31).
In contrast, the 130-nt cap-independent translation enhancer domain (TED) in
the 3′-UTR of satellite tobacco necrosis virus (STNV) RNA forms a long
bulged stem-loop, which interacts strongly with both eIF4F and eIFiso4F and
weakly with their eIF4E and eIFiso4E subunits
(32), suggesting that the TED
requires the full eIF4F or eIFiso4F for a biologically relevant interaction.
Barley yellow dwarf luteovirus (BYDV) and several other viruses, have a
different structure, called a BYDV-like CITE (BTE), in the 3′-UTR. The
BTE is characterized by a 17-nt conserved sequence incorporated in a structure
with a variable number of stem-loops radiating from a central junction
(20,
33,
34). It requires and binds the
eIF4G subunit of eIF4F and does not bind free eIF4E, eIFiso4E, or eIFiso4G,
although eIF4E slightly enhances the BTE-eIF4G interaction
(35). Other 3′ CITEs
have been identified, but the host factors with which they interact are
unknown.Here we describe unprecedented factor interactions of a CITE found in an
umbravirus and a panicovirus. Umbraviruses show strong similarity to the
Luteovirus and Dianthovirus genera in (i) the sequence of
the replication genes encoded by ORFs 1 and 2, (ii) the predicted structure of
the frameshift signals required for translation of the RNA-dependent RNA
polymerase from ORF 2 (36,
37), (iii) the absence of a
poly(A) tail, and (iv) the lack of a 5′ cap structure
(37,
38). Umbraviruses are unique
in that they encode no coat protein. For the umbravirus pea enation mosaic
virus 2 (PEMV-2), the coat protein is provided by PEMV-1, an enamovirus
(39). Uncapped PEMV-2 RNA
(PEMV RNA 2), transcribed in vitro, is infectious in pea (Pisum
sativa),4
indicating it must be translated cap-independently. The 3′-UTRs of some
umbraviruses such as Tobacco bushy top virus and Groundnut rosette virus
harbor sequences resembling BYDV-like CITEs
(BTE).5 However, no
BTE is apparent in the 3′-UTR of PEMV RNA 2. In this report we identify
a different class of CITE in the 705-nt long 3′-UTR of PEMV RNA 2,
determine its secondary structure, which may include an unusual pseudoknot,
and we show that, unlike any other natural uncapped RNA, it has a high
affinity for eIF4E, which is necessary to facilitate cap-independent
translation. 相似文献