Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3′-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3′-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.
For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones. In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.
For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two 15 N-labeled proteins and obtained 1 H-15 N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and 1 H-15 N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.Keywords: Wheat germ; cell-free; protein synthesis; HSQC; structural analysis With the increase in the available sequence information on the genomes in various cells, attention has been turned to the structures, properties, and functional activities of proteins. However, rapid progress in the area of proteomics requires the availability of sufficient amounts of proteins. Currently, three major strategies are being used for protein production: chemical synthesis, in vivo expression, and cellfree synthesis. The first two methods have severe drawbacks. Chemical synthesis is not practical for the synthesis of long peptides (Blaschke et al. 2000), and in vivo expression can produce proteins that do not have any significant effect on the physiology of the host cells (Golf and Goldberg 1987;Chrunyk et al. 1993). With a cell-free translation system, in contrast, one can synthesize larger proteins at the same or higher speed, and as accurately as ones for in vivo translation (Kurland 1982;Pavlov and Ehrenberg 1996), and express proteins that would interfere with the host cell physiology.One of the most convenient eukaryotic cell-free translation systems is based on wheat germ embryos containing all the components for translation in a concentrated dried state and ready for protein synthesis after germination. A past study has indicated that such systems are generally unstable and thus insufficient (Roberts and Paterson 1973). Recently, however, we found that plants contain endogeneous inhibiReprint requests to: Eugene Hayato Morita, Center for Gene Research, Ehime University, 3-5-7 Tarumi, Ehime 790-8566, Japan; e-mail: ehmorita@dpc.ehime-u.ac.jp; fax: 81-89-946-9968; or Toshiyuki Kohno, Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooy...
In eukaryotic DNA replication, an MCM2-7 heterohexameric complex probably functions as a replicative DNA helicase responsible for the unwinding of duplex DNA at the replication forks [1][2][3]. Recent studies suggest that during DNA replication, several other proteins, including Cdc45 and GINS, migrate along DNA with MCM proteins [4,5]. The GINS complex that may be essential for the loading of DNA polymerase e comprises the Sld5, Psf1, Psf2 and Psf3 proteins [6,7]. Consistent with this notion, it has been demonstrated that a large protein complex comprising MCM2-7, Cdc45 and GINS exhibits in vitro DNA helicase activity [8], although whether the MCM2-7 complex itself functions as a DNA helicase remains to be determined. The hetero-hexameric MCM2-7 complex that shows the DNA replication licensing activity comprises an MCM4 ⁄ 6 ⁄ 7 core complex and loosely associated MCM2 and MCM3 ⁄ 5 [9,10]. In vitro studies demonstrated that the MCM2-7 hetero-hexamer does not exhibit DNA helicase activity, but an MCM4 ⁄ 6 ⁄ 7 hexamer (dimer of an MCM4 ⁄ 6 ⁄ 7 trimer) functions as a DNA helicase [11][12][13][14]. The MCM4 ⁄ 6 ⁄ 7 complex was found to be relatively inefficient in displacing long DNA fragments in a conventional DNA helicase reaction [11]; however, its high processibility was observed when the T-stretch sequences frequently detected at replication initiation sites were present on ssDNA [15]. Both the MCM2 protein and the MCM3 ⁄ 5 complex inhibit MCM4 ⁄ 6 ⁄ 7 helicase activity by disassembling the MCM4 ⁄ 6 ⁄ 7 hexamer into the MCM2 ⁄ 4 ⁄ 6 ⁄ 7 and MCM3 ⁄ 4 ⁄ 5 ⁄ 6 ⁄ 7 complexes, respectively [16,17]. These results suggest a model in which MCM2 and MCM3 ⁄ 5 may regulate the helicase activity of MCM4 ⁄ 6 ⁄ 7. Consistent with this hypothesis, it has been shown that The molecular dissection of human MCM2, a constituent of MCM2-7 licensing factor complex, was performed to identify the region responsible for its biochemical activities. Partial digestion with trypsin dissected the MCM2 protein into a central region (148-676) containing ATPase motifs and a C-terminal region (677-895). These two fragments, along with three other fragments (148-441, 442-676 and 442-895), were produced using the wheat germ cell-free system and were examined for their ability to inhibit MCM4 ⁄ 6 ⁄ 7 helicase activity. Two fragments (442-895 and 677-895) containing the C-terminus were partly inhibitory to the activity. Further dissection revealed that one fragment (713-895) has strong inhibitory activity. The inhibitory activity of the smaller fragments derived from the C-terminal region correlated with their ability to inhibit SV40 T antigen helicase activity and also with their ability to bind to ssDNA, which has been shown by gel mobility shift analysis. These results strongly suggest that the MCM2 fragments derived from the C-terminal region inhibit DNA helicase activity through their ability to bind to ssDNA. In contrast, two fragments (148-441 and 442-676) from the central region were mainly responsible for the interaction between MCM2 and M...
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