Recombinant Expression, Purification, and Assembly of p62 Filaments

Tarafder, Abul K.; Guesdon, Audrey; Kuhm, Tanja; Sachse, Carsten

doi:10.1007/978-1-4939-8873-0_1

Cited by 3 publications

(6 citation statements)

References 14 publications

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“…Mutations in the linker region or ZZ domain of p62 both resulted in reduced RNA binding and increased presence of multimers (Figure 2D and E). To distinguish whether in these cases the loss of RNA binding resulted in multimerization or conversely multimer formation displaced the RNA, we performed EMSAs with recombinant p62 that we forced into a low-oligomeric form by MBP-tagging [19,27,35]. We observed decreased vault RNA1-1 association for recombinant MBP-p62 K7A and R21A (Figure 2F, Supplementary Figure 2C and D), confirming the direct involvement of these residues in RNA binding.…”

Section: Assignment Of Critical Riboregulatory Functions To Lys7 and Arg21mentioning

confidence: 61%

“…Yet, their RNA-binding capacity was not significantly changed (Figure 2B, Supplementary Figure 2). In addition, we used maltose-binding protein (MBP)-tagging to generate recombinant p62 with low oligomerization potential [19,27,35] and tested in vitro binding of vault RNA1-1. This analysis confirms that the binding affinity of p62 is not affected by a double mutation of residues D69 and D73…”

Section: Assignment Of Critical Riboregulatory Functions To Lys7 and Arg21mentioning

confidence: 99%

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Mechanism of riboregulation of p62 protein oligomerisation by vault RNA1-1 in selective autophagy

Buescher

Horos

Haubrich

et al. 2021

Preprint

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Macroautophagy ensures the clearance of intracellular substrates ranging from single ubiquitinated proteins to large proteotoxic aggregates and defective organelles. The selective autophagy receptor p62 binds these targets and recruits them to double-membrane vesicles, which fuse with lysosomes to degrade their content. We recently uncovered that p62 function is riboregulated by the small non-coding vault RNA1-1. Here, we present detailed insight into the underlying mechanism. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within the RNA that mediates the specific p62 interaction. Our data define molecular determinants that govern mammalian autophagy via the p62-vault RNA1-1 riboregulatory pair.

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Section: Assignment Of Critical Riboregulatory Functions To Lys7 and Arg21mentioning

confidence: 61%

Section: Assignment Of Critical Riboregulatory Functions To Lys7 and Arg21mentioning

confidence: 99%

Mechanism of riboregulation of p62 protein oligomerisation by vault RNA1-1 in selective autophagy

Buescher

Horos

Haubrich

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Mutations in the linker region or ZZ domain of p62 both resulted in reduced RNA binding and increased presence of multimers (Figure 2D and E). To distinguish whether in these cases the loss of RNA binding resulted in multimerization or conversely multimer formation displaced the RNA, we performed EMSAs with recombinant p62 that we forced into a low-oligomeric form by MBP-tagging (Horos et al 2019;Reuten et al 2016;Tarafder et al 2019). We observed decreased vault RNA1-1 association for recombinant MBP-p62 K7A and R21A (Figure 2F, Supplementary Figure 2C and D), confirming the direct involvement of these residues in RNA binding.…”

Section: Hinge Residues K7 and R21 Are Necessary For Both Rna Binding...mentioning

confidence: 61%

“…Yet, their RNA-binding capacity is not significantly changed (Figure 2B, Supplementary Figure 2). In addition to the in cellulo experiments, we used maltose-binding protein (MBP)tagging to generate recombinant p62 with low oligomerization potential (Horos et al 2019;Reuten et al 2016;Tarafder et al 2019) and tested in vitro binding of vault RNA1-1. This analysis confirms that the RNA-binding affinity of p62 is not affected by a double mutation of residues D69 and D73 (Supplementary Figure 2A).…”

Section: Hinge Residues K7 and R21 Are Necessary For Both Rna Binding...mentioning

confidence: 99%

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Vault RNA1–1 riboregulates the autophagic function of p62 by binding to lysine 7 and arginine 21, both of which are critical for p62 oligomerization

Büscher

Horos

Huppertz

et al. 2022

RNA

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Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional and post-translational mechanisms. We have recently shown that the small, non-coding vault RNA1-1 negatively riboregulates p62 oligomerisation in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1-1 inhibits p62 oligomerisation are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within vault RNA1-1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein-protein interactions critical to the activation of specific autophagy.

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Functional expression and purification of recombinant full-length human ATG7 protein with HIV-1 Tat peptide in Escherichia coli

Dong

Zhao

Guo

et al. 2021

Protein Expression and Purification

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Recombinant Expression, Purification, and Assembly of p62 Filaments

Cited by 3 publications

References 14 publications

Mechanism of riboregulation of p62 protein oligomerisation by vault RNA1-1 in selective autophagy

Mechanism of riboregulation of p62 protein oligomerisation by vault RNA1-1 in selective autophagy

Vault RNA1–1 riboregulates the autophagic function of p62 by binding to lysine 7 and arginine 21, both of which are critical for p62 oligomerization

Functional expression and purification of recombinant full-length human ATG7 protein with HIV-1 Tat peptide in Escherichia coli

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