Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6

Okerberg, Eric; Dagbay, Kevin B.; Green, Jennifer L.; Soni, Ishankumar V; Aban, Arwin; Nomanbhoy, Tyzoon; Savinov, Sergey N.; Hardy, James L.; Kozarich, John W.

doi:10.1021/acs.biochem.9b00290

Cited by 9 publications

(10 citation statements)

References 30 publications

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“…Closer in function to caspases, granzyme A and granzyme B, which can also induce cell death in a caspase-dependent (granzyme B) or independent (granzyme A) manner, bind RNA and DNA with nanomolar affinity and cleave RNA-BPs faster in the presence of nucleic acids (30). Our findings expand the body of evidence for the existence of regulatory sites on caspases, including by metallic ions (31) and small molecules like the nucleotide ATP (32). With our findings, these findings also increase the potential for design of caspase modulators targeting regions other than the canonical catalytic site and maybe achieve better selectivity.…”

Section: Discussionsupporting

confidence: 74%

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

Desroches

Denault

2019

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Discussionsupporting

confidence: 74%

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

Desroches

Denault

2019

Proc. Natl. Acad. Sci. U.S.A.

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“…However, after 24 h aging, the measurement declines to 0.15 mM and 0.13 mM, respectively. The results are consistent with previous studies, 17,30 which indicated that, without the interference of other substances in the cells, the developed method can be used for monitoring ATP concentrations in cell extracts.…”

Section: Resultssupporting

confidence: 92%

“…ATP can be generated from photosynthesis, cellular respiration, and multifunctional nucleoside. In many cellular movement and activities of metabolism and signaling, a plenty of enzymes consume ATP when the energy is moved throughout the cells in the biological processes, like muscle contractions, ions and molecules movement, biosynthesis of significant molecules, digestion and absorption of food, [14][15][16][17] etc. Additionally, during the synthesis process of DNA replication and transcription, ATP can be incorporated into nucleic acid through polymerases.…”

Section: Introductionmentioning

confidence: 99%

Label-Free Fluorescent Aptasensor for Adenosine Triphosphate Detection Using SYBR Gold as a Probe

et al. 2021

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In this experimental research, a label-free sensing strategy is developed and employed to detect adenosine triphosphate with utilization of aptamers, including exonuclease I and SYBR Gold. The conformation of aptamers bonding to the specific target molecule (ATP) is transformed into an antiparallel G-quadruplex structure from a random coil. Afterwards, considering the unfolded aptamers are the preferred substrates for exonuclease I, the addition of exonuclease I is used so as to digest unfolded aptamers in the mixture in a selective manner. In the follow-up study, in order to strengthen the fluorescence intensity, SYBR Gold is applied as a fluorescent probe. The aptasensor presents the features of high selectivity against adenosine triphosphate and the low detecting limit of concentrations (39.2 nM). In order to verify the validation of experimental procedures and the practical application of the aptasensor, the detection of adenosine triphosphate for human serum samples is performed with satisfactory success. The recovery result with the range of 93.8%–108.1% is desirable and suggests that the designed approach is applicable. The outcomes of the cellular adenosine triphosphate assay manifest that the level of adenosine triphosphate concentrations in cell extracts can be monitored without the interference of other substances in the cells. Subject to its advantageous benefits (cost-effective, easiness, rapidity, and extraordinary selectivity), the designed approach has a promising implication for adenosine triphosphate detection in the research domain of bioanalytical science and biology.

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“…31 Caspase-6 constructs were purified as previously described via the procaspase-6 (caspse-6 C163S) purification protocol. 32 Caspase-9 was purified as previously described via the caspase-9 full-length purification protocol. 31 The purity and concentrations of purified caspases were assessed by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE), and aliquots were stored at −80 °C until further usage for different assays.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence

Soni

Hardy

2021

Biochemistry

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Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4–P1′ sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4–P1′ sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.

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Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6

Cited by 9 publications

References 30 publications

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

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

Label-Free Fluorescent Aptasensor for Adenosine Triphosphate Detection Using SYBR Gold as a Probe

Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence

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