Muons penetrate much further than X-rays, they do essentially zero damage, and they are provided for free by the cosmos.
Image credit: Science Source/Digital Globe.
Contributed by Alexander Varshavsky, October 15, 2020 (sent for review September 25, 2020; reviewed by Thomas Arnesen and William P. Tansey)
Significance
The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degradation signals. In the present study, we compared wild-type human cells to their double-knockout (2-KO) counterparts that lacked the UBR1/UBR2 ubiquitin ligases of the Arg/N-degron pathway. We found that a number of specific genes were either strongly induced or strongly repressed in 2-KO cells. In addition, specific transcription factors, including glucocorticoid receptor, were identified here as physiological substrates of the Arg/N-degron pathway. We discuss the emerged role of this proteolytic system as a regulator of mammalian gene expression.
Abstract
The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degrons. Our previous work produced double-knockout (2-KO) HEK293T human cell lines that lacked the functionally overlapping UBR1 and UBR2 E3 ubiquitin ligases of the Arg/N-degron pathway. Here, we studied these cells in conjunction with RNA-sequencing, mass spectrometry (MS), and split-ubiquitin binding assays. 1) Some mRNAs, such as those encoding lactate transporter MCT2 and β-adrenergic receptor ADRB2, are strongly (∼20-fold) up-regulated in 2-KO cells, whereas other mRNAs, including those encoding MAGEA6 (a regulator of ubiquitin ligases) and LCP1 (an actin-binding protein), are completely repressed in 2-KO cells, in contrast to wild-type cells. 2) Glucocorticoid receptor (GR), an immunity-modulating transcription factor (TF), is up-regulated in 2-KO cells and also physically binds to UBR1, strongly suggesting that GR is a physiological substrate of the Arg/N-degron pathway. 3) PREP1, another TF, was also found to bind to UBR1. 4) MS-based analyses identified ∼160 proteins whose levels were increased or decreased by more than 2-fold in 2-KO cells. For example, the homeodomain TF DACH1 and the neurofilament subunits NF-L (NFEL) and NF-M (NFEM) were expressed in wild-type cells but were virtually absent in 2-KO cells. 5) The disappearance of some proteins in 2-KO cells took place despite up-regulation of their mRNAs, strongly suggesting that the Arg/N-degron pathway can also modulate translation of specific mRNAs. In sum, this multifunctional proteolytic system has emerged as a regulator of mammalian gene expression, in part through conditional targeting of TFs that include ATF3, GR, and PREP1.
Footnotes
- ↵1To whom correspondence may be addressed. Email: avarsh{at}caltech.edu.
Author contributions: T.T.M.V., D.C.M., S.P.G., and A.V. designed research; T.T.M.V. and D.C.M. performed research; T.T.M.V., D.C.M., S.P.G., and A.V. analyzed data; and T.T.M.V., D.C.M., S.P.G., and A.V. wrote the paper.
Reviewers: T.A., University of Bergen; and W.P.T., Vanderbilt University.
The authors declare no competing interest.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2020124117/-/DCSupplemental.
Data Availability.
All relevant data are included in the article and supporting information.
Published under the PNAS license.
References
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The Arg/N-degron pathway targets transcription factors and regulates specific genes
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- Biological Sciences
- Biochemistry
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