Regulation of PKR by RNA: Formation of Active and Inactive Dimers

Husain, Bushra; Hesler, Stephen; Cole, James L.

doi:10.1016/j.bpj.2015.11.1865

Cited by 5 publications

(11 citation statements)

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“…A minimum length of 30 bp of dsRNA is required to induce PKR autophosphorylation (Manche et al 1992; Lemaire et al 2008;Husain et al 2012) and dimerization of PKR on dsRNA (Husain et al 2015). Consistent with the dimerization model, dsRNAs capable of inducing PKR activation bind at least two PKR monomers (Lemaire et al 2008;Husain et al 2012) and induce formation of the kinase domain dimer (Husain et al 2015).…”

Section: Introductionmentioning

confidence: 59%

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Activation of PKR by short stem–loop RNAs containing single-stranded arms

Mayo

Wong

Lopez

et al. 2016

RNA

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Protein kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains a C-terminal kinase domain and two tandem dsRNA binding domains. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA of 30 bp is required for activation. However, short (∼15 bp) stem-loop RNAs containing flanking single-stranded tails (ss-dsRNAs) are capable of activating PKR. Activation was reported to require a 5 ′ -triphosphate. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15-nt single-stranded tails and a 15-bp stem and made systematic truncations of the tail and stem regions. Autophosphorylation assays and analytical ultracentrifugation experiments were used to correlate activation and binding affinity. PKR activation requires both 5 ′ -and 3 ′ -single-stranded tails but the triphosphate is dispensable. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. These results indicate that the single-stranded regions bind to PKR and support a model where ss-dsRNA induced dimerization is required but not sufficient to activate the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single-stranded regions may contribute to PKR activation in these cases.

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Section: Introductionmentioning

confidence: 59%

“…Therefore, we recently developed a sensitive homo-FRET assay to directly probe PKR kinase domain dimerization on RNAs (Husain et al 2015). Activating duplex RNAs containing 30 bp bind two PKRs and induce dimerization.…”

Section: Discussionmentioning

confidence: 99%

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Mayo

Wong

Lopez

et al. 2016

RNA

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“…It is interesting that cytosolic viral RNA can also serve as cGAS activating ligand by forming DNA: RNA hybrid . In general, it should be noted that some viral RNA intermediately transcribed from the cytoplasmic genome of a DNA virus can be also be recognized by cytosolic RNA sensors . Moreover, DNA reverse transcribed from genomic RNA of retroviruses can be recognized by cytosolic DNA sensors …”

Section: Pattern Recognition Receptors For Viral Nucleic Acidsmentioning

confidence: 99%

“…Protein kinase R is a cytosolic RNA‐sensing protein that is activated by viral dsRNAs of variable length. During the activation process, PKR undergoes autophosphorylation and then phosphorylates the translation initiation factor eIF2α (Figure ), leading to arrested translation of both cellular and viral mRNAs and even to apoptosis . Protein kinase R also participates in a signal transduction pathway that further upregulates IFN‐beta transcription (Figure ).…”

Section: Pattern Recognition Receptors For Viral Nucleic Acidsmentioning

confidence: 99%

Defects in interferon pathways as potential biomarkers of sensitivity to oncolytic viruses

Matveeva

Chumakov

2018

Reviews in Medical Virology

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Increased sensitivity of cancer cells to viruses is a prerequisite for the success of oncolytic virotherapy. One of the major causes of such a phenotype is the disruption of innate antiviral defenses associated with dysfunction of type 1 interferons (IFNs) that permits unlimited replication of viruses in cancer cells. Defects in IFN pathways help cancer progression by providing additional advantages to tumor cells. However, while these defects promote the survival and accelerated proliferation of malignant cells, they facilitate viral replication and thus enhance the efficiency of viral oncolysis. This review describes a broad spectrum of defects in genes that participate in IFN induction and IFN response pathways. Expression levels and/or functional activities of these genes are frequently low or absent in cancer cells, making them sensitive to virus infection. Therefore, certain specific defects in IFN signaling cascades might serve as potential biomarkers to help in identifying individual cancer patients who are likely to benefit from oncolytic virotherapy.

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“…This allows no significant limit to the concentration ranges explored, besides solubility, and has the further advantage of being useful in serum, cell, and tissue extracts. As described above, this approach also allows a constant fluorescence signal to be used and thus provides better signal/noise characteristics for data analysis (Husain et al 2015;Lyons et al 2013). Our preference is ~ 500-1000 counts (out of 4096 max), which is a function of label efficiency, but generally means 100-200 nM in typical mAb samples.…”

Section: Discussionmentioning

confidence: 99%

Analysis of nonideality: insights from high concentration simulations of sedimentation velocity data

et al. 2020

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The Aviv fluorescence detection system (Aviv-FDS) has allowed the performance of sedimentation velocity experiments on therapeutic antibodies in highly concentrated environments like formulation buffers and serum. Methods were implemented in the software package SEDANAL for the analysis of nonideal, weakly associating AUC data acquired on therapeutic antibodies and proteins (Wright et al. Eur Biophys J 47:709–722, 2018, Anal Biochem 550:72–83, 2018). This involved fitting both hydrodynamic, ks, and thermodynamic, BM1, nonideality where concentration dependence is expressed as s = so/(1 + ksc) and D = Do(1 + 2BM1c)/(1 + ksc) and so and Do are values extrapolated to c = 0 (mg/ml). To gain insight into the consequences of these phenomenological parameters, we performed simulations with SEDANAL of a monoclonal antibody as a function of ks (0–100 ml/g) and BM1 (0–100 ml/g). This provides a visual understanding of the separate and joint impact of ks and BM1 on the shape of high-concentration sedimentation velocity boundaries and the challenge of their unique determination by finite element methods. In addition, mAbs undergo weak self- and hetero-association (Yang et al. Prot Sci 27:1334–1348, 2018) and thus we have simulated examples of nonideal weak association over a wide range of concentrations (1–120 mg/ml). Here we demonstrate these data are best analyzed by direct boundary global fitting to models that account for ks, BM1 and weak association. Because a typical clinical dose of mAb is 50–200 mg/ml, these results have relevance for biophysical understanding of concentrated therapeutic proteins.

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Regulation of PKR by RNA: Formation of Active and Inactive Dimers

Cited by 5 publications

References 0 publications

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Defects in interferon pathways as potential biomarkers of sensitivity to oncolytic viruses

Analysis of nonideality: insights from high concentration simulations of sedimentation velocity data

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