Conserved Region 2 Research Articles

NMR investigation of FOXO4-DNA interaction for discriminating target and non-target DNA sequences

Forkhead box O4 (FOXO4), a human transcription factor, recognizes target DNA through its forkhead domain (FHD) while maintaining comparable binding affinity to non-target DNA. The conserved region 3 (CR3), a transactivation domain, modulates DNA binding kinetics to FHD and contributes to target DNA selection, but the underlying mechanism of this selection remains elusive. Using paramagnetic relaxation enhancement analysis, we observed a minor state of CR3 close to FHD in the presence of non-target DNA, a state absent when FHD interacts with target DNA. This minor state suggests that CR3 effectively masks the non-target DNA-binding interface on FHD. The interaction weakens significantly under high salt concentration, implying that CR3 or high salt concentrations can modulate electrostatic interactions with non-target DNA. Our 15N relaxation measurements revealed FHD’s flexibility with non-target DNA and increased rigidity with target DNA binding. Our findings offer insights into the role of FOXO4 as a transcription initiator.

Transcription factor networks drive perforin activity in the anti-bacterial immune response of tilapia

Perforin, produced by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), is one of the effectors of cell-mediated cytotoxicity (CMC) in vertebrates, playing a paramount role in killing target cells. However, whether and how perforin is involved in adaptive immune responses in early vertebrates remains unclear. Using Nile tilapia (Oreochromis niloticus) as a model, we investigated the characteristics of perforin in early vertebrates. Oreochromis niloticus perforin (OnPRF) possesses 2 conserved functional domains, membrane attack complex/perforin (MACPF) and protein kinase C conserved region 2 (C2) domains, although they share low amino acid sequence similarity with other homologs. OnPRF was widely expressed in various immune tissues and could respond to lymphocyte activation and T-cell activation in vitro at both the transcriptional and protein levels, indicating that it may be involved in adaptive immune responses. Furthermore, after infection with Edwardsiella piscicida and Aeromonas hydrophila, the mRNA and protein levels of OnPRF were significantly up-regulated within the adaptive immune response period. Additionally, we revealed that many transcription factors were involved in the transcriptional regulation of OnPRF, including p65, c-Fos, c-Jun, STAT1 and STAT4, and there was a synergy among these transcription factors. Overall, these findings demonstrate the involvement of OnPRF in T-cell activation and adaptive immune response in tilapia, thus providing new evidence for comprehending the evolution of immune response in early vertebrates.

Enhancing the interpretation of genetic observations in KCNQ1 in unselected populations: relevance to secondary findings.

Rare variants in the KCNQ1 gene are found in the healthy population to a much greater extent than the prevalence of Long QT Syndrome type 1 (LQTS1). This observation creates challenges in the interpretation of KCNQ1 rare variants that may be identified as secondary findings in whole exome sequencing.This study sought to identify missense variants within sub-domains of the KCNQ1-encoded Kv7.1 potassium channel that would be highly predictive of disease in the context of secondary findings. We established a set of KCNQ1 variants reported in over 3700 patients with diagnosed or suspected LQTS sent for clinical genetic testing and compared the domain-specific location of identified variants to those observed in an unselected population of 140 000 individuals. We identified three regions that showed a significant enrichment of KCNQ1 variants associated with LQTS at an odds ratio (OR) >2: the pore region, and the adjacent 5th (S5) and 6th (S6) transmembrane (TM) regions. An additional segment within the carboxyl terminus of Kv7.1, conserved region 2 (CR2), also showed an increased OR of disease association. Furthermore, the TM spanning S5-Pore-S6 region correlated with a significant increase in cardiac events. Rare missense variants with a clear phenotype of LQTS have a high likelihood to be present within the pore and adjacent TM segments (S5-Pore-S6) and a greater tendency to be present within CR2. This data will enhance interpretation of secondary findings within the KCNQ1 gene. Further, our data support a more severe phenotype in LQTS patients with variants within the S5-Pore-S6 region.

Characterizing non-small cell lung cancer driven by mutations in ARAF and CRAF family members.

e15085 Background: Cancer genome screening is becoming standard clinical practice, but the number of actionable mutations detected is limited. To expand on our previous report of the possibility that ARAF S214C and CRAF S257L located in the conserved region 2 (CR2) were actionable driver mutations, we investigated responses to targeted therapies in mouse models and the prevalence of these alterations across many cancer types represented in a large real-world database of molecularly profiled patient tumors. Methods: Conditional transgenic mice that expressed human ARAF S214C or CRAF S257L in their lungs were established and treated with sorafenib or trametinib. Activation of the MAPK pathway following sorafenib or trametinib treatment was assessed in vitro by phospho-ERK IHC. DNA sequencing (592-gene or whole exome) was performed for patient samples submitted to Caris Life Sciences (Phoenix, AZ), and the prevalence of ARAF/CRAF variants was determined for each cancer type. Results: Both ARAF S214C and CRAF S257L dimerized to activate the MAPK pathway. Although a low concentration of sorafenib promoted ERK phosphorylation, a high concentration of sorafenib inhibited ERK phosphorylation. Transgenic mice rapidly developed lung adenocarcinoma when the mutant proteins were expressed. Sorafenib treatment induced tumor necrosis in both ARAF- and CRAF-mutated tumors, and 3 months of sorafenib treatment prolonged the survival of CRAF-mutant mice. Pan-tumor analysis of real-world patient samples revealed that ARAF and CRAF mutations in CR2 were found in 0.125% and 0.234% of tumors, respectively. ARAF mutations in CR2 were found in 0.415% of cholangiocarcinoma, 0.132% of colorectal cancer, and 0.173% of non-small cell lung cancer (NSCLC). CRAF mutations in CR2 were found in 0.509% of colorectal cancer, 0.502% of gastric cancer, 0.303% of NSCLC, and 0.978% of melanoma. The most prevalent CR2 mutations were ARAF S214F and CRAF S257L. Conclusions: Our in vitro results suggest ARAF S214C and CRAF S257L mutations are rare driver mutations that may be effectively treated with RAF inhibitors and warrant further investigation. [Table: see text]

Molecular characterization, expression and function analysis of Epinephelus coioides PKC-ɑ response to Singapore grouper iridovirus (SGIV) infection

Protein kinase C (PKC) constitutes the main signal transduction pathway, and participates in the signal pathway of cell proliferation and movement in mammals. In this study, PKC-ɑ was obtained from Epinephelus coioides, an important marine fish cultivated in the coastal areas of southern China and Southeast Asia. The full length cDNA of PKC-ɑ was 3362 bp in length containing a 23 bp 5′UTR, a 1719 bp 3′UTR, and a 1620 bp open reading frame encoding 539 amino acids. It contains three conservative domains including protein kinase C conserved region 2 (C2), Serine/Threonine protein kinases, catalytic domain (S_TKc) and ser/thr-type protein kinases (S_TK_X). Its mRNA can be detected in all 11 tissues examined of E. coioides, and the expression was significantly upregulated response to Singapore grouper iridovirus (SGIV) infection, one of the important pathogens of marine fish. Upregulated E. coioides PKC-ɑ significantly inhibited the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and SGIV-induced cell apoptosis. The results indicated that the PKC-ɑ may play an important role in pathogenic stimulation.

USP7 regulates the ERK1/2 signaling pathway through deubiquitinating Raf-1 in lung adenocarcinoma

Ubiquitin-specific protease 7 (USP7) is one of the deubiquitinating enzymes (DUBs) in the ubiquitin-specific protease (USP) family. It is a key regulator of numerous cellular functions including immune response, cell cycle, DNA damage and repair, epigenetics, and several signaling pathways. USP7 acts by removing ubiquitin from the substrate proteins. USP7 also binds to a specific binding motif of substrate proteins having the [P/A/E]-X-X-S or K-X-X-X-K protein sequences. To date, numerous substrate proteins of USP7 have been identified, but no studies have been conducted using the binding motif that USP7 binds. In the current study, we analyzed putative substrate proteins of USP7 through the [P/A/E]-X-X-S and K-X-X-X-K binding motifs using bioinformatics tools, and confirmed that Raf-1 is one of the substrates for USP7. USP7 binds to the Pro-Val-Asp-Ser (PVDS) motif of the conserved region 2 (CR2) which contains phosphorylation sites of Raf-1 and decreased M1-, K6-, K11-, K27-, K33-, and K48-linked polyubiquitination of Raf-1. We further identified that the DUB activity of USP7 decreases the threonine phosphorylation level of Raf-1 and inhibits signaling transduction through Raf activation. This regulatory mechanism inhibits the activation of the ERK1/2 signaling pathway, thereby inhibiting the G2/M transition and the cell proliferation of lung adenocarcinoma cells. In summary, our results indicate that USP7 deubiquitinates Raf-1 and is a new regulator of the ERK1/2 signaling pathway in lung adenocarcinoma.

ChSch9 is required for infection-related morphogenesis and pathogenicity in Colletotrichum higginsianum

Colletotrichum higginsianum is an economically important anthracnose pathogen of crucifers that causes losses worldwide. For comprehensive control of anthracnose disease, it is essential to understand the mechanisms of pathogenicity in C. higginsianum. The Ser/Thr kinase Sch9 belonging to the AGC (protein kinase A, G, and C) kinase family, commonly found in eukaryotes, plays a vital role in various cell signalling pathways and development. However, it has not been studied in Colletotrichum species. Three domains, Protein kinase C conserved region 2 (C2), Serine/Threonine protein kinase catalytic (S_TKc) and Extension to Ser/Thr-type protein kinases (S_TK_X), were identified in the C. higginsianum Sch9 (ChSch9) protein. Phylogenetic analysis indicated that ChSch9 is closely related to the AGC kinase family, which is conserved in fungi. Deletion of ChSch9 caused defects in hyphal morphology, growth and conidiation. In addition, deletion of ChSch9 was associated with reduced tolerance to hyperosmotic, cell wall and oxidative stresses in C. higginsianum, and ChSch9 deletion mutants were more tolerant to heat. Furthermore, we found that ChSch9 might be involved in the pyruvate–acetaldehyde fermentation pathway. Importantly, deletion of ChSch9 led to a reduction in appressoria and infective hyphae formation, resulting in defects in virulence. Collectively, the results of this study indicate that ChSch9 is required for infection-related morphogenesis and virulence in C. higginsianum.

Molecular Analysis of the Interaction between Human PTPN21 and the Oncoprotein E7 from Human Papillomavirus Genotype 18.

Human papillomaviruses (HPVs) cause cellular hyperproliferation-associated abnormalities including cervical cancer. The HPV genome encodes two major viral oncoproteins, E6 and E7, which recruit various host proteins by direct interaction for proteasomal degradation. Recently, we reported the structure of HPV18 E7 conserved region 3 (CR3) bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, a well-defined tumor suppressor, and found that this intermolecular interaction plays a key role in E7-driven transformation and tumorigenesis. In this study, we carried out a molecular analysis of the interaction between CR3 of HPV18 E7 and the PTP domain of PTPN21, a PTP protein that shares high sequence homology with PTPN14 but is putatively oncogenic rather than tumor-suppressive. Through the combined use of biochemical tools, we verified that HPV18 E7 and PTPN21 form a 2:2 complex, with a dissociation constant of 5 nM and a nearly identical binding manner with the HPV18 E7 and PTPN14 complex. Nevertheless, despite the structural similarities, the biological consequences of the E7 interaction were found to differ between the two PTP proteins. Unlike PTPN14, PTPN21 did not appear to be subjected to proteasomal degradation in HPV18-positive HeLa cervical cancer cells. Moreover, knockdown of PTPN21 led to retardation of the migration/invasion of HeLa cells and HPV18 E7-expressing HaCaT keratinocytes, which reflects its protumor activity. In conclusion, the associations of the viral oncoprotein E7 with PTPN14 and PTPN21 are similar at the molecular level but play different physiological roles.

Abstract 2763: Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface

Abstract Ras proteins are classical members of small GTPases, controlling signal transduction pathways and promoting cell proliferation. Ras consists of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and activation is terminated by GTPase-activating proteins that induce the GTP hydrolysis. Ras has multiple partners, signals through several key pathways and fulfills critical functions in the cell life. Mutations in Ras are common in a variety of cancers; yet it is still undruggable. KRas4B is frequently mutated in cancer. Elucidation of Ras conformational ensembles at the signaling platforms in the plasma membrane including the ligand-bound conformations, membrane orientations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, and transition states are essential for deciphering Ras functions from its conformational landscapes. In the MAPK pathway, KRas4B preferentially recruits Raf-1 and activates it. Raf kinases contain Ras binding domain (RBD) and cysteine-rich domain (CRD) at conserved region 1 (CR1), and kinase domain at CR3. The high-affinity interaction of Raf RBD with Ras was solved in the absence of CRD. It is known that Raf CRD play a key role in the membrane anchoring mechanism. Recently, pivotal roles of CRD in the membrane interactions of KRas4B-Raf-1 RBD-CRD complex were reported. Here, we employ all-atom molecular dynamics (MD) simulations to investigate dimeric KRas4B-Raf-1 complex at the anionic lipid bilayer composed of DOPC and DOPS (4:1). Active KRas4B dimer modulates Raf-1 activation, promoting dimerization of Raf-1’s kinase domain. In the dimeric complex of KRas4B-Raf-1, Raf-1 CRD stably binds anionic lipid bilayers inserting its positively-charged loop into the amphipathic interface. The key basic residues at the loop are responsible for the membrane association, suggesting that CRD is an intrinsic membrane binding domain of Raf kinase. Our simulations suggest that Raf-1 CRD not only offers an additional membrane anchor for the KRas4B-Raf-1 complex, but also reduces the fluctuations of Ras-RBD, enhancing the high affinity interaction between Ras and Raf. Raf-1 CRD supports the active-state Ras orientation, promoting Ras dimerization through the allosteric lobe helical interface at the membrane. The enhanced stability of the complex at the membrane rendered by CRD promotes Raf dimerization in the MAPK signaling, which is the key Ras proliferative pathway. Here we suggest these significant roles of CRD at the membrane in the Raf activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Ruth Nussinov. Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2763.

Divergent Evolution of E1A CR3 in Human Adenovirus Species D.

Adenovirus E1A is the first viral protein expressed during infection. E1A controls critical aspects of downstream viral gene expression and cell cycle deregulation, and its function is thought to be highly conserved among adenoviruses. Various bioinformatics analyses of E1A from 38 human adenoviruses of species D (HAdV-D), including likelihood clade model partitioning, provided highly significant evidence of divergence of HAdV-Ds into two distinct groups for the conserved region 3 (CR3), present only in the E1A 13S isoform. This variance within E1A 13S of HAdV-Ds was not found in any other human adenovirus (HAdV) species. By protein sequence and structural analysis, the zinc finger motif of E1A CR3, previously shown as critical for transcriptional activation, showed the greatest differences. Subsequent codon usage bias analysis revealed substantial divergence in E1A 13S between the two groups of HAdV-Ds, suggesting that these two sub-groups of HAdV-D evolved under different cellular conditions. Hence, HAdV-D E1A embodies a previously unappreciated evolutionary divergence among HAdVs.

C-Raf participates in adaptive immune response of Nile tilapia via regulating lymphocyte activation

RAF proto-oncogene serine/threonine-protein kinase (c-Raf) is a MAP kinase kinase kinase (MAPKKK) that participates in the Erk1/2 pathway and plays an important role in lymphocyte activation. However, the study on how c-Raf regulates adaptive immunity in non-mammal is still limited. In present study, based on analysis of sequence characteristics of c-Raf from Oreochromis niloticus (On-c-Raf), we investigated its regulation roles on teleost lymphocyte activation. The On-c-Raf was highly conserved during evolution, which was composed of a Raf-like Ras-binding domain (RBD), a protein kinase C conserved region 1 (C1) domain and a serine/threonine protein kinase catalytic (S_TKc) domain. Its mRNA showed a wide distribution in tissues of O. niloticus and with the highest expression in gill. After Aeromonas hydrophila infection, during the adaptive immune stage transcription level of On-c-Raf was significantly upregulated on day 8, but came back to original level on day 16 and 30, suggesting the potential involvement of On-c-Raf in primary response but not memory formation. Furthermore, On-c-Raf mRNA in leukocytes of Nile tilapias was obviously induced by in vitro stimulation of T cell mitogen PHA. More importantly, in vitro stimulation of lymphocytes agonist PMA augmented phosphorylation level of On-c-Raf in leukocytes detected by western-blot and immunofluorescent. Thus, c-Raf regulated lymphocyte activation of Nile tilapia on both mRNA and phosphorylation level. Together, our results revealed that the c-Raf from teleost Nile tilapia engaged in adaptive immune response by regulating lymphocytes activation. Since the regulatory mechanism of lymphocyte-mediated adaptive immunity is largely unknown in teleost, our study provided important evidences to understand teleost adaptive immunity, and also shed a novel perspective for the evolution of adaptive immune system.

Ubiquitin-conjugating enzyme UBE2O regulates cellular clock function by promoting the degradation of the transcription factor BMAL1

Dysregulation of the circadian rhythm is associated with many diseases, including diabetes, obesity, and cancer. Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Arntl or Bmal1) is the only clock gene whose loss disrupts circadian locomotor behavior in constant darkness. BMAL1 levels are affected by proteasomal inhibition and by several enzymes in the ubiquitin-proteasome system, but the exact molecular mechanism remains unclear. Here, using immunoprecipitation and MS analyses, we discovered an interaction between BMAL1 and ubiquitin-conjugating enzyme E2 O (UBE2O), an E3-independent E2 ubiquitin-conjugating enzyme (i.e. hybrid E2/E3 enzyme). Biochemical experiments with cell lines and animal tissues validated this specific interaction and uncovered that UBE2O expression reduces BMAL1 levels by promoting its ubiquitination and degradation. Moreover, UBE2O expression/knockdown diminished/increased, respectively, BMAL1-mediated transcriptional activity but did not affect BMAL1 gene expression. Bioluminescence experiments disclosed that UBE2O knockdown elevates the amplitude of the circadian clock in human osteosarcoma U2OS cells. Furthermore, mapping of the BMAL1-interacting domain in UBE2O and analyses of BMAL1 stability and ubiquitination revealed that the conserved region 2 (CR2) in UBE2O significantly enhances BMAL1 ubiquitination and decreases BMAL1 protein levels. A Cys-to-Ser substitution experiment identified the critical Cys residue in the CR2 domain responsible for BMAL1 ubiquitination. This work identifies UBE2O as a critical regulator in the ubiquitin-proteasome system, which modulates BMAL1 transcriptional activity and circadian function by promoting BMAL1 ubiquitination and degradation under normal physiological conditions.

Mapping the interactions of adenoviral E1A proteins with the p160 nuclear receptor coactivator binding domain of CBP.

Many viruses deregulate the cell and force transcription of viral genes by competing with cellular proteins for binding to the transcriptional co-activators CREB-binding protein (CBP) and p300. Through its interactions with CBP/p300 and the retinoblastoma protein, the adenovirus (AdV) early region 1A (E1A) oncoprotein hijacks the cell cycle and, in rodents, transforms the cell; the mechanistic and structural basis for these effects remain unclear. In this study we compare the affinity of protein constructs from the E1A proteins from two adenovirus serotypes, non-oncogenic AdV5 and highly oncogenic AdV12, for binding to the nuclear receptor coactivator binding domain (NCBD) of CBP. NMR spectra show that the E1A constructs from both serotypes are intrinsically disordered in the free state and that each contains three homologous binding sites for the NCBD, one in the N-terminal region and two within conserved region 1 (CR1) of E1A. The binding sites in CR1 correspond to the motifs that bind the retinoblastoma protein and the TAZ2 domain of CBP/p300. The E1A and NCBD peptides fold synergistically upon complex formation. Binding affinities determined from NMR titrations show that, although the overall affinities for AdV5 and AdV12 E1A are comparable, there are significant differences between the two E1A serotypes in the relative strength with which their constituent interaction motifs bind to the NCBD. The individual E1A interaction motifs were unable to compete effectively with p53 for binding to the NCBD and both the N-terminal region and CR1 region of E1A are required for efficient competition with p53.

Dynamic Response of the C2 Domain of Protein Kinase Cα to Ca2+ Binding

Ca2+-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca2+-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy. Interactions with metal ions attenuate microsecond-timescale motions of the loop regions, indicating that preorganization of the metal-binding loops occurs before membrane insertion. Binding of a full complement of Ca2+ ions has a profound effect on the millisecond-timescale dynamics of the N- and C-terminal regions of C2α. We propose that Ca2+ binding allosterically destabilizes the terminal regions of C2α and thereby facilitates the conformational rearrangement necessary for full membrane insertion and activation of protein kinase Cα.

APKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization

SummaryAtypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.

Abstract 4049: Conserved region 2 of Human Papilloma Virus 18 protein E7 is required for E7 binding to centromere protein C

Abstract The molecular genetics of cervical cancer have not been thoroughly elucidated, despite the fact that cervical cancer is responsible for the deaths of over 250,000 women annually worldwide. Infection with human papillomaviruses (HPVs) is linked to the pathogenesis of a variety of human diseases, including genital warts, laryngeal papillomas, and most notably, cervical cancer. Recently HPV infection was reported to be associated with head and neck cancers.High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. HPV infection leads to aneuploidy, a numerical chromosomal aberration caused by dysregulation of chromosomal segregation. We found that high-risk HPV18 and HPV58 E7 proteins bind to centromere protein C (CENP-C), a component of the kinetochore that is essential for proper chromosomal segregation. Low-risk HPV4, HPV6, and HPV11 E7s do not bind to CENP-C. The PxDLLCxE sequence in conserved region 2 (CR2) of HPV18 E7 is required for E7 binding to CENP-C. Our results indicate that differences in the ability of high- and low-risk HPV E7s to bind to CENP-C reflect the different oncogenic potentials of high- and low-risk type HPVs.We demonstrated that the E7 proteins from low-risk HPVs do not bind to CENP-C, and that the PxDLLCxE sequence in conserved region 2 of the HPV18 E7 protein is required for E7 binding to CENP-C. CENP-C is a component of the inner kinetochore, and as such, it plays an essential role in proper chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Therefore, we hypothesize that differences in the capability of various E7s from low- and high-risk type HPVs to bind to CENP-C reflect the different oncogenic potential of high- and low-risk type HPVs. Citation Format: Aoi Tokuda, Shoko Takahashi, Masafumi Yoshimoto, Yuji Yaginuma. Conserved region 2 of Human Papilloma Virus 18 protein E7 is required for E7 binding to centromere protein C. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4049.

The Cellular Protein Complex Associated with a Transforming Region of E1A Contains c-MYC.

The cell-transforming activity of human adenovirus 5 (hAd5) E1A is mediated by the N-terminal half of E1A, which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400, and pRb family members. Among these protein interactions, the interaction of pRb family proteins with conserved region 2 (CR2) of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning amino acid residues 26 to 35 which also interacted with TRRAP and p400. Small interfering RNA (siRNA)-mediated depletion of TRRAP reduced c-MYC interaction with E1A, while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7, and ATXN7L3 via TRRAP. Acute short hairpin RNA (shRNA)-mediated depletion of c-MYC reduced the E1A transforming activity, while depletion of ENY2 and MAX did not. These results suggested that the association of c-MYC with E1A may, at least partially, play a role in the E1A transformation activity, independently of MAX. The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2, promotes cell proliferation, through inactivating the activities of the pRb family proteins, is better understood than the activities of the other domains. Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb.

Function analysis of fish Tollip gene in response to virus infection.

Toll-interacting protein (Tollip) is one of the important regulatory proteins of Toll-like receptor (TLR) signaling pathways. In previous studies, a Tollip sequence of grouper (Epinephelus coioides) was identified and the signal transduction functions of Tollip were studied. However, the response of Tollip to virus infection has not been characterized from grouper. In the present paper, the Tollip homolog (EtTollip) from grouper (Epinephelus tauvina) was cloned and its immune response to Singapore grouper iridovirus (SGIV) was investigated. EtTollip shares significant similarities to other mammalian Tollips, which contain a centrally localized protein kinase C conserved region 2 (C2) domain and a C-terminal CUE domain. After challenging with SGIV, the expression levels of EtTollip were altered in the spleen and head kidney of grouper. EtTollip mainly aggregated in the cytoplasm in a condensed state and was also distributed on the membranes of GS cells. EtTollip significantly inhibited the activities of NF-κB and IFN-β luciferase reporter when transfected into grouper spleen (GS) cells. SGIV can increase the activities of NF-κB and IFN-β luciferase reporter, especially to IFN-β. When transfected EtTollip with EcMyd88, the activity of NF-κB was increased, while transfected EtTollip with EcIRF3, the activity of IFN-β was significantly increased. Over-expressed EtTollip inhibited the transcription of SGIV genes significantly in GS cells, and silencing of EtTollip with siRNA led to increase of SGIV genes loads. Taken together, the results provide new insights in to the importance of Tollip as evolutionarily conserved molecule for grouper innate immunity against virus infection.

Transcriptional activation of OCT4 by the ETS transcription factor PEA3 in NCCIT human embryonic carcinoma cells

We examined the molecular mechanism of OCT4 gene regulation by polyomavirus enhancer activator 3 (PEA3) in NCCIT cells. Endogenous PEA3 and OCT4 were significantly elevated in undifferentiated cells and reduced upon differentiation. PEA3 knockdown led to a reduction in OCT4 levels. OCT4 promoter activity was significantly up-regulated by dose-dependent PEA3 overexpression. Deletion and site-directed mutagenesis of the OCT4 promoter revealed a putative binding site within the conserved region 2 (CR2). PEA3 interacted with the binding element within CR2 in NCCIT cells. This study reveals the molecular details of the mechanism by which the oncogenic factor PEA3 regulates OCT4 gene expression as a transcriptional activator.

Transcriptional regulation of OCT4 by the ETS transcription factor ESE-1 in NCCIT human embryonic carcinoma cells

The epithelium-specific ETS transcription factor-1 (ESE-1) is physiologically important in the pathogenesis of various diseases. Recently, OCT4, a transcription factor involved in stem cell pluripotency, has been implicated in tumorigenesis. In this study, we invested the molecular mechanism by which ESE-1 regulates transcription of OCT4 in NCCIT human embryonic carcinoma cells. Real-time PCR analysis revealed that OCT4 levels were high in undifferentiated NCCIT cells but significantly decreased upon retinoic acid-mediated differentiation, concomitant with up-regulation of ESE-1 expression. OCT4 mRNA level rose following shRNA-mediated knockdown of ESE-1, but declined when ESE-1 was overexpressed, suggesting that the expression levels of OCT4 and ESE-1 may be coordinated in an opposite manner. Promoter-reporter assays revealed that induced OCT4 promoter activity in NCCIT cells was significantly down-regulated by ESE-1 overexpression in a dose-dependent manner. The inhibitory effect of ESE-1 on OCT4 promoter activity was relieved by co-expression of an ESE-1 mutant lacking the transactivation domain, but not by mutants lacking other domains. Serial deletion and site-directed mutagenesis of the OCT4 promoter revealed that a potential ETS binding site (EBS) is present in the conserved region 2 (CR2). ESE-1 interacted with the EBS element in CR2 and enrichment of CR2 significantly increased upon RA-mediated differentiation of NCCIT cells, suggesting that this binding is likely to be involved in ESE-1-mediated repression of OCT4 promoter activity upon differentiation. Taken together, the results of this study reveal the molecular details of the mechanism by which the oncogenic factor ESE-1 regulates expression of the stem cell transcription factor OCT4 in pluripotent NCCIT cells.