Epitope-Based Specific Antibody Modifications.

Modified antibodies have essential roles in analytic, diagnostic, and therapeutic uses, and thus, these antibodies are required to have optimal physical and biological properties. Consequently, the development of methods for site-selective antibody modification is crucial. Herein, we used epitope-based affinity labeling to introduce a Fab region-selective antibody modification method. Although labeling that exploits the high affinity between an antibody and its epitope may appear straightforward, it remains challenging probably because of the loss of target affinity caused by modification around the epitope-binding site. By thoroughly screening the modifying agent structure, reaction conditions, and purification methods, we developed an efficient method for the selective modification of the Fab region of the antibody while maintaining the high affinity for the epitope.

UVA causes specific mutagenic DNA damage through ROS production, rather than CPD formation, in Drosophila larvae.

Evidence is accumulating that ultraviolet A (UVA) plays an important role in photo-carcinogenesis. However, the types of DNA damage involved in the resulting mutations remain unclear. Previously, using Drosophila, we found that UVA from light-emitting diode (LED-UVA) induces double-strand breaks in DNA through oxidative damage in an oxidative damage-sensitive (urate-null) strain. Recently, it was proposed that cyclobutane pyrimidine dimers (CPDs), which also are induced by UVA irradiation, might play a significant role in the induction of mutations. In the present study, we investigated whether reactive oxygen species (ROS) and CPDs are produced in larval bodies following LED-UVA irradiation. In addition, we assessed the somatic cell mutation rate in urate-null Drosophila induced by monochromatic UVA irradiation. The production of ROS through LED-UVA irradiation was markedly higher in the urate-null strain than in the wild-type Drosophila. CPDs were detected in the DNA of both of UVA- and UVB-irradiated larvae. The level of CPDs was unexpectedly higher in the wild-type strain than in urate-null flies following UVA irradiation, whereas this parameter was expectedly similar between the urate-null and wild-type Drosophila following UVB irradiation. The somatic cell mutation rate induced by UVA irradiation was higher in the urate-null strain than in the wild-type strain. These results suggest that mutations induced by UVA-specific pathways occur through ROS production, rather than via CPD formation.

Mapping of the regions implicated in nuclear localization of multi-functional DNA repair endonuclease XPF-ERCC1.

The structure-specific endonuclease XPF-ERCC1 is a multi-functional heterodimer that participates in a variety of DNA repair mechanisms for maintaining genome integrity. Both subunits contain C-terminal tandem helix-hairpin-helix (HhH2 ) domains, which are necessary for not only their dimerization but also enzymatic activity as well as protein stability. However, the interdependency of both subunits in their nuclear localization remains poorly understood. In this study, we have analyzed the region(s) that affects the subcellular localization of XPF and ERCC1 using various deletion mutants. We first identified the nuclear localization signal (NLS) in XPF, which was essential for its nuclear localization under the ERCC1-free condition, but dispensable in the presence of ERCC1 (probably as XPF-ERCC1 heterodimer). Interestingly, in the NLS-independent and ERCC1-dependent XPF nuclear localization, the physical interaction between XPF and ERCC1 via C-terminal HhH2 domains was not needed. Instead, the amino acid regions 311-469 of XPF and 216-260 of ERCC1 are required for the nuclear localization. Furthermore, we found that the 311-469 region of XPF interacts with ERCC1 in a co-immunoprecipitation assay. These results suggest that the nuclear localization of XPF-ERCC1 heterodimer is regulated at multiple levels in an interdependent manner.

Camptothecin compromises transcription recovery and cell survival against cisplatin and ultraviolet irradiation regardless of transcription-coupled nucleotide excision repair.

DNA-damaging anti-cancer drugs are used clinically to induce cell death by causing DNA strand breaks or DNA replication stress. Camptothecin (CPT) and cisplatin are commonly used anti-cancer drugs, and their combined use enhances the anti-tumour effects. However, the mechanism underlying this enhanced effect has not been well studied. In this study, we analysed the combined effect of CPT and cisplatin or ultraviolet (UV) and found that CPT suppresses transcription recovery after UV damage and induces the disappearance of the Cockayne syndrome group B (CSB) protein, a transcription-coupled nucleotide excision repair (TC-NER) factor. This CPT-induced disappearance of CSB expression was suppressed by proteasome and transcription inhibitors. Moreover, CSB ubiquitination was detected after CPT treatment in a transcription-dependent manner, suggesting that the transcription stress caused by CPT induces CSB ubiquitination, resulting in CSB undetectability. However, Cockayne syndrome group A (CSA) and CUL4A were not involved in the CPT-induced CSB undetectability, suggesting that CSB ubiquitination caused by CPT is regulated differently from the UV response. However, cisplatin or UV sensitivity was enhanced by CPT even in CSB- or CSA-knockout cells. Furthermore, the excessive CSB expression, which suppressed CSB ubiquitination, did not cancel the combined effect of CPT. These results suggest that CPT blocks the repair of cisplatin or UV-induced DNA damage regardless of TC-NER status. CPT possibly compromised the alternative repair pathways other than TC-NER, leading to the suppression of transcription recovery and enhancement of cell killing.

CD28 confers CD4+ T cells with resistance to cyclosporin A and tacrolimus but to different degrees.

BACKGROUND: Cyclosporin A (CSA) and tacrolimus (TAC) suppress T-cell activation and subsequent proliferation by inhibiting calcineurin. Though they have the same target, CSA and TAC have quite different molecular structures, indicating quantitative and/or qualitative differences in their effects. OBJECTIVE: CD28 is a costimulatory molecule that enhances T-cell activation. It has also been shown to attenuate calcineurin inhibitors. In this study, we compared the CD28-mediated resistance of CD4+ T cells to those calcineurin inhibitors and tried to predict CD28's impact on infectious diseases. METHODS: CD4+ T-cell proliferation was induced with anti-CD3 mAb in the presence or absence of anti-CD28 mAb in vitro. CSA or TAC was added at various concentrations, and the half-maximal inhibitory concentration on CD4+ T-cell proliferation was determined. Effects of lipopolysaccharide (LPS) on dendritic cells (DCs) and CD4+ T-cell proliferation were also evaluated in vitro. RESULTS: Anti-CD28 mAb conferred CD4+ T cells with resistance to both CSA and TAC, and CD28's effect on the latter was approximately twice that on the former. LPS induced expression of CD28 ligands CD80/86 on DCs. The addition of LPS to culture containing DCs seemed to make CD4+ T cells slightly resistant to TAC but not to CSA. However, its effect on the former was very weak under our experimental conditions. CONCLUSIONS: CD28 attenuated TAC more strongly than CSA. Although LPS did not demonstrate strong enough resistance in our in vitro model, TAC might maintain a better antibacterial immune response than CSA in clinical use.

Topoisomerase I-driven repair of UV-induced damage in NER-deficient cells.

Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.

DCAF7 is required for maintaining the cellular levels of ERCC1-XPF and nucleotide excision repair.

The ERCC1-XPF heterodimer is a structure-specific endonuclease and plays multiple roles in various DNA repair pathways including nucleotide excision repair and also telomere maintenance. The dimer formation, which is mediated by their C-terminal helix-hairpin-helix regions, is essential for their endonuclease activity as well as the stability of each protein. However, the detailed mechanism of how a cellular level of ERCC1-XPF is regulated still remains elusive. Here, we report the identification of DDB1- and CUL4-associated factor 7 (DCAF7, also known as WDR68/HAN11) as a novel interacting protein of ERCC1-XPF by mass spectrometry after tandem purification. Immunoprecipitation experiments confirmed their interaction and suggested dominant association of DCAF7 with XPF but not ERCC1. Interestingly, siRNA-mediated knockdown of DCAF7, but not DDB1, attenuated the cellular level of ERCC1-XPF, which is partly dependent on proteasome. The depletion of TCP1α, one of components of the molecular chaperon TRiC/CCT known to interact with DCAF7 and promote its folding, also reduced ERCC1-XPF level. Finally, we show that the depletion of DCAF7 causes inefficient repair of UV-induced (6-4) photoproducts, which can be rescued by ectopic overexpression of XPF or ERCC1-XPF. Altogether, our results strongly suggest that DCAF7 is a novel regulator of ERCC1-XPF protein level and cellular nucleotide excision repair activity.

PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching.

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.

Spironolactone-induced XPB degradation depends on CDK7 kinase and SCFFBXL18 E3 ligase.

The multisubunit complex transcription factor IIH (TFIIH) has dual functions in transcriptional initiation and nucleotide excision repair (NER). TFIIH is comprised of two subcomplexes, the core subcomplex (seven subunits) including XPB and XPD helicases and the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex (three subunits) containing CDK7 kinase. Recently, it has been reported that spironolactone, an anti-aldosterone drug, inhibits cellular NER by inducing proteasomal degradation of XPB and potentiates the cytotoxicity of platinum-based drugs in cancer cells, suggesting possible drug repositioning. In this study, we have tried to uncover the mechanism underlying the chemical-induced XPB destabilization. Based on siRNA library screening and subsequent analyses, we identified SCFFBXL18 E3 ligase consisting of Skp1, Cul1, F-box protein FBXL18 and Rbx1 responsible for spironolactone-induced XPB polyubiquitination and degradation. In addition, we showed that CDK7 kinase activity is required for this process. Finally, we found that the Ser90 residue of XPB is essential for the chemical-induced destabilization. These results led us to propose a model that spironolactone may trigger the phosphorylation of XPB at Ser90 by CDK7, which promotes the recognition and polyubiquitination of XPB by SCFFBXL18 for proteasomal degradation.

Activation of disulfide bond cleavage triggered by hydrophobization and lipophilization of functionalized dihydroasparagusic acid.

Concisely synthesized and functionalized dihydroasparagusic acid (DHAA) derivatives were used to show that the introduction of a hydrophobic functional group dramatically reduced air oxidation activity at the dithiol moieties and dominantly activated the cleavage of S-S bonds in proteins, presumably due to the hydrophobization and lipophilization. Notably, the reaction sites of water-reactive dithiol moieties behaved similarly to hydrophobic and lipophilic functional groups, which suggests impersonation of the reaction site.

Aquarius is required for proper CtIP expression and homologous recombination repair.

Accumulating evidence indicates that transcription is closely related to DNA damage formation and that the loss of RNA biogenesis factors causes genome instability. However, whether such factors are involved in DNA damage responses remains unclear. We focus here on the RNA helicase Aquarius (AQR), a known R-loop processing factor, and show that its depletion in human cells results in the accumulation of DNA damage during S phase, mediated by R-loop formation. We investigated the involvement of Aquarius in DNA damage responses and found that AQR knockdown decreased DNA damage-induced foci formation of Rad51 and replication protein A, suggesting that Aquarius contributes to homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Interestingly, the protein level of CtIP, a DSB processing factor, was decreased in AQR-knockdown cells. Exogenous expression of Aquarius partially restored CtIP protein level; however, CtIP overproduction did not rescue defective HR in AQR-knockdown cells. In accordance with these data, Aquarius depletion sensitized cells to genotoxic agents. We propose that Aquarius contributes to the maintenance of genomic stability via regulation of HR by CtIP-dependent and -independent pathways.

Rapid G0/1 transition and cell cycle progression in CD8+ T cells compared to CD4+ T cells following in vitro stimulation.

T-cell population consists of two major subsets, CD4+ T cells and CD8+ T cells, which can be distinguished by the expression of CD4 or CD8 molecules, respectively. Although they play quite different roles in the immune system, many of their basic cellular processes such as proliferation following stimulation are presumably common. In this study, we have carefully analyzed time-course of G0/1 transition as well as cell cycle progression in the two subsets of quiescent T-cell population following in vitro growth stimulation. We found that CD8+ T cells promote G0/1 transition more rapidly and drive their cell cycle progression faster compared to CD4+ T cells. In addition, expression of CD25 and effects of its blockade revealed that IL-2 is implicated in the rapid progression, but not the earlier G0/1 transition, of CD8+ T cells.

Rapid proliferation of activated lymph node CD4(+) T cells is achieved by greatly curtailing the duration of gap phases in cell cycle progression.

Peripheral T cells are in G0 phase and do not proliferate. When they encounter an antigen, they enter the cell cycle and proliferate in order to initiate an active immune response. Here, we have determined the first two cell cycle times of a leading population of CD4(+) T cells stimulated by PMA plus ionomycin in vitro. The first cell cycle began around 10 h after stimulation and took approximately 16 h. Surprisingly, the second cell cycle was extremely rapid and required only 6 h. T cells might have a unique regulatory mechanism to compensate for the shortage of the gap phases in cell cycle progression. This unique feature might be a basis for a quick immune response against pathogens, as it maximizes the rate of proliferation.

Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells.

Histone H2A variant H2AX is phosphorylated at Ser(139) in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0 phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to an earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1, and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical nonproliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB.

The scaffold protein JLP plays a key role in regulating ultraviolet B-induced apoptosis in mice.

The ultraviolet B (UVB) component of sunlight can cause severe damage to skin cells and even induce skin cancer. Growing evidence indicates that the UVB-induced signaling network is complex and involves diverse cellular processes. In this study, we investigated the role of c-Jun NH2 -terminal kinase-associated leucine zipper protein (JLP), a scaffold protein for mitogen-activated protein kinase (MAPK) signaling cascades, in UVB-induced apoptosis. We found that UVB-induced skin epidermal apoptosis was prevented in Jlp knockout (KO) as well as in keratinocyte-specific Jlp KO mice. Analysis of the repair of UVB-induced DNA damage over time showed no evidence for the involvement of JLP in this process. In contrast, UVB-stimulated p38 MAPK activation in the skin was impaired in both Jlp KO and keratinocyte-specific Jlp KO mice. Moreover, topical treatment of UVB-irradiated mouse skin with a p38 inhibitor significantly suppressed the epidermal apoptosis in wild-type mice, but not in Jlp KO mice. Our findings suggest that JLP in skin basal keratinocytes plays an important role in UVB-induced apoptosis by modulating p38 MAPK signaling pathways. This is the first study to show a critical role for JLP in an in vivo response to environmental stimulation.

Concanavalin A-mediated T cell proliferation is regulated by herpes virus entry mediator costimulatory molecule.

T cell activation is regulated by two distinct signals, signals one and two. Concanavalin A (ConA) is an antigen-independent mitogen and functions as signal one inducer, leading T cells to polyclonal proliferation. CD28 is known to be one of major costimulatory receptors and to provide signal two in the ConA-induced T cell proliferation. Here, we have studied the implication of other costimulatory pathways in the ConA-mediated T cell proliferation by using soluble recombinant proteins consisting of an extracellular domain of costimulatory receptors and Fc portion of human IgG. We found that T cell proliferation induced by ConA, but not PMA plus ionomycin or anti-CD3 mAb, is significantly inhibited by herpes virus entry mediator (HVEM)-Ig, even in the presence of CD28 signaling. Moreover, the high concentration of HVEM-Ig molecules almost completely suppressed ConA-mediated T cell proliferation. These results suggest that HVEM might play more important roles than CD28 in ConA-mediated T cell proliferation.

Proteasome inhibitors and knockdown of SMG1 cause accumulation of Upf1 and Upf2 in human cells.

The ubiquitin-proteasome system (UPS) is one of the most promising anticancer drug targets of the century. However, the involved molecular mechanisms are still unclear. The nonsense-mediated mRNA decay (NMD) pathway is a highly conserved pathway which degrades nonsense mutation­containing mRNA selectively and efficiently. In this pathway, the SMG1-Upf1-eRF (SURF) complex binds to Upf2 on the exon junction complex and finally causes degradation of nonsense-containing mRNA. To reveal the relationship between the UPS and NMD pathways, we analyzed the effects of proteasome inhibitors on Upf1 and Upf2. The data showed that treatment with proteasome inhibitors caused the accumulation of the Upf1 and Upf2 proteins in A549 cells. In addition, we found that knockdown of SMG1 also caused the upregulation of Upf1 and Upf2 proteins, which was confirmed by different target sequences of siRNA. SMG1 and UPS appear to participate in different pathways of the degradation of Upf1 and Upf2, since simultaneous treatment with both of them caused additive effects. This study demonstrated the quantitative regulation of Upf1 and Upf2 proteins by UPS and SMG1.

Establishment of a microplate-formatted cell-based immunoassay for rapid analysis of nucleotide excision repair ability in human primary cells.

DNA photolesions induced by UV, cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP), are repaired by nucleotide excision repair (NER) in human cells. Various immunoassays using monoclonal antibodies specific for the photolesions have been developed and widely used for the analysis of cellular NER activity. In this study, we have newly developed a microplate-formatted cell-based immunoassay, based on indirect immunofluorescence staining with lesion-specific antibodies combined with an infrared imaging system. Using this assay, we show the repair kinetics of CPD and 6-4PP in various fibroblasts from newborn and adult donors with no age-related difference. Furthermore, epidermal keratinocytes and melanocytes exhibit comparable NER activity, and calcium ion-induced differentiation of keratinocytes has no significant impacts on their NER activity. We also evaluated the effects of a proteasome inhibitor, MG132, and a histone deacetylase inhibitor, sodium butyrate, on NER efficiency using this assay. All these results suggest that the new assay is highly useful for the rapid and quantitative analysis of NER activity in various primary cells with limited growth activity and is applicable to a screening system for drugs affecting NER efficiency.

Role of interaction of XPF with RPA in nucleotide excision repair.

Nucleotide excision repair (NER) is a very important defense system against various types of DNA damage, and it is necessary for maintaining genomic stability. The molecular mechanism of NER has been studied in considerable detail, and it has been shown that proper protein-protein interactions among NER factors are critical for efficient repair. A structure-specific endonuclease, XPF-ERCC1, which makes the 5' incision in NER, was shown to interact with a single-stranded DNA binding protein, RPA. However, the biological significance of this interaction was not studied in detail. We used the yeast two-hybrid assay to determine that XPF interacts with the p70 subunit of RPA. To further examine the role of this XPF-p70 interaction, we isolated a p70-interaction-deficient mutant form of XPF that contains a single amino acid substitution in the N-terminus of XPF by the reverse yeast two-hybrid assay using randomly mutagenized XPF. The biochemical properties of this RPA-interaction-deficient mutant XPF-ERCC1 are very similar to those of wild-type XPF-ERCC1 in vitro. Interestingly, expression of this mutated form of XPF in the XPF-deficient Chinese hamster ovary cell line, UV41, only partially restores NER activity and UV resistance in vivo compared to wild-type XPF. We discovered that the RPA-interaction-deficient XPF is not localized in nuclei and the mislocalization of XPF-ERCC1 prevents the complex from functioning in NER.

SCFß(TrCP) mediates stress-activated MAPK-induced Cdc25B degradation.

Cdc25A, which is one of the three mammalian CDK-activating Cdc25 protein phosphatases (Cdc25A, B and C), is degraded through SCF(ßTrCP)-mediated ubiquitylation following genomic insult; however, the regulation of the stability of the other two Cdc25 proteins is not well understood. Previously, we showed that Cdc25B is primarily degraded by cellular stresses that activate stress-activated MAPKs, such as Jun NH(2)-terminal kinase (JNK) and p38. Here, we report that Cdc25B was ubiquitylated by SCF(ßTrCP) E3 ligase upon phosphorylation at two Ser residues in the ßTrCP-binding-motif-like sequence D(94)AGLCMDSPSP(104). Point mutation of these Ser residues to alanine (Ala) abolished the JNK-induced ubiquitylation by SCF(ßTrCP), and point mutation of DAG to AAG or DAA eradicated both ßTrCP binding and ubiquitylation. Further analysis of the mode of ßTrCP binding to this region revealed that the PEST-like sequence from E(82)SS to D(94)AG is crucially involved in both the ßTrCP binding and ubiquitylation of Cdc25B. Furthermore, the phospho-mimetic replacement of all 10 Ser residues in the E(82)SS to SPSP(104) region with Asp resulted in ßTrCP binding. Collectively, these results indicate that stress-induced Cdc25B ubiquitylation by SCF(ßTrCP) requires the phosphorylation of S(101)PS(103)P in the ßTrCP-binding-motif-like and adjacent PEST-like sequences.