SJS/TEN 2019: From science to translation.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are potentially life-threatening, immune-mediated adverse reactions characterized by widespread erythema, epidermal necrosis, and detachment of skin and mucosa. Efforts to grow and develop functional international collaborations and a multidisciplinary interactive network focusing on SJS/TEN as an uncommon but high burden disease will be necessary to improve efforts in prevention, early diagnosis and improved acute and long-term management. SJS/TEN 2019: From Science to Translation was a 1.5-day scientific program held April 26-27, 2019, in Vancouver, Canada. The meeting successfully engaged clinicians, researchers, and patients and conducted many productive discussions on research and patient care needs

Identification of novel picornavirus proteinase substrates using terminal amine isotopic labeling of substrates

Viruses have exploited strategies of proteolysis for the purposes of processing viral proteins and manipulating cellular processes to direct synthesis of new virions and subvert host antiviral responses. Many viruses encode proteases within their genome, of which many have been well studied among the family of positive-sense single-stranded RNA picornaviruses. A subset of host proteins have already been identified as targets of picornaviral proteinases; however, the full repertoire of targets is not known. In this thesis, a novel proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) was used to conduct a global analysis of protease-generated N-terminal peptides by mass spectrometry and identify novel substrates of the 3C (3Cpro) and 2A (2Apro) proteinases from poliovirus and coxsackievirus type B3 (CVB3). TAILS was performed on HeLa cell extracts subjected to purified poliovirus 3Cpro or CVB3 2Apro, and on mouse HL-1 cardiomyocyte extracts subjected to purified CVB3 3Cpro. A list of high confidence candidate substrates for all three proteinases was generated, which included a peptide corresponding to the known poliovirus 3Cpro substrate polypyrimide tract binding protein at a known cleavage site, thus validating this approach. Furthermore, three identical peptides in both the poliovirus and CVB3 3Cpro list of high confidence substrates were identified, suggesting that cleavage of these substrates may contribute to general strategy of picornaviral infection. A total of seven high confidence substrates were validated as novel targets of 3Cpro in vitro and during virus infection. Moreover, mutations in the TAILS-identified cleavage sites for these candidates blocked cleavage in vitro and during infection. Depletion of these proteins by siRNAs modulated virus infection, suggesting that cleavage of these substrates either promotes or inhibits virus infection. In summary, an in vitro TAILS assay can be utilized to identify novel substrates of viral proteinases that are cleave during infection. Moreover, TAILS can identify common substrates of viral proteinases between different viral species, revealing general strategies of infection utilized by related viruses. Finally, the identification of novel host substrates provides new insights the viral-host interactions mediated by viral proteinases that are required for successful infection.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Protein-protein interactions mediating hypoxia-inducible factor 1 complex function

The hypoxia-inducible factor 1 (HIF1) complex, comprised of a HIF1α and aryl hydrocarbon receptor translocator (ARNT/HIF1β) heterodimer, regulates the transcription of genes promoting tumour progression under hypoxia. Preliminary data suggests that the retinoblastoma protein (RB) attenuates HIF1-mediated transcription directly through interaction with the co-activator thyroid hormone receptor/retinoblastoma interacting protein 230 (TRIP230) to inhibit invasiveness of MCF7 human breast cancer cells. The objective of this study was to further characterize the invasive phenotype in MCF7 cells following loss of RB. Loss of RB resulted in a hypoxic-dependent increase in expression of HIF1 target genes involved in tumour progression. Protein-protein interaction analysis demonstrated that RB exists in complex with ARNT and TRIP230. Moreover, this complex dissociated in the presence of an ARNT-interacting peptide. Lastly, the C-terminal region of the TRIP230 RB-interaction domain has been identified as the minimal interaction domain. Altogether, these results further establish RB as an attenuator of HIF1-mediated transcription

A TRIP230-Retinoblastoma Protein Complex Regulates Hypoxia-Inducible Factor-1α-Mediated Transcription and Cancer Cell Invasion

Localized hypoxia in solid tumors activates transcriptional programs that promote the metastatic transformation of cells. Like hypoxia-inducible hyper-vascularization, loss of the retinoblastoma protein (Rb) is a trait common to advanced stages of tumor progression in many metastatic cancers. However, no link between the role of Rb and hypoxia-driven metastatic processes has been established. We demonstrated that Rb is a key mediator of the hypoxic response mediated by HIF1α/β, the master regulator of the hypoxia response, and its essential co-activator, the thyroid hormone receptor/retinoblastoma-interacting protein (TRIP230). Furthermore, loss of Rb unmasks the full co-activation potential of TRIP230. Using small inhibitory RNA approaches in vivo, we established that Rb attenuates the normal physiological response to hypoxia by HIF1α. Notably, loss of Rb results in hypoxia-dependent biochemical changes that promote acquisition of an invasive phenotype in MCF7 breast cancer cells. In addition, Rb is present in HIF1α-ARNT/HIF1β transcriptional complexes associated with TRIP230 as determined by co-immuno-precipitation, GST-pull-down and ChIP assays. These results demonstrate that Rb is a negative modulator of hypoxia-regulated transcription by virtue of its direct effects on the HIF1 complex. This work represents the first link between the functional ablation of Rb in tumor cells and HIF1α-dependent transcriptional activation and invasion

Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

<div><p>Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the <i>Picornaviridae</i> family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3C^pro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.</p></div

Loss of Rb promotes hypoxia-dependent invasiveness in MCF7 cells in a Matrigel Invasion Assay - MCF7 cells were transfected with scrambled siRNA (SCX) or Rb siRNA as described above.

<p>Twenty-four hours after siRNA transfection, the cells were subjected to the Matrigel invasion assay. Plates were incubated in normoxic (20% O₂) or hypoxic conditions (1% O₂) at 37°C for 24 h and invading cells were fixed and visualized with toluidine blue. (<b>A</b>) Photomicrographs of matrigel-embedded MCF7 cells. (<b>B</b>) Numerical representation of relative invasion of matrigel-embedded MCF7 cells after treatment with SCX or siRb and exposure to normoxic or hypoxic conditions (n = 6), (<b>C</b>) Knock-down of Rb in MCF7 cells does not alter cell proliferation in response to CoCl₂. Cells were transfected with siRNA’s as described above. Twenty-four h after transfection, cells were treated with vehicle or 100 µM CoCl₂ to activate HIF1α and cells were counted at 0, 6, 12, 24, 36 48, and 72 h later. Error bars represent ± S.E.M. *p<0.01.</p

Rb represses HIF1-regulated target gene mRNA in MCF7 human breast cancer cells and LNCaP human prostate cancer cells.

<p>MCF7 cells (<b>A, B, C and D</b>) and LNCaP cells (<b>E</b>) were transfected with either scrambled siRNA (SCX) or Rb siRNAs siRb 1, and siRb 2. Twenty-four hours after transfection cells were either maintained under normoxic conditions or 1% O₂ for a further 24 h. Gene expression was determined by quantitative real-time PCR after isolation and reverse transcription of total RNA. VEGF, CXCR4, PLOD2 and Rb expression were normalized to constitutively active 36B4 gene expression. Open bars represent normoxia (20% O₂) and closed (grey) bars represent hypoxia (1% O₂). Error bars represent ± S.D. *p<0.05.</p

Rb mediates its transcriptional effects on hypoxia-inducible gene regulation through an ARNT-TRIP230-Rb complex.

<p>(<b>A</b>) Immuno-blot of complexes precipitated using either a mouse monoclonal antibody directed to TRIP230 or mouse control IgG from the nuclear extracts of MCF7 cells. Blots were probed for the presence of TRIP30, ARNT and Rb. The left hand lane of each blot contains nuclear extract representing 10% of input. (<b>B</b>) GST-ARNT-PAS-B is capable of pulling down TRIP230 and Rb. Immuno-blot analysis of GST-ARNT-PAS-B pull-down and GST pull-down of TRIP230 and Rb from MCF7 nuclear extracts. GST moieties were fixed to glutathione-agarose beads and incubated for 90 min at 4 degrees C with 500 µg of MCF7 cell nuclear extract. Input lanes were loaded with 250 µg of nuclear extract. Complete blots for panels A and B can be found in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099214#pone.0099214.s001" target="_blank">Figure S1</a>. GST-ARNT-PAS-B is capable of pulling down phosphorylated Rb. (<b>C</b>) Immuno-blot analysis of GST-ARNT-PAS-B pull-down and GST pull-down of Rb-phospho-serine⁷⁸⁰ (Rb-pS⁷⁸⁰) from MCF7 nuclear extracts harvested from cells left at normoxia (N) or treated with 1% O₂ for 6 h (H). (<b>D</b>) Chromatin immuno-precipitation assay of EPO enhancer and VEGF promoter regions in MCF7 cells using control or Rb-phospho-serine⁷⁸⁰ antibodies. Cells were treated as described above. Rb attenuates TRIP230-mediated co-activation of ARNT-dependent transcriptional activity. The Rb- and ARNT-interaction domains are indicated. Hepa-1c1c7 cells (<b>E</b>) and MCF7 cells (<b>F</b>) were transfected with a hypoxia responsive 4xHRE-driven luciferase construct as a reporter, expression plasmids for TRIP230, TRIP230ΔRB and Rb as indicated and subjected to 1% O₂ or atmospheric (20%) O₂ for 24 h. Whole cell lysates were assayed for luciferase activity. (<b>G</b>) A schematic of the TRIP230ΔRB mutant. (<b>H</b>) TRIP230 protein levels are unaffected by transfection of Rb expression plasmid into Hepa1c1c7 cells. Whole cell lysates were analyzed by immuno-blot and membranes were probed with affinity-purified antibodies to TRIP230, Rb and α-tubulin. *p<0.05.</p

ARNT and TRIP230 are essential for Rb-regulation of HIF1.

<p>(<b>A</b>) MCF7 cells were transfected with either scrambled siRNA (SCX) or Rb siRNAs siRb 1, and siRb 2, or a combination of TRIP230 siRNA and siRb1. Twenty-four hours after transfection cells were either maintained under normoxic conditions or 1% O₂ for a further 24 h. Gene expression was determined by quantitative real-time PCR after isolation and reverse transcription of total RNA. CXCR4 expression was normalized to constitutively active 36B4 gene expression. (<b>B</b>) Immuno-blot analysis of TRIP230 and Rb after siRNA transfection with either scrambled siRNA, or a combination of siTRIP230 and siRb. Alpha-tubulin (α-tubulin) was used as a loading control. (<b>C</b>) MCF7 cells were transfected with either scrambled siRNA (SCX) or Rb siRNAs siRb 1, and siRb 2, or ARNT, or DP1 siRNA or a combination of ARNT/Rb1 siRNA and treated as described above. (<b>D</b>) Immuno-blot of ARNT and α-TUB after transfection with either scrambled control of siRNA directed to ARNT. (<b>E</b>) Immuno-blot of DP1, TRIP230 and α-tubulin (α-tubulin). MCF7 cells were transfected with either scrambled control (SCX) or siRNA directed to DP1. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099214#pone-0099214-g003" target="_blank">figure 3A and 3C</a> were analyzed using a two-way-ANOVA. *p<0.01.</p

Illustration describing the transcriptional regulation of HIF-target genes in cells either expressing or lacking Rb.

<p>(<b>A</b>) In cells that are Rb-positive, the full transcriptional activation capacity of the HIF1-TRIP230 complex is repressed or muted resulting in regulated expression of HIF1 target genes. (<b>B</b>) In cells lacking Rb, gene expression mediated by HIF1 becomes uncontrolled.</p