scholarly journals Caspase-7 uses RNA to enhance proteolysis of poly(ADP-ribose) polymerase 1 and other RNA-binding proteins

2019 ◽  
Vol 116 (43) ◽  
pp. 21521-21528 ◽  
Author(s):  
Alexandre Desroches ◽  
Jean-Bernard Denault
Keyword(s):  
Binding Proteins ◽  
Caspase 3 ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Pocket ◽  
Rnase A ◽  
Amino Acid Sequences ◽  
Critical Feature ◽  
Caspase 7 ◽  
Parp 1

To achieve swift cell demise during apoptosis, caspases cleave essential proteins for cell survival and removal. In addition to the binding of preferred amino acid sequences to its substrate-binding pocket, caspase-7 also uses exosites to select specific substrates. 4 lysine residues (K38KKK) located in the N-terminal domain of caspase-7 form such an exosite and promote the rapid proteolysis of the poly(ADP-ribose) polymerase 1 (PARP-1), but the mechanism of recognition remains mostly unknown. In this study, we show that the overall positive charge of the exosite is the critical feature of this evolutionarily conserved binding site. Additionally, interaction with the caspase-7 exosite involves both the Zn3 and BRCT domains of PARP-1 and is mediated by RNA. Indeed, PARP-1 proteolysis efficacy is sensitive to RNase A and promoted by added RNA. Moreover, using affinity chromatography and gel shift assays, we demonstrate that caspase-7, but not caspase-3 or a caspase-7 with a mutated exosite, binds nucleic acids. Finally, we show that caspase-7 prefers RNA-binding proteins (RNA-BPs) as substrates compared to caspase-3 and that RNA enhances proteolysis by caspase-7 of many of these RNA-BPs. Thus, we have uncovered an unusual way by which caspase-7 selects and cleaves specific substrates.

scholarly journals Characterization of caspase-7 interaction with RNA

2021 ◽  
Author(s):  
Alexandre Desroches ◽  
Jean-Bernard Denault
Keyword(s):  
Cell Death ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Amino Acid Sequences ◽  
Binding Assays ◽  
Rna Molecules ◽  
Caspase 7

Apoptosis is a regulated form of cell death essential to the removal of unwanted cells. At its core, a family of cysteine peptidases named caspases cleaves key proteins allowing cell death to occur. To do so, each caspase catalytic pocket recognizes preferred amino acid sequences resulting in proteolysis, but some also use exosites to select and cleave important proteins efficaciously. Such exosites have been found in a few caspases, notably caspase-7 that has a lysine patch (K38KKK) that binds RNA which acts as a bridge to RNA-binding proteins favoring proximity between the peptidase and its substrates resulting in swifter cleavage. Although caspase-7 interaction with RNA has been identified, in-depth characterization of this interaction is lacking. In this study, using in vitro cleavage assays, we determine that RNA concentration and length affect the cleavage of RNA-binding proteins. Additionally, using binding assays and RNA sequencing, we found that caspase-7 binds RNA molecules regardless of their type, sequence, or structure. Moreover, we demonstrate that the N-terminal peptide of caspase-7 reduces the affinity of the peptidase for RNA which translates into slower cleavages of RNA-binding proteins. Finally, employing engineered heterodimers, we show that a caspase-7 dimer can use both exosites simultaneously to increase its affinity to RNA because a heterodimer with only one exosite has reduced affinity for RNA and cleavage efficacy. These findings shed light on a mechanism that furthers substrate recognition by caspases and provides potential insight into its regulation during apoptosis.

scholarly journals Trypanosoma brucei RNA Binding Proteins p34 and p37 Mediate NOPP44/46 Cellular Localization via the Exportin 1 Nuclear Export Pathway

2007 ◽  
Vol 6 (12) ◽  
pp. 2206-2213 ◽  
Author(s):  
Kristina Hellman ◽  
Kimberly Prohaska ◽  
Noreen Williams
Keyword(s):  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Nuclear Export ◽  
Cellular Localization ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Export Signal ◽  
Amino Acid Sequences ◽  
Protein Levels

ABSTRACT We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.

scholarly journals Sequence-Based Prediction of RNA-Binding Proteins Using Random Forest with Minimum Redundancy Maximum Relevance Feature Selection

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Xin Ma ◽  
Jing Guo ◽  
Xiao Sun
Keyword(s):  
Feature Selection ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Accurate Method ◽  
Amino Acid Sequences ◽  
Evolutionary Information ◽  
Sequence Information ◽  
Minimum Redundancy Maximum Relevance ◽  
Accuracy Of Prediction

The prediction of RNA-binding proteins is one of the most challenging problems in computation biology. Although some studies have investigated this problem, the accuracy of prediction is still not sufficient. In this study, a highly accurate method was developed to predict RNA-binding proteins from amino acid sequences using random forests with the minimum redundancy maximum relevance (mRMR) method, followed by incremental feature selection (IFS). We incorporated features of conjoint triad features and three novel features: binding propensity (BP), nonbinding propensity (NBP), and evolutionary information combined with physicochemical properties (EIPP). The results showed that these novel features have important roles in improving the performance of the predictor. Using the mRMR-IFS method, our predictor achieved the best performance (86.62% accuracy and 0.737 Matthews correlation coefficient). High prediction accuracy and successful prediction performance suggested that our method can be a useful approach to identify RNA-binding proteins from sequence information.

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