scholarly journals Identification of herboxidiene features that mediate conformation-dependent SF3B1 interactions to inhibit splicing

2020 ◽  
Author(s):  
Adriana Gamboa Lopez ◽  
Srinivasa Rao Allu ◽  
Patricia Mendez ◽  
Guddeti Chandrashekar Reddy ◽  
Hannah M. Maul-Newby ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Growth ◽  
Spliceosome Assembly ◽  
Relationship Analysis ◽  
Branch Point Sequence ◽  
U2 Snrnp ◽  
Before And After ◽  
Multiple Conformations

ABSTRACTSmall molecules that target the spliceosome SF3B complex are potent inhibitors of cancer cell growth. The compounds affect an early stage of spliceosome assembly when U2 snRNP first engages the branch point sequence of an intron. Recent cryo-EM models of U2 snRNP before and after intron recognition suggest several large-scale rearrangements of RNA and protein interactions involving SF3B. Employing an inactive herboxidiene analog as a competitor with SF3B inhibitors, we present evidence for multiple conformations of SF3B in the U2 snRNP, only some of which are available for productive inhibitor interactions. We propose that both thermodynamics and an ATP-binding event promote the conformation conducive to SF3B inhibitor interactions. However, SF3B inhibitors do not impact an ATP-dependent rearrangement in U2 snRNP that exposes the branch binding sequence for base pairing. We also report extended structure activity relationship analysis of herboxidiene, which identified features of the tetrahydropyran ring that mediate its interactions with SF3B and its ability to interfere with splicing. In combination with structural models of SF3B interactions with inhibitors, our data leads us to extend the model for early spliceosome assembly and inhibitor mechanism. We postulate that interactions between a carboxylic acid substituent of herboxidiene and positively charged SF3B1 sidechains in the inhibitor binding channel are required to maintain inhibitor occupancy and counteract the SF3B transition to a closed state that is promoted by stable U2 snRNA interactions with the intron.

scholarly journals U2 snRNA-Protein Contacts in Purified Human 17S U2 snRNPs and in Spliceosomal A and B Complexes

2006 ◽  
Vol 26 (7) ◽  
pp. 2803-2816 ◽  
Author(s):  
Olexandr Dybkov ◽  
Cindy L. Will ◽  
Jochen Deckert ◽  
Nastaran Behzadnia ◽  
Klaus Hartmuth ◽  
...  
Keyword(s):  
Branch Point ◽  
Protein Interactions ◽  
Cross Linking ◽  
Molecular Architecture ◽  
Point Selection ◽  
Stem Loop ◽  
U2 Snrna ◽  
Branch Site ◽  
Branch Point Sequence ◽  
U2 Snrnp

ABSTRACT The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5′ end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5′ end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.

scholarly journals Myocardial Infarction Biomarker Discovery with Integrated Gene Expression, Pathways and Biological Networks analysis

2020 ◽  
Author(s):  
Abdulrahman MUJALLI ◽  
Babajan Banaganapalli ◽  
Noor A. Shaik ◽  
Ramu Elango ◽  
Jumana Y. Al-Aama
Keyword(s):  
Gene Expression ◽  
Myocardial Infarction ◽  
Protein Interactions ◽  
Biological Networks ◽  
Large Scale ◽  
Biomarker Discovery ◽  
Early Stage ◽  
Receptor Interaction ◽  
Disease Development ◽  
Genes And Environment

Abstract Background Myocardial infarction (MI) is the most prevalent coronary atherosclerotic heart disease caused by the complex molecular interactions between multiple genes and environment. Molecular exploration of gene expression changes in MI patients is very crucial not just to understand the molecular basis of disease development but also to identify potential therapeutic targets. Therefore, we aim to identify potential biomarkers for the disease development mechanisms and for prognosis of MI using extensive integrated biological network analysis. Methodology Gene expression datasets (GSE66360) generated from 51 healthy controls and 49 endothelial cell samples from patients experiencing acute MI were used to analyze the differentially expressed genes (DEG), protein-protein interactions (PPI), gene network-clusters to annotate the candidate pathways relevant to MI pathogenesis. Results Bioinformatic analysis revealed 810 DEGs, between control and MI samples, with 574 up- and 236 down- regulated genes. Their functional annotations with Gene Ontology (GO) has captured several MI targeting biological processes like immune response, inflammation and platelets degranulation. Most significantly DEGs enriched KEGG pathways are related to the following functions: Cytokine-cytokine receptor interaction, TNF and NFkB signaling. By constructing the PPI network using STRING and CytoHubba, seventeen hub and bottleneck genes were found, whose involvement in MI was further confirmed by DisGeNET data. Search in the Open Target Platform reveal unique bottleneck genes as potential target for MI. Conclusion Our integrative bioinformatics analysis of large-scale gene expression data has identified several potential genetic biomarkers associated with early stage MI providing a new insight into molecular mechanism underlying the disease.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Protein Protein Interactions ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

A Target-Based Method for Designing Heterochiral Cyclic Peptide Binders: De Novo Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
Cyclic Peptide ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Protein Protein Interactions ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

Konkurrenz zwischen Stieleiche und Buche auf Lothar-Sturmflächen | Competition between pedunculate oak and European beech on Lothar windthrow areas

2009 ◽  
Vol 160 (5) ◽  
pp. 114-123 ◽  
Author(s):  
Daniel Otto ◽  
Sven Wagner ◽  
Peter Brang
Keyword(s):  
Large Scale ◽  
Early Stage ◽  
Tree Height ◽  
European Beech ◽  
Threshold Value ◽  
Test Methods ◽  
Permanent Plots ◽  
Pedunculate Oak ◽  
Competitive Pressure ◽  
Competition Index

The competitive pressure of naturally regenerated European beech (Fagus sylvatica) saplings on planted pedunculate oak (Quercus robur) was investigated on two 1.8 ha permanent plots near Habsburg and Murten (Switzerland). The plots were established with the aim to test methods of artificial oak regeneration after large-scale windthrow. On both plots, 80 oaks exposed to varying levels of competitive pressure from at most 10 neighbouring beech trees were selected. The height of each oak as well as stem and branch diameters were measured. The competitive pressure was assessed using Schütz's competition index, which is based on relative tree height, crown overlap and distance from competing neighbours. Oak trees growing without or with only slight competition from beech were equally tall, while oaks exposed to moderate to strong competition were smaller. A threshold value for the competition index was found above which oak height decreased strongly. The stem and branch diameters of the oaks started to decrease even if the competition from beech was slight, and decreased much further with more competition. The oak stems started to become more slender even with only slight competition from beech. On the moderately acid beech sites studied here, beech grow taller faster than oak. Thus where beech is competing with oak and the aim is to maintain the oak, competitive pressure on the oak must be reduced at an early stage. The degree of the intervention should, however, take the individual competitive interaction into account, with more intervention if the competition is strong.

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