p97 Composition Changes Caused by Allosteric Inhibition Are Suppressed by an On-Target Mechanism that Increases the Enzyme's ATPase Activity

Her, Nam-Gu; Toth, Julia I.; Ma, Chen‐Ting; Yang, Wei; Motamedchaboki, Khatereh; Sergienko, Eduard; Petroski, Matthew D.

doi:10.1016/j.chembiol.2016.03.012

Cited by 36 publications

(56 citation statements)

References 40 publications

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“…An analysis of the effects of NMS873 on VCP-interacting proteins appeared while we were finalizing our manuscript (55). Although their mass spectrometry findings are generally consistent with our own (see Fig.…”

Section: Discussionsupporting

confidence: 75%

Valosin-containing protein (VCP)–Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor

Xue

Blythe

Freiberger

et al. 2016

Molecular & Cellular Proteomics

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Protein quality control (PQC) plays an important role in stemming neurodegenerative diseases and is essential for the growth of some cancers. Valosin-containing protein (VCP)/p97 plays a pivotal role in multiple PQC pathways by interacting with numerous adaptors that link VCP to specific PQC pathways and substrates and influence the post-translational modification state of substrates. However, our poor understanding of the specificity and architecture of the adaptors, and the dynamic properties of their interactions with VCP hinders our understanding of fundamental features of PQC and how modulation of VCP activity can best be exploited therapeutically. In this study we use multiple mass spectrometry-based proteomic approaches combined with biophysical studies to characterize the interaction of adaptors with VCP. Our results reveal that most VCP-adaptor interactions are characterized by rapid dynamics that in some cases are modulated by the VCP inhibitor NMS873. These findings have significant implications for both the regulation of VCP function and the impact of VCP inhibition on different VCP-adaptor complexes.

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Section: Discussionsupporting

confidence: 75%

Valosin-containing protein (VCP)–Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor

Xue

Blythe

Freiberger

et al. 2016

Molecular & Cellular Proteomics

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“…We expressed and purified p97-harboring mutations identified from CB-5083-resistant (P472L, Q473P, G481A, N660K, or T688A) and NMS-873-resistant (A530T, R567H, or L639F) HCT116 cells ( Fig. 1A) (35,39). Mutants previously shown to disrupt the NMS-873 analog binding in vitro (K615V or N616F (37)), MSP-1 mutants harboring R95G or R155H (12, 26, 33, Figure 1.…”

Section: P472l Mutation Increases P97 Atpase Activity and Desensitizementioning

confidence: 99%

“…NMS-873 exclusively targets D2 activity but relies on an allosteric mechanism requiring D2 with ATP bound to inhibit cooperative D2 ATP hydrolysis (37). We and others have found that prolonged exposure of cancer cell lines to cytotoxic concentrations of these inhibitors results in the emergence of resistant cells (35,38,39). The resistant cells harbor single amino acid mutations in D2 and the D1-D2 linker of p97 and are distinct from those found in MSP-1 patients ( Fig.…”

mentioning

confidence: 94%

Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors

Yang

Toth

Blanco

et al. 2018

Journal of Biological Chemistry

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The AAA ؉ ATPase p97 regulates ubiquitin-dependent protein homeostasis and has been pursued as a cancer drug target. The ATP-competitive inhibitor CB-5083 and allosteric inhibitor NMS-873 are the most advanced p97 inhibitors described to date. Previous studies have reported that their cytotoxicity can be readily overcome and involves single p97 mutations in the linker between the D1 and D2 ATPase domains and within D2. We report here that the proline 472 to leucine (P472L) mutation, in the D1-D2 linker and identified in CB-5083-resistant cells, desensitizes p97 to both inhibitor classes. This mutation does not disrupt the distinct D2-binding sites of the inhibitors. Instead, P472L changes ATPase domain communication within the p97 hexamer. P472L enhances cooperative D2 ATP binding and hydrolysis. This mechanism alters the function of the D1-D2 linker in the control of D2 activity involving the ATP-bound state of D1. Although increased D2 activity is sufficient to desensitize the P472L mutant to NMS-873, the mutant's desensitization to CB-5083 also requires D1 ATPase domain function. Our study highlights the remarkable adaptability of p97 ATPase domain communication that enables escape from mechanistically distinct classes of cytotoxic p97 inhibitors. . 2 The abbreviations used are: MSP-1, multisystem proteinopathy type-1; ITC, isothermal titration calorimetry; PDB, Protein Data Bank; PAM, protospacer adjacent motif; TR-FRET, time-resolved fluorescence energy transfer.

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“…With p97 inhibitor CB-5083 entering the realm of clinical trials, multiple studies have evaluated the resistance mechanisms towards different classes of p97 inhibitors. Her et al reported that a single heterozygous missense mutation in p97 (A530T) is sufficient to produce resistance to the p97 inhibitor NMS-873 in HCT116 cells [1]. Likewise, Anderson et al reported that single homozygous missense mutations in p97 are sufficient to produce resistance to the p97 inhibitor CB-5083 in HCT116 cells [2].…”

Section: Introductionmentioning

confidence: 99%

Heterozygous mutations in p97 and resistance to p97 inhibitors

Bastola

Minn

Chien

2018

Preprint

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In recent years, multiple studies including ours have reported on the mechanism of resistance towards p97 inhibitors. While all these studies outline target alteration via mutations in p97 as the primary mechanism of resistance, discrepancies persist in the current literature due to the occurrence of both heterozygous and homozygous mutations when using HCT116 cells. Here, we report a pre-existing heterozygous frameshift mutation at codon 616 (N616fs*) in one of the p97 alleles in HCT116 cells and show that this mutant allele is subjected to nonsense-mediated decay. Furthermore, we independently generated p97 inhibitor (CB-5083) resistant HCT116 cells, and we observed a single heterozygous mutation at codon 526 (L526S) in genomic DNA sequencing but a homozygous L526S mutation in complementary DNA sequencing, indicating that the missense mutation (L526S) occurs in the allele that does not harbor the frameshift N616fs* mutation. Our results underscore the importance of performing simultaneous genomic and complementary DNA sequencing when confirming mutations in p97.

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p97 Composition Changes Caused by Allosteric Inhibition Are Suppressed by an On-Target Mechanism that Increases the Enzyme's ATPase Activity

Cited by 36 publications

References 40 publications

Valosin-containing protein (VCP)–Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor

Valosin-containing protein (VCP)–Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor

Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors

Heterozygous mutations in p97 and resistance to p97 inhibitors

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