Table_5_PCBP-1 Regulates the Transcription and Alternative Splicing of Inflammation and Ubiquitination-Related Genes in PC12 Cell.XLS

PCBP-1, a multifunctional RNA binding protein, is expressed in various human cell/tissue types and involved in post-transcriptional gene regulation. PCBP-1 has important roles in cellular Iron homeostasis, mitochondrial stability, and other cellular activities involved in the pathophysiological process of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). However, it remains enigmatic whether PCPB-1 is associated with the pathogenesis of PD. In this study, we cloned and constitutively overexpressed PCBP-1 in rat PC12 cells (PC12 cell is the common cell line studying neurodegenerative disease include PD). RNA-seq was performed to analyze PCBP-1-regulated differentially expressed genes (DEGs) and alternative splicing events (ASEs) between control and PCBP1-overexpressed cells. GO and KEGG pathway analyses were performed to identify functional DEGs and alternatively spliced genes. Consequently, we validated PCBP-1-regulated genes using RT-qPCR. Finally, we downloaded CLIP-seq data from GEO (GSE84700) to analyze the mechanisms of PCBP-1’s regulation of gene expression and ASEs by revealing the binding profile of PCBP-1 on its target pre-mRNAs. Overexpression of PCBP-1 partially regulated the ASE and expression of genes enriched in neuroinflammation and protein ubiquitination, which were also associated with PD pathogenesis. Moreover, RT-qPCR assay verified the PCBP-1-modulated expression of neuroinflammatory genes, like LCN-2, and alternative splicing (AS) of ubiquitination-related gene WWP-2. Finally, CLIP-seq data analysis indicated that the first UC motif was the critical site for PCBP-1 binding to its targets. In this study, we provided evidence that PCBP-1 could regulate the expression of LCN-2 gene expression associated with neuroinflammation and AS of WWP-2 in relation to protein ubiquitination. These findings thus provided novel insights into the potential application of PCBP-1 as the disease pathophysiological or therapeutic target for neurodegenerative disease.</p

Image_1_PCBP-1 Regulates the Transcription and Alternative Splicing of Inflammation and Ubiquitination-Related Genes in PC12 Cell.TIFF

PCBP-1, a multifunctional RNA binding protein, is expressed in various human cell/tissue types and involved in post-transcriptional gene regulation. PCBP-1 has important roles in cellular Iron homeostasis, mitochondrial stability, and other cellular activities involved in the pathophysiological process of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). However, it remains enigmatic whether PCPB-1 is associated with the pathogenesis of PD. In this study, we cloned and constitutively overexpressed PCBP-1 in rat PC12 cells (PC12 cell is the common cell line studying neurodegenerative disease include PD). RNA-seq was performed to analyze PCBP-1-regulated differentially expressed genes (DEGs) and alternative splicing events (ASEs) between control and PCBP1-overexpressed cells. GO and KEGG pathway analyses were performed to identify functional DEGs and alternatively spliced genes. Consequently, we validated PCBP-1-regulated genes using RT-qPCR. Finally, we downloaded CLIP-seq data from GEO (GSE84700) to analyze the mechanisms of PCBP-1’s regulation of gene expression and ASEs by revealing the binding profile of PCBP-1 on its target pre-mRNAs. Overexpression of PCBP-1 partially regulated the ASE and expression of genes enriched in neuroinflammation and protein ubiquitination, which were also associated with PD pathogenesis. Moreover, RT-qPCR assay verified the PCBP-1-modulated expression of neuroinflammatory genes, like LCN-2, and alternative splicing (AS) of ubiquitination-related gene WWP-2. Finally, CLIP-seq data analysis indicated that the first UC motif was the critical site for PCBP-1 binding to its targets. In this study, we provided evidence that PCBP-1 could regulate the expression of LCN-2 gene expression associated with neuroinflammation and AS of WWP-2 in relation to protein ubiquitination. These findings thus provided novel insights into the potential application of PCBP-1 as the disease pathophysiological or therapeutic target for neurodegenerative disease.</p

Actual Structure, Thermodynamic Driving Force, and Mechanism of Benzofuranone-Typical Compounds as Antioxidants in Solution

5,7-Ditert-butyl-3-(3,4-dimethylphenyl)benzofuran-2(3H)-one (HP-136) (1H) and its 30 analogues (2H−5H) as benzofuranone-typical antioxidants were synthesized. The structures of the benzofuranones in solid and solution were examined by using experimental and theoretical methods. The results show that the dominant structure is the lactone form rather than the enol form both in solid and solution. The thermodynamic driving forces of the 31 benzofuranone-typical compounds to release protons [ΔGPD(XH)], hydrogen atoms [ΔGHD(XH)], and electrons [Eox(XH)] and the thermodynamic driving forces of the anions (X−) of the benzofuranones to release electrons [Eox(X−)] were determined for the first time in DMSO. The ΔGHD(XH) scale of these compounds in DMSO ranges from 65.2 to 74.1 (kcal/mol) for 1H−4H and from 73.8 to 75.0 (kcal/mol) for 5H, respectively, which are all smaller than that of the most widely used commercial antioxidant BHT (2,6-ditert-butyl-4-methylphenol, 81.6 kcal/mol), suggesting that the 31 XH could be used as good hydrogen-atom-donating antioxidants. The ΔGPD(XH) were observed to range from 11.5 to 16.0 (kcal/mol) for 1H−4H and from 18.6 to 22.4 (kcal/mol) for 5H, indicating that benzofuranones (1H−4H) are good proton donors, and their analogues (5H) should belong to middle-strong proton donors. Eox(XH) of the 31 XH to release an electron vary from 1.346 to 1.962 (V versus Fc+/0), implying that the 31 XH are weak electron donors, whereas the quite negative Eox(X−) show that X− are good electron donors. The Gibbs free-energy changes of the radical cations (XH+•) to release protons [ΔGPD(XH+•)] were evaluated according to the corresponding thermodynamic cycle, and the results reveal that XH+• are good proton donors. Further inspection of our experimental results showed the ΔGHD(XH), ΔGPD(XH), ΔGPD(XH+•), Eox(XH), and Eox(X−) of the five chemical and electrochemical processes are all linearly dependent on the sum of Hammett substituent parameters σ with very good correlation coefficients, indicating that for any one- or multisubstituted species at the para- and/or meta-position of benzofuranones and their various reaction intermediates, the five thermodynamic driving force parameters all can be easily and safely estimated from the corresponding Hammett substituent parameters. The rates of hydrogen atom transfer from XH to DPPH• were determined by using the UV−vis absorption spectroscopy technique. Combining these important thermodynamic parameters and dynamic determination results, the mechanism of hydrogen transfer from HP-136 and its analogues to DPPH• was studied. The results suggest that the hydrogen transfer from HP-136 and its analogues 2H to DPPH• actually includes two steps, proton transfer and the following electron transfer, but the proton transfer is rate-determined

Actual Structure, Thermodynamic Driving Force, and Mechanism of Benzofuranone-Typical Compounds as Antioxidants in Solution

5,7-Ditert-butyl-3-(3,4-dimethylphenyl)benzofuran-2(3H)-one (HP-136) (1H) and its 30 analogues (2H−5H) as benzofuranone-typical antioxidants were synthesized. The structures of the benzofuranones in solid and solution were examined by using experimental and theoretical methods. The results show that the dominant structure is the lactone form rather than the enol form both in solid and solution. The thermodynamic driving forces of the 31 benzofuranone-typical compounds to release protons [ΔGPD(XH)], hydrogen atoms [ΔGHD(XH)], and electrons [Eox(XH)] and the thermodynamic driving forces of the anions (X−) of the benzofuranones to release electrons [Eox(X−)] were determined for the first time in DMSO. The ΔGHD(XH) scale of these compounds in DMSO ranges from 65.2 to 74.1 (kcal/mol) for 1H−4H and from 73.8 to 75.0 (kcal/mol) for 5H, respectively, which are all smaller than that of the most widely used commercial antioxidant BHT (2,6-ditert-butyl-4-methylphenol, 81.6 kcal/mol), suggesting that the 31 XH could be used as good hydrogen-atom-donating antioxidants. The ΔGPD(XH) were observed to range from 11.5 to 16.0 (kcal/mol) for 1H−4H and from 18.6 to 22.4 (kcal/mol) for 5H, indicating that benzofuranones (1H−4H) are good proton donors, and their analogues (5H) should belong to middle-strong proton donors. Eox(XH) of the 31 XH to release an electron vary from 1.346 to 1.962 (V versus Fc+/0), implying that the 31 XH are weak electron donors, whereas the quite negative Eox(X−) show that X− are good electron donors. The Gibbs free-energy changes of the radical cations (XH+•) to release protons [ΔGPD(XH+•)] were evaluated according to the corresponding thermodynamic cycle, and the results reveal that XH+• are good proton donors. Further inspection of our experimental results showed the ΔGHD(XH), ΔGPD(XH), ΔGPD(XH+•), Eox(XH), and Eox(X−) of the five chemical and electrochemical processes are all linearly dependent on the sum of Hammett substituent parameters σ with very good correlation coefficients, indicating that for any one- or multisubstituted species at the para- and/or meta-position of benzofuranones and their various reaction intermediates, the five thermodynamic driving force parameters all can be easily and safely estimated from the corresponding Hammett substituent parameters. The rates of hydrogen atom transfer from XH to DPPH• were determined by using the UV−vis absorption spectroscopy technique. Combining these important thermodynamic parameters and dynamic determination results, the mechanism of hydrogen transfer from HP-136 and its analogues to DPPH• was studied. The results suggest that the hydrogen transfer from HP-136 and its analogues 2H to DPPH• actually includes two steps, proton transfer and the following electron transfer, but the proton transfer is rate-determined

Table_1_PCBP-1 Regulates the Transcription and Alternative Splicing of Inflammation and Ubiquitination-Related Genes in PC12 Cell.DOCX

PCBP-1, a multifunctional RNA binding protein, is expressed in various human cell/tissue types and involved in post-transcriptional gene regulation. PCBP-1 has important roles in cellular Iron homeostasis, mitochondrial stability, and other cellular activities involved in the pathophysiological process of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). However, it remains enigmatic whether PCPB-1 is associated with the pathogenesis of PD. In this study, we cloned and constitutively overexpressed PCBP-1 in rat PC12 cells (PC12 cell is the common cell line studying neurodegenerative disease include PD). RNA-seq was performed to analyze PCBP-1-regulated differentially expressed genes (DEGs) and alternative splicing events (ASEs) between control and PCBP1-overexpressed cells. GO and KEGG pathway analyses were performed to identify functional DEGs and alternatively spliced genes. Consequently, we validated PCBP-1-regulated genes using RT-qPCR. Finally, we downloaded CLIP-seq data from GEO (GSE84700) to analyze the mechanisms of PCBP-1’s regulation of gene expression and ASEs by revealing the binding profile of PCBP-1 on its target pre-mRNAs. Overexpression of PCBP-1 partially regulated the ASE and expression of genes enriched in neuroinflammation and protein ubiquitination, which were also associated with PD pathogenesis. Moreover, RT-qPCR assay verified the PCBP-1-modulated expression of neuroinflammatory genes, like LCN-2, and alternative splicing (AS) of ubiquitination-related gene WWP-2. Finally, CLIP-seq data analysis indicated that the first UC motif was the critical site for PCBP-1 binding to its targets. In this study, we provided evidence that PCBP-1 could regulate the expression of LCN-2 gene expression associated with neuroinflammation and AS of WWP-2 in relation to protein ubiquitination. These findings thus provided novel insights into the potential application of PCBP-1 as the disease pathophysiological or therapeutic target for neurodegenerative disease.</p

Table_4_PCBP-1 Regulates the Transcription and Alternative Splicing of Inflammation and Ubiquitination-Related Genes in PC12 Cell.XLSX

PCBP-1, a multifunctional RNA binding protein, is expressed in various human cell/tissue types and involved in post-transcriptional gene regulation. PCBP-1 has important roles in cellular Iron homeostasis, mitochondrial stability, and other cellular activities involved in the pathophysiological process of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). However, it remains enigmatic whether PCPB-1 is associated with the pathogenesis of PD. In this study, we cloned and constitutively overexpressed PCBP-1 in rat PC12 cells (PC12 cell is the common cell line studying neurodegenerative disease include PD). RNA-seq was performed to analyze PCBP-1-regulated differentially expressed genes (DEGs) and alternative splicing events (ASEs) between control and PCBP1-overexpressed cells. GO and KEGG pathway analyses were performed to identify functional DEGs and alternatively spliced genes. Consequently, we validated PCBP-1-regulated genes using RT-qPCR. Finally, we downloaded CLIP-seq data from GEO (GSE84700) to analyze the mechanisms of PCBP-1’s regulation of gene expression and ASEs by revealing the binding profile of PCBP-1 on its target pre-mRNAs. Overexpression of PCBP-1 partially regulated the ASE and expression of genes enriched in neuroinflammation and protein ubiquitination, which were also associated with PD pathogenesis. Moreover, RT-qPCR assay verified the PCBP-1-modulated expression of neuroinflammatory genes, like LCN-2, and alternative splicing (AS) of ubiquitination-related gene WWP-2. Finally, CLIP-seq data analysis indicated that the first UC motif was the critical site for PCBP-1 binding to its targets. In this study, we provided evidence that PCBP-1 could regulate the expression of LCN-2 gene expression associated with neuroinflammation and AS of WWP-2 in relation to protein ubiquitination. These findings thus provided novel insights into the potential application of PCBP-1 as the disease pathophysiological or therapeutic target for neurodegenerative disease.</p

Table_2_PCBP-1 Regulates the Transcription and Alternative Splicing of Inflammation and Ubiquitination-Related Genes in PC12 Cell.XLS

PCBP-1, a multifunctional RNA binding protein, is expressed in various human cell/tissue types and involved in post-transcriptional gene regulation. PCBP-1 has important roles in cellular Iron homeostasis, mitochondrial stability, and other cellular activities involved in the pathophysiological process of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). However, it remains enigmatic whether PCPB-1 is associated with the pathogenesis of PD. In this study, we cloned and constitutively overexpressed PCBP-1 in rat PC12 cells (PC12 cell is the common cell line studying neurodegenerative disease include PD). RNA-seq was performed to analyze PCBP-1-regulated differentially expressed genes (DEGs) and alternative splicing events (ASEs) between control and PCBP1-overexpressed cells. GO and KEGG pathway analyses were performed to identify functional DEGs and alternatively spliced genes. Consequently, we validated PCBP-1-regulated genes using RT-qPCR. Finally, we downloaded CLIP-seq data from GEO (GSE84700) to analyze the mechanisms of PCBP-1’s regulation of gene expression and ASEs by revealing the binding profile of PCBP-1 on its target pre-mRNAs. Overexpression of PCBP-1 partially regulated the ASE and expression of genes enriched in neuroinflammation and protein ubiquitination, which were also associated with PD pathogenesis. Moreover, RT-qPCR assay verified the PCBP-1-modulated expression of neuroinflammatory genes, like LCN-2, and alternative splicing (AS) of ubiquitination-related gene WWP-2. Finally, CLIP-seq data analysis indicated that the first UC motif was the critical site for PCBP-1 binding to its targets. In this study, we provided evidence that PCBP-1 could regulate the expression of LCN-2 gene expression associated with neuroinflammation and AS of WWP-2 in relation to protein ubiquitination. These findings thus provided novel insights into the potential application of PCBP-1 as the disease pathophysiological or therapeutic target for neurodegenerative disease.</p

Table_3_PCBP-1 Regulates the Transcription and Alternative Splicing of Inflammation and Ubiquitination-Related Genes in PC12 Cell.XLSX

PCBP-1, a multifunctional RNA binding protein, is expressed in various human cell/tissue types and involved in post-transcriptional gene regulation. PCBP-1 has important roles in cellular Iron homeostasis, mitochondrial stability, and other cellular activities involved in the pathophysiological process of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). However, it remains enigmatic whether PCPB-1 is associated with the pathogenesis of PD. In this study, we cloned and constitutively overexpressed PCBP-1 in rat PC12 cells (PC12 cell is the common cell line studying neurodegenerative disease include PD). RNA-seq was performed to analyze PCBP-1-regulated differentially expressed genes (DEGs) and alternative splicing events (ASEs) between control and PCBP1-overexpressed cells. GO and KEGG pathway analyses were performed to identify functional DEGs and alternatively spliced genes. Consequently, we validated PCBP-1-regulated genes using RT-qPCR. Finally, we downloaded CLIP-seq data from GEO (GSE84700) to analyze the mechanisms of PCBP-1’s regulation of gene expression and ASEs by revealing the binding profile of PCBP-1 on its target pre-mRNAs. Overexpression of PCBP-1 partially regulated the ASE and expression of genes enriched in neuroinflammation and protein ubiquitination, which were also associated with PD pathogenesis. Moreover, RT-qPCR assay verified the PCBP-1-modulated expression of neuroinflammatory genes, like LCN-2, and alternative splicing (AS) of ubiquitination-related gene WWP-2. Finally, CLIP-seq data analysis indicated that the first UC motif was the critical site for PCBP-1 binding to its targets. In this study, we provided evidence that PCBP-1 could regulate the expression of LCN-2 gene expression associated with neuroinflammation and AS of WWP-2 in relation to protein ubiquitination. These findings thus provided novel insights into the potential application of PCBP-1 as the disease pathophysiological or therapeutic target for neurodegenerative disease.</p

Actual Structure, Thermodynamic Driving Force, and Mechanism of Benzofuranone-Typical Compounds as Antioxidants in Solution

5,7-Ditert-butyl-3-(3,4-dimethylphenyl)benzofuran-2(3H)-one (HP-136) (1H) and its 30 analogues (2H−5H) as benzofuranone-typical antioxidants were synthesized. The structures of the benzofuranones in solid and solution were examined by using experimental and theoretical methods. The results show that the dominant structure is the lactone form rather than the enol form both in solid and solution. The thermodynamic driving forces of the 31 benzofuranone-typical compounds to release protons [ΔGPD(XH)], hydrogen atoms [ΔGHD(XH)], and electrons [Eox(XH)] and the thermodynamic driving forces of the anions (X−) of the benzofuranones to release electrons [Eox(X−)] were determined for the first time in DMSO. The ΔGHD(XH) scale of these compounds in DMSO ranges from 65.2 to 74.1 (kcal/mol) for 1H−4H and from 73.8 to 75.0 (kcal/mol) for 5H, respectively, which are all smaller than that of the most widely used commercial antioxidant BHT (2,6-ditert-butyl-4-methylphenol, 81.6 kcal/mol), suggesting that the 31 XH could be used as good hydrogen-atom-donating antioxidants. The ΔGPD(XH) were observed to range from 11.5 to 16.0 (kcal/mol) for 1H−4H and from 18.6 to 22.4 (kcal/mol) for 5H, indicating that benzofuranones (1H−4H) are good proton donors, and their analogues (5H) should belong to middle-strong proton donors. Eox(XH) of the 31 XH to release an electron vary from 1.346 to 1.962 (V versus Fc+/0), implying that the 31 XH are weak electron donors, whereas the quite negative Eox(X−) show that X− are good electron donors. The Gibbs free-energy changes of the radical cations (XH+•) to release protons [ΔGPD(XH+•)] were evaluated according to the corresponding thermodynamic cycle, and the results reveal that XH+• are good proton donors. Further inspection of our experimental results showed the ΔGHD(XH), ΔGPD(XH), ΔGPD(XH+•), Eox(XH), and Eox(X−) of the five chemical and electrochemical processes are all linearly dependent on the sum of Hammett substituent parameters σ with very good correlation coefficients, indicating that for any one- or multisubstituted species at the para- and/or meta-position of benzofuranones and their various reaction intermediates, the five thermodynamic driving force parameters all can be easily and safely estimated from the corresponding Hammett substituent parameters. The rates of hydrogen atom transfer from XH to DPPH• were determined by using the UV−vis absorption spectroscopy technique. Combining these important thermodynamic parameters and dynamic determination results, the mechanism of hydrogen transfer from HP-136 and its analogues to DPPH• was studied. The results suggest that the hydrogen transfer from HP-136 and its analogues 2H to DPPH• actually includes two steps, proton transfer and the following electron transfer, but the proton transfer is rate-determined