Temporal Proteomic and Phosphoproteomic Analysis of EV-A71-Infected Human Cells

Human enterovirus A71 (EV-A71), a member of the Picornaviridae family, is one of the main etiological viruses that lead to hand, foot, and mouth disease (HFMD). We utilized a multiplex tandem mass tag-based quantitative proteomic technique to monitor the alternation of the whole cell proteome and phosphoproteome of human rhabdomyosarcoma cells over the course of EV-A71 infection. We successfully quantified more than 7000 host proteins and 17,000 phosphosites, of which 80 proteins and nearly 1700 phosphosites were significantly regulated upon viral infection. We found that Myc proto-oncogene protein level decreased significantly, benefiting EV-A71 replication. Multiple signaling pathways were regulated in phosphorylation events that converge for protein translation, cell cycle control, and cell survival. Numerous host factors targeted by virus proteins are phosphoproteins. These factors are involved in host translational initiation, unfolded protein response, endoplasmic reticulum stress, and stress granule formation, and their phosphorylation may play key roles in the virus life cycle. Notably, we identified three conserved phosphorylation sites on viral polyproteins that have not been previously reported. Our study provides valuable resources for a systematic understanding of the interaction between the host cells and the EV-A71 at the protein and the post-translational level

Temporal Proteomic and Phosphoproteomic Analysis of EV-A71-Infected Human Cells

Human enterovirus A71 (EV-A71), a member of the Picornaviridae family, is one of the main etiological viruses that lead to hand, foot, and mouth disease (HFMD). We utilized a multiplex tandem mass tag-based quantitative proteomic technique to monitor the alternation of the whole cell proteome and phosphoproteome of human rhabdomyosarcoma cells over the course of EV-A71 infection. We successfully quantified more than 7000 host proteins and 17,000 phosphosites, of which 80 proteins and nearly 1700 phosphosites were significantly regulated upon viral infection. We found that Myc proto-oncogene protein level decreased significantly, benefiting EV-A71 replication. Multiple signaling pathways were regulated in phosphorylation events that converge for protein translation, cell cycle control, and cell survival. Numerous host factors targeted by virus proteins are phosphoproteins. These factors are involved in host translational initiation, unfolded protein response, endoplasmic reticulum stress, and stress granule formation, and their phosphorylation may play key roles in the virus life cycle. Notably, we identified three conserved phosphorylation sites on viral polyproteins that have not been previously reported. Our study provides valuable resources for a systematic understanding of the interaction between the host cells and the EV-A71 at the protein and the post-translational level

Temporal Proteomic and Phosphoproteomic Analysis of EV-A71-Infected Human Cells

Human enterovirus A71 (EV-A71), a member of the Picornaviridae family, is one of the main etiological viruses that lead to hand, foot, and mouth disease (HFMD). We utilized a multiplex tandem mass tag-based quantitative proteomic technique to monitor the alternation of the whole cell proteome and phosphoproteome of human rhabdomyosarcoma cells over the course of EV-A71 infection. We successfully quantified more than 7000 host proteins and 17,000 phosphosites, of which 80 proteins and nearly 1700 phosphosites were significantly regulated upon viral infection. We found that Myc proto-oncogene protein level decreased significantly, benefiting EV-A71 replication. Multiple signaling pathways were regulated in phosphorylation events that converge for protein translation, cell cycle control, and cell survival. Numerous host factors targeted by virus proteins are phosphoproteins. These factors are involved in host translational initiation, unfolded protein response, endoplasmic reticulum stress, and stress granule formation, and their phosphorylation may play key roles in the virus life cycle. Notably, we identified three conserved phosphorylation sites on viral polyproteins that have not been previously reported. Our study provides valuable resources for a systematic understanding of the interaction between the host cells and the EV-A71 at the protein and the post-translational level

Discovery of a New Class of Uracil Derivatives as Potential Mixed Lineage Kinase Domain-like Protein (MLKL) Inhibitors

Necroptosis is a form of programmed cell death. Mixed lineage kinase domain-like protein (MLKL) is the necroptosis executor, and it is involved in various diseases such as tissue damage and neurodegeneration-related diseases. Here, we report the development of novel MLKL inhibitors with a uracil nucleus through scaffold morphing from our previously reported xanthine MLKL inhibitor TC13172. After a rational structure–activity relationship study, we obtained the highly potent compounds 56 and 66. Mechanism studies revealed that these compounds partially inhibited MLKL oligomerization and significantly inhibited MLKL translocation to the membrane. Compared with TC13172, 56 and 66 have a different mode of action and, importantly, their reaction rate with glutathione is more than 150-fold lower. This reduction in potential off-target effects and cell toxicity makes this series an attractive starting point for further drug development for MLKL-related disease treatments

Discovery of a New Class of Uracil Derivatives as Potential Mixed Lineage Kinase Domain-like Protein (MLKL) Inhibitors

Necroptosis is a form of programmed cell death. Mixed lineage kinase domain-like protein (MLKL) is the necroptosis executor, and it is involved in various diseases such as tissue damage and neurodegeneration-related diseases. Here, we report the development of novel MLKL inhibitors with a uracil nucleus through scaffold morphing from our previously reported xanthine MLKL inhibitor TC13172. After a rational structure–activity relationship study, we obtained the highly potent compounds 56 and 66. Mechanism studies revealed that these compounds partially inhibited MLKL oligomerization and significantly inhibited MLKL translocation to the membrane. Compared with TC13172, 56 and 66 have a different mode of action and, importantly, their reaction rate with glutathione is more than 150-fold lower. This reduction in potential off-target effects and cell toxicity makes this series an attractive starting point for further drug development for MLKL-related disease treatments

Second Generation TQ-Ligation for Cell Organelle Imaging

Bioorthogonal ligations play a crucial role in labeling diverse types of biomolecules in living systems. Herein, we describe a novel class of <i>ortho</i>-quinolinone quinone methide (<i>o</i>QQM) precursors that show a faster kinetic rate in the “click cycloaddition” with thio-vinyl ether (TV) than the first generation TQ-ligation in both chemical and biological settings. We further demonstrate that the second generation TQ-ligation is also orthogonal to the widely used strain-promoted azide–alkyne cycloaddition (SPAAC) both <i>in vitro</i> and <i>in vivo</i>, revealing that these two types of bioorthogonal ligations could be used as an ideal reaction pair for the simultaneous tracking of multiple elements within a single system. Remarkably, the second generation TQ-ligation and SPAAC are effective for selective and simultaneous imaging of two different cell organelles in live cells

Silencing of <i>35S-NPTII</i> transgene and endogenous transposable elements and other loci is affected in the <i>mms19</i> mutant.

<p>(A) The effect of <i>mms19</i> on the silencing of <i>RD29A-LUC</i> and <i>35S-NPTII</i> transgenes. Each genotype harboring the <i>RD29A-LUC</i> and <i>35S-NPTII</i> transgenes was grown on MS medium for 14 days followed by cold treatment for 2 days at 4 °C. The treated seedlings were sprayed with luciferin for luminescence imaging. The seedlings were grown on MS medium supplemented with 150 mg/L kanamycin for 20 days and photographed. (B) The <i>mms19</i> and <i>abo4</i> mutants release the silencing of transposable elements. The transcript levels of the transposable element genes <i>TSI</i>, <i>AT2G11780</i>, and <i>AT3G32195</i> were detected in the wild type, <i>mms19-2</i> and its complementation line, and <i>abo4</i> by quantitative RT-PCR. <i>ACT7</i> was used as an internal control for normalization. Quantitative RT-PCR experiments were biologically repeated three times with similar results. Showing is the result of three technical replicates from one representative experiment.</p

The <i>mms19</i> mutants cause an early flowering phenotype by affecting the expression of flowering-related genes.

<p>(A) The early flowering phenotype of <i>mms19</i> mutants in standard long-day conditions (16-h-light and 8-h-dark at 22°C). (B) The early-flowering phenotype was restored by the <i>MMS19-Myc</i> construction in <i>mms19-2</i>. (C) The statistics of total leaf numbers upon flowering under long-day conditions in the wild type, the <i>mms19</i> mutants and the complementation lines. T2-5 and T2-6 were two randomly selected individual <i>MMS19-MYC</i> transgenic lines in T2 generation. At least 30 individual plants were counted. Error bars stand for SD. Asterisks indicate significant difference as determined by the <i>t</i>-test (P<0.05). Numbers of rosette and cauline leaves are indicated by blue and red bars, respectively. (D) Leaf numbers under long-day conditions with or without vernalization. (E) Leaf numbers under short-day conditions. (F), (G) and (H) The effect of <i>mms19</i>, <i>abo4</i>, and <i>icu2</i> on the expression of flowering-related genes as determined by quantitative RT-PCR. <i>ACT7</i> was amplified as an internal control. The quantitative RT-PCR experiments were biologically repeated for three times and indicated similar results. A representative repetition is shown. Error bars represent SD. Asterisks show significant difference as determined by the <i>t</i>-test (P<0.05).</p

The Cytosolic Iron-Sulfur Cluster Assembly Protein MMS19 Regulates Transcriptional Gene Silencing, DNA Repair, and Flowering Time in <i>Arabidopsis</i>

<div><p>MMS19 is an essential component of the cytoplasmic iron-sulfur (Fe-S) cluster assembly complex in fungi and mammals; the <i>mms19</i> null mutant alleles are lethal. Our study demonstrates that MMS19/MET18 in <i>Arabidopsis thaliana</i> interacts with the cytoplasmic Fe-S cluster assembly complex but is not an essential component of the complex. We find that MMS19 also interacts with the catalytic subunits of DNA polymerases, which have been demonstrated to be involved in transcriptional gene silencing (TGS), DNA repair, and flowering time regulation. Our results indicate that MMS19 has a similar biological function, suggesting a functional link between MMS19 and DNA polymerases. In the <i>mms19 </i>null mutant, the assembly of Fe-S clusters on the catalytic subunit of DNA polymerase α is reduced but not blocked, which is consistent with the viability of the mutant. Our study suggests that MMS19 assists the assembly of Fe-S clusters on DNA polymerases in the cytosol, thereby facilitating transcriptional gene silencing, DNA repair, and flowering time control.</p></div

The effect of <i>mms19</i> on the transcriptome as determined by RNA-seq analyses.

<p>(A) Differentially expressed genes in <i>mms19</i> and <i>abo4</i> mutants relative to the wild type are shown by Venn diagrams. (B) Differentially expressed genes in <i>mms19</i> and <i>abo4</i> mutants relative to the wild type are shown by heat maps. (C) Differentially expressed TEs in <i>mms19</i> and <i>abo4</i> mutants relative to the wild type are shown by Venn diagrams. (D) Gene Ontology (GO) analysis of co-upregulated genes in <i>mms19</i> and <i>abo4</i> mutants. The lengths of bars represent statistical values of gene enrichment in the indicated biological processes. The biological processes are listed only when their genes are significantly (P<0.05) enriched.</p