Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: Mpro is an obligate homodimeric cysteine protease (PDB 7BB2). (a) Each monomer can be broken up into three regions: Domain I (residues 1-101; yellow/orange); Domain II (102-184; light violet/magenta); and Domain III (201-301; pale green/forest green). (b) The active site in each monomer is created from the interface between Domains I and II, whereby the catalytic dyad’s H41 and C145 are derived from Domains I and II, respectively. In the WT structure, the active-site cysteine (C145) is located ∼12 Å from C117, the cysteine involved in the disulfide bond in the H163A Mpro structure. (c) Surface representation of the WT Mpro with a focus on the active-site cleft. The enzyme’s S2 to S4 pockets are denoted by the black line. The key residue of interest, H163, is located in a pocket laterally connected to this active site groove (denoted by “*”). (d) The surface representation from (c) is rotated 90° counterclockwise to show this H163 lateral pocket from a head-on perspective. Side chains that make up the lateral pocket and the catalytic dyad are rendered as cylinders in both (c) and (d). All molecular representations in this paper were generated in CCP4MG.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Derivative Assay, Residue, Generated

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: (a) The general fold of Mpro is conserved when comparing the WT (grey; PDB 7TGR) and H163A mutant structures in complex with the covalent inhibitor GC376 (PDB 8DD6). Cα RMSD values were calculated using Chimera. Only one monomer is depicted as the other monomer comprising the dimer is crystallographically identical. (b) The pose of GC376 is also nearly identical between the WT (grey) and H163A mutant structures, although there are slight differences in the ring puckering and rotameric conformation of some inhibitor moieties, particularly the phenyl ring of GC376. These changes are supported by 2F o -F c density at 1.2 σ and can be attributed to the slightly different inhibitor-enzyme interactions between the two structures ( Supplementary Fig. 1 ). (c) The carbonyl on the γ-lactam moiety makes a hydrogen bond with the H163 imidazole ring in the WT enzyme (grey). Upon mutation to alanine, a water molecule compensates for the loss of the imidazole ring by making hydrogen bonds with GC376 and the backbone carbonyl of M165. This is supported by 2F o -F c density at 1.0 σ.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: (a) The apo H163A Mpro structure contains a disulfide bond between the active-site nucleophile, C145, and the previously distant cysteine C117 . This disulfide bond is not completely formed as there is 2F o -F c density at 1.2 σ that supports an alternate, non-disulfide-bonded conformation of C117. The side chain of N28 also takes on two conformations, one seen in both WT (grey) and mutant structures and the other only seen in the mutant. There is a concomitant structural change between the formation of the disulfide bond and the rotation of the N28 side chain as the N28 side chain in its WT conformation sterically hinders the disulfide bond from forming. The beta strand containing the active-site nucleophile can take on two conformations depending on whether or not the disulfide bond is present. (b) When the disulfide bond between C145 and C117 is formed, the C145 beta strand runs antiparallel to the C117 beta strand (2F o -F c density shown at 1.2 σ). (c) When the disulfide bond is broken, the C145 beta strand relaxes to a second conformation (F o -F c density shown at 4.0 σ), aligning almost exactly to the WT conformation of the strand when the structures are superimposed. The flexibility in the loop N-terminal to the C145 beta strand (K137-G143) allows for this relaxation to occur. Despite there being crystallographic evidence for both conformational states, only the disulfide-bonded conformation was modelled as density corresponding to the second conformation could not be accurately modelled with only one alternate conformation. (b) and (c) depict the two conformations of chain B of the H163A structure.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: In addition to the local restructuring of the active site, structural changes are also seen distally in Domain I. An NOS bridge between C22 and K61 is captured in chain B (a) (2F o -F c density shown at 1.2 σ) but not in chain A (b) (2F o -F c density shown at 1.0 σ). (c) This structural asymmetry is also seen when comparing the N-termini of the two monomers, where 2F o -F c density is only seen for the N-terminus of chain B (shown at 1.4 σ) but not chain A. (d) When comparing the positions of the N-termini between the WT and H163A mutant, the four most N-terminal residues are drastically rotated approximately 90° to fit into an alternate pocket. This is due to the movement of the F140 loop in the H163A mutant structure, which occupies the space previously held by the WT N-terminus. There is no density to support a single conformation of the N-terminus in the other protomer.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: Many of the structural differences seen between the apo WT and apo H163A Mpro structures can be attributed to the movement of the F140 side chain. (a) F140 is normally found in an “in” conformation within the core of the enzyme (WT structure is in grey), where it is stabilized by a face-to-face π-stacking interaction with the side chain of H163. When the H163 side chain is mutated, F140 flips to an energetically favored “out” conformation in an ∼14 Å motion to situate itself close to the C-terminus of the other monomer. (b) The “out” conformation in the H163A mutant results in the formation of a new hydrogen-bonding network in the space previously occupied by the F140 side chain (2F o -F c density shown at 1.3 σ). This network is formed from two new water molecules (W73 and W179) and the side chains of Y118, Y126, S147, and H172. (c) A hypothetical structural rearrangement of this F140 pocket is shown with arrows indicating the motion of these side chains from their start (WT; grey) to end (H163A) conformations.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: Free-energy surfaces describing the transitions from state A (WT conformation) to state B (conformation closer to H163A crystal structure) in the WT (a) , H163A (c) , and F140A model (e) are shown. The surfaces were explored for the changes in two CVs, distance between Cα atoms of amino acids at 140 and 163 positions and the sidechain dihedral rotation of N28. The resultant surfaces are depicted in a red to blue spectrum that correspond to high- and low-energy structures, respectively, and the states along the path are marked. The comparison of the 1D free energy profiles corresponding to the minimum energy paths connecting states A and B in the WT, H163A, and F140A models are shown in (b) . The superimposed structures of the H163A crystal structure against the initial and the final states from the metadynamics simulation of H163A model are described in (d) . In the initial state of the H163A model, the side chain of F140 (green cyan) was present in the ‘in’ conformation whereas, at the end of the simulation, the active site loop was dislocated and the F140 sidechain was exposed to the surface – a conformation similar (light cyan) to that seen in the mutant crystal structure. The sidechain rotation of N28 (purple) is also shown. (f) Free-energy profile for the N28A model shows a “free fall” from state A to state B due to a lack of any significant barrier in its path.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Comparison, Mutagenesis

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: MicroRNA-139-5p inhibits bladder cancer proliferation and self-renewal by targeting the Bmi1 oncogene.

doi: 10.1177/1010428317718414

Figure Lengend Snippet: Figure 5. MiR-139-5p inhibits c-MYC and Wnt signaling pathway via downregulation of Bmi1. (a) Expression of Bmi1 and c-MYC levels in T24 and 5637 cells was determined by western blotting. (b) Expression of Wnt signaling pathway was measured by TOP/ FOP detection in T24 and 5637 cells (*p < 0.05 compared with NC treatment (scrambled miRNA; Student’s t test)).

Article Snippet: TOP Flash (Addgene plasmid # 12456) and FOP Flash (TOP Flash mutant; Addgene plasmid # 12457) were gifted by Randall Moon.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Pulmonary Neoplasms in Patients with Birt-Hogg-Dubé Syndrome: Histopathological Features and Genetic and Somatic Events

doi: 10.1371/journal.pone.0151476

Figure Lengend Snippet: Summary of histology, gene mutations and immunostaining.

Article Snippet: For immunostaining, the following reagents were used: rabbit polyclonal antibodies against phospho-mTOR (p-mTOR) (Ser2448) (Cell Signaling Technology, Danvers, MA), phospho-S6 ribosomal protein (p-S6) (Ser235/236) (Cell Signaling Technology), phospho-Akt (p-Akt) (Ser473) (Cell Signaling Technology), and FLCN (ab93196, Abcam, Cambridge, UK); rabbit monoclonal mutant/deleted-specific antibody against EGFR (L858R and E746-A750 deletion) (Cell Signaling Technology); rabbit monoclonal antibody against ROS1 (clone D4D6, Cell Signaling Technology); and mouse monoclonal antibody against ALK (clone 5A4, Abcam).

Techniques: Immunostaining, Mutagenesis, Labeling

Journal: PLoS ONE

Article Title: Pulmonary Neoplasms in Patients with Birt-Hogg-Dubé Syndrome: Histopathological Features and Genetic and Somatic Events

doi: 10.1371/journal.pone.0151476

Figure Lengend Snippet: (A) Germline and somatic FLCN status in 3 representative cases with different mutation patterns are shown. Control normal sequences are shown on the left. Germline mutations are shown on the middle. The somatic status of FLCN in microdissected neoplasms are shown on the right. (B) The papillary adenocarcinoma (PAC) had a heterozygous missense mutation (L858R) in EGFR (indicated by an arrow). (C) The micropapillary adenocarcinoma (MPAC) had a heterozygous missense mutation (G12D) in KRAS (right, indicated by an arrow).

Article Snippet: For immunostaining, the following reagents were used: rabbit polyclonal antibodies against phospho-mTOR (p-mTOR) (Ser2448) (Cell Signaling Technology, Danvers, MA), phospho-S6 ribosomal protein (p-S6) (Ser235/236) (Cell Signaling Technology), phospho-Akt (p-Akt) (Ser473) (Cell Signaling Technology), and FLCN (ab93196, Abcam, Cambridge, UK); rabbit monoclonal mutant/deleted-specific antibody against EGFR (L858R and E746-A750 deletion) (Cell Signaling Technology); rabbit monoclonal antibody against ROS1 (clone D4D6, Cell Signaling Technology); and mouse monoclonal antibody against ALK (clone 5A4, Abcam).

Techniques: Mutagenesis, Control

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) TAGLN2 mRNA was expressed at significantly higher levels in IDH1/2 WT tumors compared to IDH1/2 mutant tumors in both our institutional (p-value=7.73×10 −5 ; FDR=0.053) and the validation TCGA (p-value <0.0001; FDR < 0.0001) cohorts. (B) Mass spectrometry identified higher TAGLN2 protein expression in IDH1/2 WT compared to IDH1/2 mutant LGG from our institutional cohort. Six different peptides corresponding to TAGLN2 protein were expressed at significantly up-regulated in IDH1/2 WT compared to IDH1/2 mutant tumors. (C) Publicly available TAGLN2 mRNA expression of all Grade II (G2, n=249), III (G3, n=265) and IV (GBM, n=153) gliomas from the TCGA database are significantly different (p<0.0001). (D) TAGLN2 expression in IDH1/2 WT Grade II (n=21), III (n=52) and IV (n=133) tumors from TCGA data was significantly different (p=5.136×10 −20 ).

Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

Techniques: Mutagenesis, Biomarker Discovery, Mass Spectrometry, Expressing

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) Promoter methylation was detected in IDH1/2 mutant tumors (n=54, cyan) and IDH1/2 WT tumors (n=8, salmon) from our institutional cohort using 15 CpG TAGLN2 promoter methylation sites included on the Illumina HM-450K array. IDH1/2 mutant showed significantly higher levels of methylation (FDR<0.05) in the majority of CpG islands corresponding to TAGLN2 (n=11), as demonstrated by the heat map. Low methylation levels are denoted in green and high methylation levels are denoted in red. (B) Methylation results were validated using methylation data from the publicly available TCGA cohort.

Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

Techniques: Methylation, Mutagenesis

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: Clinical-pathological characteristics of patients analyzed for TAGLN2 mRNA expression in institutional and TCGA LGG cohorts

Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

Techniques: Expressing, Mutagenesis

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: Multi-variable analysis of clinical-pathological factors with OS from low(er) grade gliomas in the TCGA cohort

Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) GBM30 neurospheres stably expressing TAGLN2 shRNA and corresponding scrambled shRNA control were generated and the level of stable TAGLN2 knock-down detected by Western blot is shown. (B) GBM30 neurospheres with stable knock-down of TAGLN2 or scrambled shRNA control were counted at 24, 72, and 1120 hours after plating. Knock-down of TAGLN2 resulted in significantly decreased cell counts (p<0.05). (C) GBM30 neurospheres and (D) U87 MG glioma cells stably overexpressing TAGLN2 and corresponding vector control were generated and the level of stable TAGLN2 overexpression was detected by Western blot. Of note, endogenous TAGLN2 (22 Kda) and exogenous TAGLN2 -myc (28 kDa) are shown. (E) GBM30 neurospheres stably overexpressing TAGLN2 resulted in significantly increased cell proliferation compared to vector control at 72 and 120 hours (p<0.05). (F) U87 MG cells stably overexpressing TAGLN2 resulted in increased cell proliferation compared to the vector alone. Experiments were performed twice with six replicates each. * , statistically significant difference in proliferation.

Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

Techniques: Stable Transfection, Expressing, shRNA, Control, Generated, Knockdown, Western Blot, Plasmid Preparation, Over Expression

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: Since TAGLN2 has been shown to play a role in invasion and metastases of other cancer types, the invasive ability of (A) GBM30 neurospheres with stable shRNA-mediated knock-down of TAGLN2 were compared to their respective scrambled shRNA control. GBM30 cells showed a decrease in average number of cells invading through the matrix after knock-down of TAGLN2 compared to control. In contrast, (B) GBM30 neurospheres and (C) U87 MG glioma cells with stable overexpression of TAGLN2 showed an increase in average number of cells invading through the matrix compared to vector control. Experiments were performed three times with triplicate invasion assays. * , statistically significant difference in invading cells (p<0.05). Photographs are representative images at 40x and 100x magnification.

Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

Techniques: shRNA, Knockdown, Control, Over Expression, Plasmid Preparation

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) TAGLN2 protein levels were compared in U87 MG IDH1/2 WT parental cells and a commercially available U87 MG isogenic cell line overexpressing IDH1 with a heterozygous R132H mutation by Western blot analysis. TAGLN2 protein was decreased in IDH1 mutant cells compared to IDH1/2 WT cells. (B) U87 MG IDH1 mutant cells were treated with increasing concentrations of 5-azacytidine (5-AZA) demethylating agent and TAGLN2 protein was evaluated by Western blot. 5-AZA resulted in increasing TAGLN2 protein levels expression in a dose-dependent manner.

Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

Techniques: Mutagenesis, Western Blot, Expressing

Journal: Cell chemical biology

Article Title: HTiP: High-throughput immunomodulator phenotypic screening platform to reveal IAP antagonists as anti-cancer immune enhancers

doi: 10.1016/j.chembiol.2018.11.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-KRAS (G12V specific) antibody , Cell signaling , Cat# 14412S.

Techniques: Neutralization, Control, Recombinant, Viability Assay, Enzyme-linked Immunosorbent Assay, Software, Selection