Dataset Information

Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor.

ABSTRACT: The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers. We report the first crystal structures of the human HDGF PWWP domain (residues 1-100) in a complex with SMYD1 of 10?bp at 2.84?Å resolution and its apo form at 3.3?Å, respectively. The structure of the apo PWWP domain comprises mainly four ?-strands and two ?-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable ?-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions.

SUBMITTER: Chen LY

PROVIDER: S-EPMC5762634 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor.

Chen Li-Ying LY Huang Yen-Chieh YC Huang Shih-Tsung ST Hsieh Yin-Cheng YC Guan Hong-Hsiang HH Chen Nai-Chi NC Chuankhayan Phimonphan P Yoshimura Masato M Tai Ming-Hong MH Chen Chun-Jung CJ

Scientific reports 20180110 1

The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers. We report the first crystal structures of the human HDGF PWWP domain (residues 1-100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWW ...[more]

PMID: 29321480

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Project description:Hepatoma-derived growth factor (HDGF) is a nuclear protein with both mitogenic and angiogenic activity that is highly expressed in the developing heart and vasculature. To date, the mechanism underlying the function of HDGF is unknown. Oligonucleotide microarray analysis was used to gain insights into HDGF function. Adenoviral expression of HDGF significantly (> or =2-fold) downregulated a large group (66) of genes, and increased expression of a relatively small number of genes (9). Two groups of target genes that are involved in cardiovascular development and transcriptional regulation, including the skeletal/cardiac muscle specific SET and MYND domain containing 1 (SMYD1) gene, were validated by real time PCR. This suggested that HDGF could function as a transcriptional repressor. In a one-hybrid system, GBD-HDGF significantly repressed reporter gene activity in a dose-dependent manner. This demonstrated that HDGF has transcriptional repressive activity. Moreover, in G-7 myoblast cells, over-expression of a GFP-HDGF fusion specifically downregulated SMYD1 mRNA expression and the activity of the human SMYD1 promoter. HDGF repressed SMYD1 gene transcription through interaction with a transcriptional corepressor C-terminal binding protein (CtBP). Over-expression of CtBP potentiated the trans-repressive activity of HDGF; on the other hand, knocking down CtBP attenuated the trans-repressive effect of HDGF. HDGF binds CtBP through a non-canonical binding motif (PKDLF) within the PWWP domain, as mutation of DL to AS abolished HDGF and CtBP interaction, and diminished the trans-repressive effect of HDGF without affecting DNA binding. Finally, fluorescent microscopy showed that HDGF induced the nuclear accumulation of CtBP, suggesting that HDGF forms a transcriptional complex with CtBP. Taken together, our data demonstrate that HDGF functions as a transcriptional repressor of the SMYD1 gene through interaction with the transcriptional corepressor CtBP. Because of moderate conservation of the CtBP binding motif in HDGF family members, trans-repressive activity mediated by CtBP may be a common function among HDGF proteins.

Project description:Hepatoma-derived growth factor (HDGF) is a mitogen that promotes endothelial proliferation and neuronal survival. Using a unique technology of ligandomics, we recently identified HDGF as a retinal endothelial binding protein. The purpose of this study is to examine the role of HDGF in regulating ocular vasculature and the expression of HDGF in the retina.HDGF expression in the retinal was analyzed with western blot and immunohistochemistry. Angiogenic activity was investigated in human retinal microvascular endothelial cells (HRMVECs) with in vitro endothelial proliferation, migration, and permeability assays. In vivo angiogenic activity was quantified with a corneal pocket assay. The Evans blue assay and western blot using anti-mouse albumin were performed to detect the capacity of HDGF to induce retinal vascular leakage.Immunohistochemistry revealed that HDGF is expressed in the retina with a distinct pattern. HDGF was detected in retinal ganglion cells and the inner nuclear layer but not in the inner plexiform layer, suggesting that HDGF is expressed in the nucleus, but not in the cytoplasm, of retinal neurons. In contrast to family member HDGF-related protein 3 (HRP-3) that has no expression in photoreceptors, HDGF is also present in the outer nuclear layer and the inner and outer segments of photoreceptors. This suggests that HDGF is expressed in the nucleus as well as the cytoplasm of photoreceptors. In vitro functional assays showed that HDGF induced the proliferation, migration, and permeability of HRMVECs. Corneal pocket assay indicated that HDGF directly stimulated angiogenesis in vivo. Intravitreal injection of HDGF significantly induced retinal vascular leakage.These results suggest that HDGF is an angiogenic factor that regulates retinal vasculature in physiologic and pathological conditions. Identification of HDGF by ligandomics and its independent characterization in this study also support the validity of this new technology for systematic identification of cellular ligands, including angiogenic factors.

Project description:BackgroundBreast cancer is a common cancer among women in the world. However, its pathogenesis is still to be determined. The role and molecular mechanism of Nucleosome Assembly Protein 1 Like 1 (NAP1L1) in breast cancer have not been reported. Elucidation of molecular mechanism might provide a novel therapeutic target for breast cancer treatment.MethodsA bioinformatics analysis was conducted to determine the differential expression of NAP1L1 in breast cancer and find the potential biomarker that interacts with NAP1L1 and hepatoma-derived growth factor (HDGF). The expression of NAP1L1 in tissues was detected by using immunohistochemistry. Breast cancer cells were transfected with the corresponding lentiviral particles and siRNA. The efficiency of transfection was measured by RT-qPCR and western blotting. Then, MTT, Edu, plate clone formation, and subcutaneous tumorigenesis in nude mice were used to detect the cell proliferation in breast cancer. Furthermore, coimmunoprecipitation (Co-IP) assay and confocal microscopy were performed to explore the detailed molecular mechanism of NAP1L1 in breast cancer.ResultsIn this study, NAP1L1 protein was upregulated based on the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Consistent with the prediction, immunohistochemistry staining showed that NAP1L1 protein expression was significantly increased in breast cancer tissues. Its elevated expression was an unfavorable factor for breast cancer clinical progression and poor prognosis. Stably or transiently knocking down NAP1L1 reduced the cell growth in vivo and in vitro via repressing the cell cycle signal in breast cancer. Furthermore, the molecular basis of NAP1L1-induced cell cycle signal was further studied. NAP1L1 interacted with the HDGF, an oncogenic factor for tumors, and the latter subsequently recruited the key oncogenic transcription factor c-Jun, which finally induced the expression of cell cycle promoter Cyclin D1(CCND1) and thus the cell growth of breast cancer.ConclusionsOur data demonstrated that NAP1L1 functions as a potential oncogene via interacting with HDGF to recruit c-Jun in breast cancer.

Dataset Information

Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor.

Publications

Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor.

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