Characterization of mammalian N-degrons and development of heterovalent inhibitors of the N-end rule pathway

Jiang, Yanxialei; Pore, Subrata K.; Lee, Jung Hoon; Sriram, Shashi; Khanh, Binh; Han, Dong Hoon; Agarwalla, Pritha; Zakrzewska, Adriana; Kim, Yong-Ho; Banerjee, Rajkumar; Lee, Seung-Han; Lee, Minjae

doi:10.1039/c3sc51059j

Cited by 11 publications

(20 citation statements)

References 37 publications

(45 reference statements)

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“…Herein, we focus on recent efforts to develop small-molecule modulators of the mammalian N-end rule pathway through rational design, high-throughput screening (HTS), and chemical engineering. Of note is a recently identified N-end rule inhibitor, para -chloroamphetamine (PCA), which potently delays the degradation of RGS4 (regulator of G protein signaling 4), a bona fide in vivo N-end rule substrate in the mouse brain [30, 31]. Given that the components in the pathway and their functions are emerging as novel therapeutic targets, we also review recent findings about the biochemical mechanisms and physiological functions of the mammalian N-end rule pathway.…”

Section: The N-end Rule Pathwaymentioning

confidence: 99%

“…This conjecture was experimentally tested using RF-C11, a synthetic heterovalent compound that has both an N-terminal Arg (type 1) and an N-terminal Phe (type 2) chemically linked by two C11 hydrocarbon chains (Figure 3B) [30, 100]. Compared with homovalent controls such as RR-C11 (with two N-terminal Arg) and FF-C11 (with two N-terminal Phe) and monovalent dipeptides such as Arg-Ala and Phe-Ala, RF-C11 showed significantly higher inhibitory efficacy for in vitro ubiquitination and degradation of model N-end rule substrates.…”

Section: Chemical Modulation Of the N-end Rule Pathwaymentioning

confidence: 99%

“…When the efficacy to inhibit the in vitro degradation of Arg-nsP4 was measured, the IC 50 value of RF-C11 was determined to be 16 μM as compared with the homovalent control RR-C11 (67 μM) and the monovalent control Arg-Ala (283 μM) [25]. Similarly, the IC 50 value of RF-C11 for Tyr-nsP4 degradation was determined to be 2.7 μM, which was markedly lower than those of FF-C11 (151 μM) and Phe-Ala (21 μM) [30]. RF-C11 also effectively inhibited the degradation of RGS4, a physiological N-end rule substrate, in living cells without noticeable cytotoxicity [30].…”

Section: Chemical Modulation Of the N-end Rule Pathwaymentioning

confidence: 99%

“…Similarly, the IC 50 value of RF-C11 for Tyr-nsP4 degradation was determined to be 2.7 μM, which was markedly lower than those of FF-C11 (151 μM) and Phe-Ala (21 μM) [30]. RF-C11 also effectively inhibited the degradation of RGS4, a physiological N-end rule substrate, in living cells without noticeable cytotoxicity [30]. In vivo efficacy of RF-C11 was further supported by the finding that RF-C11 delays cell-autonomous hypertrophism in cultured primary cardiomyocytes, identifying a previously unknown function of the N-end rule pathway [100].…”

Section: Chemical Modulation Of the N-end Rule Pathwaymentioning

confidence: 99%

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Pharmacological Modulation of the N-End Rule Pathway and Its Therapeutic Implications

Lee

Jiang

Kwon

et al. 2015

Trends in Pharmacological Sciences

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The N-end rule pathway is a proteolytic system in which single N-terminal amino acids of short-lived substrates determine their metabolic half-lives. Substrates of this pathway have been implicated in the pathogenesis of many diseases, including malignancies, neurodegeneration, and cardiovascular disorders. This review provides a comprehensive overview of current knowledge about the mechanism and functions of the N-end rule pathway. Pharmacological strategies for the modulation of target substrate degradation are also reviewed, with emphasis on their in vivo implications. Given the rapid advances in structural and biochemical understanding of the recognition components (N-recognins) of the N-end rule pathway, small-molecule inhibitors and activating ligands of N-recognins emerge as therapeutic agents with novel mechanisms of action.

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Section: The N-end Rule Pathwaymentioning

confidence: 99%

Section: Chemical Modulation Of the N-end Rule Pathwaymentioning

confidence: 99%

Section: Chemical Modulation Of the N-end Rule Pathwaymentioning

confidence: 99%

Section: Chemical Modulation Of the N-end Rule Pathwaymentioning

confidence: 99%

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Pharmacological Modulation of the N-End Rule Pathway and Its Therapeutic Implications

Lee

Jiang

Kwon

et al. 2015

Trends in Pharmacological Sciences

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“…[11][12][13] In a previous study, we confirmed that the L-conformation, protonated a-amine group, and hydrophobic side chains are essential N-degron structural components required for direct interaction with N-recognins. 14 Based on this conformational information, several small, biocompatible Arg/N-end rule inhibitors have been developed to target both the UBR box and the N-domain simultaneously. 7 Among these inhibitors, the sympathomimetic amine derivative, para-chloroamphetamine (PCA) is a potent N-end rule inhibitor, which not only blocks degradation of N-end rule reporter substrates in cultured cells, but also effectively delays degradation of its physiological substrates such as RGS4 (regulator of G-protein signaling 4) in mouse brain tissues.…”

Section: Introductionmentioning

confidence: 99%

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

Jiang

Lee

et al. 2016

Autophagy

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The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.

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N‐end rule pathway inhibitor sensitizes cancer cells to antineoplastic agents by regulating XIAP and RAD21 protein expression

Pore

Ganguly

et al. 2019

J of Cellular Biochemistry

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Anticancer drugs exert their effects on cancer cells by deregulating many pathways linked to cell cycle, apoptosis, etc. but cancer cells gradually become resistive against anticancer drugs, thereby necessitating the development of newer generation anticancer molecules. N‐end rule pathway has been shown to be involved in the degradation of many cell cycle and apoptosis‐related proteins. However, the involvements of this pathway in cancer are not well established. Recently, we developed a non‐peptide‐based N‐end rule pathway inhibitor, RF‐C11 for type 1 and 2 recognition domains of E3 ubiquitin ligases. The inhibitor significantly increased the half‐life of potential N‐degrons leading to significant physiological changes in vivo. We hypothesized RF‐C11 may be used to decipher the N‐end rule pathway's role in cancer towards the development of anticancer therapeutics. In this study, we showed that RF‐C11, barring noncancer cells, significantly sensitizes cancer cells towards different anticancer agents tested. We further find that the profound cellular sensitization to anticancer drugs was affected by (a) downregulation of X‐linked inhibitor of apoptosis protein, an antiapoptotic protein and (b) by stabilization of RAD21, and thereby inhibiting metaphase to anaphase promotion. The study shows that RF‐C11 or its analogs may be used as a novel additive in combination therapy against cancer.

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Characterization of mammalian N-degrons and development of heterovalent inhibitors of the N-end rule pathway

Cited by 11 publications

References 37 publications

Pharmacological Modulation of the N-End Rule Pathway and Its Therapeutic Implications

Pharmacological Modulation of the N-End Rule Pathway and Its Therapeutic Implications

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

N‐end rule pathway inhibitor sensitizes cancer cells to antineoplastic agents by regulating XIAP and RAD21 protein expression

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